CN112899233A - Method for constructing acute lymphocytic leukemia mouse model - Google Patents

Abstract

The invention discloses a method for constructing an acute lymphocytic leukemia mouse model, which comprises the following steps: preparing a leukemia cell line suspension with high ERG expression, using a shERG packaged by lentivirus to knock down the ERG protein expression of the cell line, transfecting Luciferase into wild type and shERG cell lines by lentivirus, inoculating the cell line to an immune deficient mouse, evaluating the tumor process by a small animal imaging system, evaluating the treatment effect of a specific medicament on the acute lymphocytic leukemia with high ERG expression by the small animal imaging system and a flow analysis method, and the like. The model evaluates the efficacy of a particular drug against a subtype of acute lymphocytic leukemia. The disease model construction method provided by the invention can directly and non-invasively evaluate the tumor development process of the model animal and has very high success rate. In addition, the disease models established according to the methods of the present application have high clinical relevance.

Description

A method for constructing a mouse model of acute lymphoblastic leukemia

technical field

The invention relates to the field of leukemia disease models and construction methods thereof, in particular to a construction method of an acute lymphoblastic leukemia mouse model.

Background technique

Acute Lymphoblastic Leukemia (ALL) is a common and severe life-threatening malignancy in children. The incidence in children less than 10 years old is about 3/100000-4/100000). At present, it can be divided into B cell type (B-ALL) and T cell type (T-ALL) according to their different cell types. The current treatment regimens for ALL include risk-adapted treatment regimens, including combination chemotherapy, targeted therapy, and hematopoietic stem cell transplantation. Combination chemotherapy mainly uses vinblastine, cytarabine, methotrexate and so on. Due to the continuous improvement of treatment technology, the prognosis of ALL has been greatly improved, and the 5-year survival rate of ALL patients has reached 80%. However, there are still 20% of ALL patients, especially those in the high-risk group, whose disease is difficult to relieve or easy to relapse. Therefore, it is still urgent to explore the relapse mechanism and new treatment methods for these ALL patients. These require reliable and highly anthropomorphic models of leukemia to support. The biological characteristics of leukemia cells cultured in vitro are usually different from those of leukemia cells in vivo, and experiments in humans will lead to extremely high costs and risks. Therefore, the corresponding animal model is an in-depth study of leukemia. important and indispensable part.

Mice are an ideal animal model commonly used in leukemia research. Corresponding mouse models can be obtained for malignant tumors such as myeloid and lymphoid systems of the human blood system. However, the existing humanized mouse models still have a low success rate, Due to the lack of clinical relevance and other defects, the construction of a leukemia model with high specificity and high anthropomorphism is extremely important for leukemia research.

SUMMARY OF THE INVENTION

The invention provides a method for constructing a mouse model of acute lymphoblastic leukemia. The disease model construction method provided by the present invention is a method for a specific subtype of acute lymphoblastic leukemia, and has a very high success rate. Furthermore, disease models established according to the methods of the present invention are highly specific and clinically relevant.

A method for constructing a mouse model of acute lymphoblastic leukemia, comprising the following steps:

a) Acute lymphoblastic leukemia cell lines with high ERG expression were purchased from the cell bank, and after the identification of the cell lines, the cells were expanded and cultured, and the cells were extracted for protein. Lymphocytic leukemia cell line, the identified cell line is amplified and cultured, the leukemia cell suspension to be inoculated is collected, and the concentration is adjusted to obtain the cell line;

In step a), in the cell line, the final density of leukemia cells in the leukemia cell suspension to be inoculated is adjusted to be 0.5× ^{10 7} to 2× ^{10 7} /ml, preferably 1× ^{10 7} /ml.

b) lentiviral transfection to construct the luciferase-labeled cell line described in a), after transfection, measure the bioluminescence value of the cell suspension to determine the success rate of transfection;

In step b), use the steady-Glo luciferase assay systemc to measure the bioluminescence value of the cell suspension.

c) Packaging and transfection of shRNA ERG recombinant lentivirus Step b) The obtained acute lymphoblastic leukemia cell line with high expression of ERG, and subsequent identification was performed to obtain an acute lymphoblastic leukemia cell line with silent ERG protein, flow cytometry The successfully transfected cells were sorted by cytometer and continued to be cultured; the wild-type cell line was cultured at the same time, and the proliferation rate of the two cells was compared;

d) Determine the success rate of transfection of sorted cells: observe the proportion of cells with GPF green fluorescence under a fluorescence microscope; expand sorted cells and collect cells to extract cytosol and nuclear proteins, and detect target knockout The expression of protein ERG, if the expression of ERG protein is lower than 70% of wild-type cells, can be used for subsequent experiments; the identified leukemia cells are expanded and cultured, the leukemia cell suspension to be inoculated is collected, and the concentration is adjusted to obtain the target cell suspension. liquid;

In step d), the final density of leukemia cells in the leukemia cell suspension to be inoculated is adjusted to be 0.5× ^{10 7} to 2× ^{10 7} /ml, preferably 1× ^{10 7} /ml, to obtain the target cell suspension.

e) Select immunodeficient mice according to the type of model to be prepared, and construct a mouse model of acute lymphoblastic leukemia.

In step e), the immunodeficient mice are selected according to needs, such as preparing an in situ acute lymphoblastic leukemia mouse model, a severe combined immunodeficiency model such as NSG, etc. is selected; if preparing an ectopic acute lymphoblastic leukemia mouse model The mouse model is selected from immune-deficient mice such as nude mice or Scid.

For the acute lymphoblastic leukemia model, use the identified wild-type leukemia cells obtained in a) to prepare the leukemia cell suspension to be inoculated; for the study of the effect of ERG protein on the tumor progression such as nesting, proliferation and metastasis during the onset of leukemia , using the shERG leukemia cells obtained from d) to prepare a leukemia cell suspension to be inoculated.

Construction of acute lymphoblastic leukemia mouse model, including:

Inoculate the cell line obtained in step a) and the target cell suspension obtained in step d) into the peripheral blood of e) immunodeficiency mice or immunodeficiency mice of different degrees to prepare in situ or ectopic models .

The cells with green fluorescence under the fluorescence microscope are leukemia cells with knockdown of ERG protein expression.

The leukemia type is acute lymphoblastic leukemia, and the immunodeficient mouse is NSG mouse, Scid or nude mouse.

The method further includes monitoring the tumor processes such as nesting, proliferation, and metastasis in the mice of the human leukemia cells (1a, 1d) with bioluminescence in the peripheral blood of the mice injected and inoculated by a small animal imaging system .

A mouse humanized leukemia model prepared by the method.

A leukemia drug screening method, comprising:

a) obtaining the mouse humanized leukemia model;

b) The small animal imaging system monitors the fluorescein value of the humanized cells of the mouse humanized leukemia model. In the in situ model, when the abdomen or bilateral femurs of the mouse are imaged in the abdominal position, fluorescence appears, and the maximum value is greater than 500 , and administer the candidate drug to the model; in the ectopic model, the candidate drug can be administered to the model when fluorescence occurs at the inoculation site.

c) After the drug candidate is administered, the small animal imaging system continues to regularly monitor the changes in the intensity and position of fluorescein in the model humanized leukemia cells to evaluate the efficacy of the drug candidate.

After the drug candidate is administered, the small animal imaging system monitors the changes in the fluorescence value of the humanized leukemia cells within 2 to 4 weeks of administration, and collects the bone marrow and peripheral blood of the mice whose fluorescence value increases slowly or stops. Flow cytometry analysis of the proportion of human (hu) and murine (mu) CD45+ cells when the proportion of huCD45+ cells in the peripheral blood cells of the model remains below 1% for at least 1 week , determine that the candidate drug is effective.

A leukemia drug screening system, comprising the leukemia humanized disease model.

The drug screening system further includes one or more candidate drugs.

In one aspect, the application provides a method for preparing a humanized disease model of leukemia, the method comprising:

a) Acute lymphoblastic leukemia cell lines with high expression of ERG were purchased from the cell bank, and after identification of the cell lines, the cells were expanded and cultured, and the cells were collected to extract protein, and the expression of ERG protein was identified by western blotting;

b) Amplifying and culturing the identified cell line, collecting the leukemia cell suspension to be inoculated, and adjusting the final density of leukemia cells in the leukemia cell suspension to be inoculated to 1× ^{10 7} /ml.

c) Packaging of shRNA ERG recombinant lentivirus and transfection of acute lymphoblastic leukemia cell lines with high expression of ERG, and subsequent identification to obtain acute lymphoblastic leukemia cell lines with silent ERG protein.

d) Sort the successfully transfected cells in c) by flow cytometry, and continue to culture; at the same time, the wild-type cell line is cultured, and the proliferation rate of the two cells is compared.

e) Judgment d) Transfection success rate of sorted cells: the proportion of cells with GPF green fluorescence observed under a fluorescence microscope; amplify sorted cells and collect cells to extract cytosol and nuclear proteins, WB detection The expression of the target knockout protein ERG, the ERG protein expression is lower than 70% of the wild-type cells can be used for subsequent experiments; the identified leukemia cells are expanded and cultured, the leukemia cell suspension to be inoculated is collected, and the The final density of leukemia cells in the leukemia cell suspension to be inoculated is 1× ^{10 7} /ml.

f) Select appropriate immunodeficient mice according to the type of model to be prepared, such as preparing an in situ acute lymphoblastic leukemia mouse model, select a severe combined immunodeficiency model such as NSG, etc.; such as preparing an ectopic acute lymphoblastic leukemia The mouse model is selected from immune-deficient mice such as nude mice or Scid. The leukemia cell suspension to be inoculated obtained from a and d) is injected into the immunodeficiency mice of different degrees described in e) to prepare an orthotopic or ectopic model.

In certain embodiments, the successfully transfected leukemia cells are leukemia cells with green fluorescence under a fluorescence microscope, and the final density of leukemia cells in the leukemia cell suspension to be inoculated is adjusted to be 1× ^{10 7} /ml.

In certain embodiments, the leukemia type is acute lymphoblastic leukemia, and the immunodeficient mice are NSG mice, Scid or nude mice, all of which are 4-6 week old mice.

In certain embodiments, the method comprises monitoring tumor processes such as nesting, proliferation, metastasis, etc., of the bioluminescent human leukemia cells in the peripheral blood of the mice injected and inoculated by a small animal imaging system. , the monitoring time points are 3 days, 7 days, 10 days, 14 days, 17 days, 21 days, 24 days, and 28 days after vaccination.

In certain embodiments, the mouse model is a humanized leukemia model, divided into an orthotopic humanized mouse model and an ectopic humanized mouse model.

and When the maximum value is greater than 500, the candidate drug is administered to the model; in the ectopic model, the candidate drug can be administered to the model when fluorescence occurs at the inoculation site.

In certain embodiments, after administration of the drug candidate, the small animal imaging system continues to regularly monitor changes in the intensity and location of fluorescein in the model humanized leukemia cells to assess the efficacy of the drug candidate, and the monitoring time point is the administration of 1 day, 3 days, 7 days, 10 days, 14 days, 17 days, 21 days, 24 days, 28 days after the drug.

In certain embodiments, after administration of the drug candidate, a small animal imaging system monitors the change in the fluorescence value of the humanized leukemia cells within 2 weeks to 4 weeks of administration, and collects the group of mice whose fluorescence value increases slowly or stops. The proportion of human (hu) and murine (mu) CD45 ⁺ cells was analyzed by flow cytometry, when the proportion of huCD45 ⁺ cells in the peripheral blood cells of the model remained below 1%, When the ratio is maintained for at least 1 week, the candidate drug is determined to be effective.

In certain embodiments, the drug screening system further includes one or more candidate drugs.

In certain embodiments, the candidate drug is selected from one or more of the following group: ERG-inhibiting small molecule polypeptide drug EIP and antitumor drug cytarabine, vincristine, tranexamic acid, dexamethasone Metasone, doxorubicin, L-asparagine, topotecan, clofarabine, carfilzomib, temsirolimus, dasatinib, bortezomib, SAR3419, all-trans retinoic acid, SAHA, su Nitinib, cyclophosphamide, and retinolamine. A single drug or a combination of a small molecule polypeptide drug that can inhibit the expression of ERG protein and a classic anti-leukemia drug can be selected. For example, the drug candidate may include a combination of small molecule polypeptide EIP and cytarabine, and the drug is administered to the model in the following manner: administered by intraperitoneal injection, and the amount of each administration is: the EIP group is administered according to 10 mg/ The dose of kg is administered 5 times a week for 2-4 consecutive weeks; cytarabine is administered at a dose of 250 mg/kg, twice a week for 2 consecutive weeks; combined administration In the group, the two drugs were co-administered on a group of mice according to their respective doses. For example, the drug candidate may comprise a combination of small molecule polypeptide EIP and SAHA, and the drug is administered to the model in the following manner: by intraperitoneal injection, each administration amount is: EIP group at a dose of 10 mg/kg Administration, 5 times a week, continuous administration for 2-4 weeks; SAHA is administered at a dose of 100 mg/kg, 5 times a week, continuous administration for 2-4 weeks; the combined administration group has two The drugs were administered in combination to a group of mice at their respective doses. For example, the drug candidate may include doxorubicin administered alone, and the doxorubicin administered to the model by intravenous injection at 0.5-3 mg/kg once a week and Continue administration for 4-6 weeks. For example, the drug candidate may include L-asparagine, and the L-asparagine is administered to the model by intraperitoneal injection in an amount of 500-2000 KU/kg once daily and Continue administration for 4-6 weeks. In certain embodiments, the drug candidate comprises topotecan, and the topotecan is administered to the model by intraperitoneal injection in an amount of 0.1-5 mg/kg, administered daily Once administered for 4-8 weeks, with two weeks of dosing followed by one week off. In certain embodiments, the drug candidate comprises temsirolimus, and the temsirolimus is administered to the model by intraperitoneal injection in an amount of 5-50 mg/kg per administration , once daily for 1-3 weeks. In certain embodiments, the drug candidate comprises dasatinib, and the dasatinib is administered to the model by oral administration in an amount of 5-50 mg/kg daily Administer once and continue for 4-6 weeks. In certain embodiments, the drug candidate comprises bortezomib, and the bortezomib is administered to the model by intraperitoneal injection in an amount of 0.1-20 mg/kg, administered weekly 2 times for 4-8 weeks. In certain embodiments, the drug candidate comprises SAR3419, and the SAR3419 is administered to the model by intraperitoneal injection in an amount of 0.5-50 mg/kg, administered once a week and continuously Administer for 4-8 weeks. In certain embodiments, the drug candidate comprises all-trans retinoic acid, and the all-trans retinoic acid is administered to the model by intraperitoneal injection in an amount of 0.5-10 mg/kg per administration , administered once a week and continued for 4-6 weeks. In certain embodiments, in certain embodiments, the drug candidate comprises cyclophosphamide, and the cyclophosphamide is administered to the model by intraperitoneal injection in an amount of 30- 300 mg/kg administered once daily for 4-6 weeks. In certain embodiments, the drug candidate comprises retinoid, and the retinoid is administered to the model by intraperitoneal injection in an amount of 20-200 mg/kg per administration , once daily for 4-6 weeks.

Compared with the prior art, the present invention has the following advantages:

The model of the invention evaluates the efficacy of a specific drug on a subtype of acute lymphoblastic leukemia, and uses the constructed method to evaluate the drug efficacy of a specific drug on a subtype of acute lymphoblastic leukemia.

The method includes: preparing a suspension of a leukemia cell line with high expression of ERG, knocking down the ERG protein expression of the aforementioned cell line with lentivirus-packaged shERG, transfecting Luciferase with lentivirus into wild-type and shERG cell lines, and inoculating the cell lines on Immunodeficiency mice, small animal imaging system to evaluate tumor progression, small animal imaging system and flow analysis method to evaluate the therapeutic effect of specific drugs on this type of acute lymphoblastic leukemia with high ERG expression.

The disease model construction method provided by the present invention can directly and non-invasively evaluate the tumor development process of the model animal, and has a very high success rate. Furthermore, disease models established according to the methods of the present application have high clinical relevance.

Description of drawings

Figure 1 is an example, lentivirus stably transfected leukemia cell line;

Figure 2 is an example, the polypeptide drug obviously inhibits the expression of ERG protein in HAL-01 cell line;

Fig. 3 is the result of transfection of shRNA ERG recombinant lentivirus in one example;

Figure 4 is an example of observing tumor processes such as nesting, invasion and metastasis of human cells in mice by non-invasive small animal imaging methods;

Figure 5 is an example, flow cytometry analysis of human-derived cells and mouse-derived cells found that human-derived leukemia cells mainly nest in mouse bone marrow;

Fig. 6 is an example, inoculation of HAL-01 cell line carrying stable shRNA ERG in NSG mice delays the tumor progression of leukemia model;

Figure 7 is an example, inoculation of NSG mice with combined administration of HAL-01 cell line delays the tumor progression of the leukemia model.

Detailed ways

Other aspects and advantages of the present disclosure can be readily appreciated by those skilled in the art from the following detailed description. Only exemplary embodiments of the present disclosure are shown and described in the following detailed description. As those skilled in the art will recognize, this disclosure enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention to which this application relates. Accordingly, the drawings and descriptions in the specification of the present application are only exemplary and not restrictive.

The invention to which this application relates is set forth with particularity characteristic of the appended claims. The features and advantages of the inventions involved in this application can be better understood by reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the drawings is as follows:

The embodiments of the invention of the present application are described below with specific specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the contents disclosed in this specification.

In this application, the term "humanization" generally refers to making a non-human organism or its tissues, cells, gene expression products, genes, etc. to carry human genes, gene expression products, cells and/or tissues.

In this application, the term "disease model" generally refers to an individual with disease-mimicking manifestations, which may be, for example, animals, plants, microorganisms, and the like. In certain embodiments, the disease model is formed by an animal (eg, mouse, rat, etc.).

In this application, the term "leukemia" generally refers to a disease caused by the massive proliferation and accumulation of leukemia cells, usually starting in the bone marrow.

In this application, the term "acute lymphoblastic leukemia (ALL)" generally refers to hematological malignancies caused by abnormal proliferation of lymphocytes in the body.

In this application, the term "cell suspension" generally refers to a suspension of cells formed by one or more cells dispersed in a liquid, wherein the cells may be separated as individuals or in groups of no more than about 50, no more than about In clumps of 40, no more than about 30, no more than about 20, no more than about 10 or fewer cells.

In this application, the term "leukemia cell" generally refers to immature leukocytes resulting from the pathological differentiation of hematopoietic stem cells. For example, leukemia cells usually do not have the functions of normal cells and can also have a strong ability to proliferate.

In this application, the term "nude mouse" generally refers to a mutant mouse with congenital thymus defect, a mutant mouse formed due to the homozygosity of the nude locus gene in the VIII linkage group. The lymphocytes of nude mice are special, so the animals have no thymus, which leads to the disorder of T cell production. Although the precursors of T cells and B cells are normal, normal T cells cannot be generated due to the lack of the thymus gland.

In this application, the term "NSG mouse" generally refers to NOD-Scid interleukin 2 receptor gamma knockout mice, which lack mature T cells, B cells and natural killer cells.

In this application, the term "stock solution" generally refers to a solution having a concentration higher than that in conventional use. For example, the concentration of the stock solution can be 1 to 3 orders of magnitude higher than the use concentration.

In this application, the term "liquid nitrogen, generally refers to liquid nitrogen gas.

In this application, the term "drug candidate" generally refers to some substances that may be used as drugs, and these substances can be evaluated by appropriate methods to obtain information on their biological activities, pharmacological effects, and efficacy.

In this application, the term "bone marrow sample" generally refers to a sample of bone marrow tissue from a model animal.

In this application, the term "separation" generally refers to utilizing the differences in physical or chemical properties of the components in the mixture to distribute the components to different spatial regions or sequentially at different times through appropriate methods. to the same spatial region.

In this application, the term "small animal imaging" generally refers to the application of imaging methods for qualitative and quantitative studies of biological processes in a living state at the cellular and molecular levels.

In this application, the term "immunodeficient mouse" generally refers to a mouse that is deficient in one or more components of the immune system, which may be caused by congenital genetic mutation and/or artificial means.

In the present application, the term "inoculation by injection" generally refers to the process of infusing an immunogen or immune effector substance into a subject to be injected through an injection device. In certain embodiments of the present application, the immunogen or immune effector substance is a solution.

In this application, the term "antibody" is used herein in its broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bi specific antibodies) and their antigen-binding fragments, etc.

In this application, the term "small molecule polypeptide drug EIP" refers to a series of polypeptide fragments that can specifically bind to ERG protein obtained through 4 rounds of enrichment screening using phage display library. The specific binding to ERG protein was confirmed by reverse phage ELISA. We performed sequence analysis on 57 phage clones and found that the polypeptides obtained by screening all contained a conserved fragment of 7 amino acids, and the enrichment of this fragment increased significantly with the screening process. We used the ForteBioOctet Red multi-channel biomolecular interaction system to detect the binding force of these 7 amino acid conserved fragments to ERG protein, and then we covalently linked the HIV-TAT fragment to the polypeptide fragment to help the polypeptide fragment penetrate the cell membrane Therefore, it can combine with the transcription factor ERG protein to synthesize peptide drugs (ERG Inhibitory Peptides, EIP). The peptide drugs we administer are synthesized by Shanghai Aibo Biotechnology Co., Ltd.

In this application, the term "flow cytometry (FCM)" generally refers to a method of counting and sorting tiny particles (eg, cells) suspended in a fluid. For example, single cells or other biological particles in suspension can be sorted by flow cytometry, and high-speed, cell-by-cell quantification and sorting can be achieved, eg, by detecting a labeled signal (eg, a fluorescent signal).

In this application, the term "about" generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.

Method for preparing humanized disease model of leukemia

In one aspect, the present application provides a method for preparing a humanized disease model of leukemia. The method may include purchasing an acute lymphoblastic leukemia cell line with high expression of ERG from a cell bank, and after identifying the cell line, expanding and culturing it, Collect cell extract protein, add lysate, homogenize on ice, centrifuge at 12,000 rpm at 4°C for 15 minutes, take the supernatant for protein quantification and carry out SDS-PAGE; after electrophoresis, the samples are electrotransferred to nitrocellulose membrane, and then subjected to immunoreaction, followed by Block at room temperature for 1 hour, incubate with primary antibody overnight at 4°C, incubate with secondary antibody at room temperature for 2 hours, wash the membrane with ^TBST for ² hours, add ECL solution, and incubate for exactly 1 minute; Company) development, use image lab ^TM software to read the gray value of the band, use GAPDH as an internal reference for statistical analysis, determine the expression of ERG protein, and obtain a wild-type acute lymphoblastic leukemia cell line with high expression of ERG protein. The identified cell line is expanded and cultured, the leukemia cell suspension to be inoculated is collected, and the final density of leukemia cells in the leukemia cell suspension to be inoculated is adjusted to 1× ^{10 7} /ml.

The described method for preparing a leukemia humanized disease model may also include: lentivirus transfection to construct a luciferase-labeled acute lymphoblastic leukemia cell line with high ERG expression, and the cells of a certain amount of cells are collected by centrifugation in a 1.5ml EP tube. , and then dilute the cell pellet with 100-200 μl of serum-free culture medium, based on the cell being completely immersed in the medium; calculate the number of virus particles required according to the MOI, add the virus solution to the cells, and place a 1.5ml EP tube at 37 Incubate in a ℃ incubator for 30 minutes; aspirate the mixed solution in the tube and add it to the petri dish; add a sufficient amount of fresh culture medium and add 6 μg/ml Polybrene to improve the transfection efficiency; change the medium after 48 hours to increase the concentration of puromycin , for resistance screening, the increasing concentration gradient can be 0.1 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 4 μg/ml, 8 μg/ml, and the cells obtained by screening are positive cells. The positive rate of cells was observed after 96 hours. The specific method was as follows: put the cells to be tested at room temperature, add 100 ml of the cell suspension to 100 ml of the steady-Glo luciferase assay systemc measurement solution, mix well and let stand for five minutes to fully lyse the cells. Measure the bioluminescence value using a multifunctional microplate reader.

(sc-134220)的混合物，最终浓度为5μg/ml。从板孔中取出培养基，并每孔用1ml的这种Polybrene/培养基混合物替换(对于12孔板)。注意：聚丙烯是一种中和电荷相互作用以增加假病毒衣壳与细胞膜之间结合的聚阳离子。Polybrene的最佳浓度取决于细胞类型，可能需要凭经验确定(通常在2-10μg/ml的范围内)。过度暴露于Polybrene(>12hr)可能对某些细胞有毒。在室温下解冻慢病毒颗粒，并在使用前轻轻混合。通过将shRNA慢病毒颗粒添加到培养物中来感染细胞。轻轻旋转板以混合并孵育过夜。病毒颗粒的使用量根据所用细胞系的特性而有很大差异。注意：将融化的shRNA慢病毒颗粒放在冰上。反复的冻融循环和颗粒长时间暴露于环境温度可能会导致病毒滴度降低。注意：首次将shRNA慢病毒构建体转导到细胞中时，我们建议使用一定数量的shRNA慢病毒颗粒原液。此外，我们建议将一孔与对照shRNA慢病毒颗粒(sc-108080)转导的细胞包括在一起。第3天除去培养基，并用1ml完全培养基(不含Polybrene)代替。将细胞孵育过夜。第4天要选择表达shRNA的稳定克隆，请根据细胞类型将细胞从1：3分离到1：5，并继续在完全培养基中孵育24-48小时。第5-6天及以后通过卡那霉素抗性选择来选择表达shRNA的稳定克隆。对于卡那霉素的选择，请使用足以杀死未转导细胞的量。卡那霉素的浓度通常在2到10μg/ml之间就足够了，但是在使用新的细胞系时建议使用卡那霉素。每3-4天用新鲜的含卡那霉素的培养基替换培养基，直到可以鉴定出耐药菌落为止。挑选几个菌落，扩增它们并测定其稳定的shRNA表达。注意：由于慢病毒构建体随机整合到细胞基因组中，因此产生的卡那霉素抗性克隆可能具有不同水平的shRNA表达。注意：要通过Western Blot进行shRNA表达分析，请按以下步骤制备细胞裂解液：用PBS洗涤细胞一次。轻轻摇动12孔板或上下吸液，以100μl1：1比例的2x电泳样品缓冲液(sc-24945)和RIPA裂解缓冲液(sc-24948)的混合物裂解细胞。如有必要，在冰上超声处理裂解物。对于通过RT-PCR进行的shRNA表达分析，使用P.Chomczynski和N.Sacchi(1987.通过硫氰酸胍盐-苯酚-氯仿提取的RNA分离的单步方法)分离RNA或市售的RNA分离试剂盒。The method for preparing a humanized disease model of leukemia may further include: packaging and transfection of shRNA ERG recombinant lentivirus: the packaged lentivirus is provided by Santa Cruz Biotechnology (Shanghai) Co., Ltd.; 24 hours before virus infection, Target cells were seeded in 12-well plates. 1 ml of complete optimal medium (containing serum and antibiotics) was added and cells were incubated overnight. On the day of infection (day 2), cells should be approximately 50% confluent. NOTE: Other plate formats can also be used for transduction. In this case, the number of cells should be adjusted according to the growth area of the well or plate. Prepare complete medium on day 2 with

(sc-134220) at a final concentration of 5 μg/ml. Media was removed from plate wells and replaced with 1 ml of this Polybrene/media mixture per well (for 12-well plates). Note: Polypropylene is a polycation that neutralizes charge interactions to increase binding between the pseudoviral capsid and the cell membrane. The optimal concentration of Polybrene depends on the cell type and may need to be determined empirically (usually in the range of 2-10 μg/ml). Overexposure to Polybrene (>12hr) may be toxic to some cells. Thaw lentiviral particles at room temperature and mix gently before use. Cells are infected by adding shRNA lentiviral particles to the culture. Gently swirl the plate to mix and incubate overnight. The amount of viral particles used varies widely depending on the characteristics of the cell line used. Note: Keep thawed shRNA lentiviral particles on ice. Repeated freeze-thaw cycles and prolonged exposure of the particles to ambient temperature may result in reduced viral titers. NOTE: When transducing shRNA lentiviral constructs into cells for the first time, we recommend using a certain amount of shRNA lentiviral particle stock. Additionally, we recommend including a well with cells transduced with control shRNA lentiviral particles (sc-108080). On day 3 the medium was removed and replaced with 1 ml of complete medium (without Polybrene). Cells were incubated overnight. Day 4 To select stable clones expressing shRNA, dissociate cells from 1:3 to 1:5 depending on cell type and continue to incubate in complete medium for 24-48 hours. Stable clones expressing shRNA were selected by kanamycin resistance selection on days 5-6 and beyond. For kanamycin selection, use an amount sufficient to kill untransduced cells. Concentrations of kanamycin between 2 and 10 μg/ml are usually sufficient, but kanamycin is recommended when working with new cell lines. Replace the medium with fresh kanamycin-containing medium every 3-4 days until resistant colonies can be identified. Several colonies were picked, expanded and assayed for stable shRNA expression. NOTE: Since the lentiviral constructs randomly integrate into the cellular genome, the resulting kanamycin-resistant clones may have different levels of shRNA expression. NOTE: For shRNA expression analysis by Western Blot, prepare cell lysate as follows: Wash cells once with PBS. Cells were lysed with 100 μl of a 1:1 mixture of 2x electrophoresis sample buffer (sc-24945) and RIPA lysis buffer (sc-24948) by gently shaking the 12-well plate or pipetting up and down. If necessary, sonicate the lysate on ice. For shRNA expression analysis by RT-PCR, RNA was isolated using P. Chomczynski and N. Sacchi (1987. One-step method for RNA isolation by guanidinium thiocyanate-phenol-chloroform extraction) or commercially available RNA isolation reagents box.

The method for preparing a leukemia humanized disease model may also include: selecting an appropriate immunodeficiency mouse according to the difference of the tested drug and the malignancy of the leukemia cell line. The immunodeficient mice can be partially immunodeficient or combined with severe immunodeficiency, and all kinds of mice do not need pre-irradiation with sub-lethal doses. In the method of the present application, an appropriate immunodeficiency mouse is selected according to the difference of the tested drug and the malignancy of the leukemia cell line, thereby improving the success rate of establishing the model. Selection of suitable immunodeficient mice can also improve the clinical relevance of the established models. For example, when the leukemia type is acute lymphoblastic leukemia, and the leukemia cell line is extremely malignant, the administered drug is a non-classical anti-leukemia drug with unknown efficacy, and the immunodeficient mouse An ectopic acute lymphoblastic leukemia model can be established in nude mice or SCID mice. When the type of leukemia is acute lymphoblastic leukemia, the degree of malignancy of the leukemia cell line is not high, and the administered drug is a well-defined or classic anti-leukemia drug, the immunodeficiency The mice can be SG mice, NSG-SGM mice, NSI mice, and/or NRG mice to establish an orthotopic acute lymphoblastic leukemia model. The immunodeficient mouse can be about 2-10 weeks old, eg, about 3-9 weeks, about 4-8 weeks, about 5-7 weeks.

The method for preparing a leukemia humanized disease model may also include: injecting the leukemia cell suspension to be inoculated obtained above into the different immunodeficient mice. In certain embodiments of the present application, the immunodeficient mouse can also be irradiated with infrared light and/or heated before the leukemia cell suspension to be inoculated. For example, no more than about 200 watts (eg, no more than about 190 watts, no more than about 180 watts, no more than about 170 watts, no more than about 160 watts, no more than about 150 watts, no more than The mice are irradiated and heated with an infrared light of about 140 watts, no more than about 130 watts, no more than about 120 watts, etc.). Appropriate irradiation intensity and time should prevent the mice from being burned by excessive temperature.

The method for preparing a leukemia humanized disease model may further include: monitoring the growth of the human leukemia cells from the cell line in the mouse, that is, the tumor progression. Those skilled in the art can select appropriate methods to monitor the growth of leukemia cells from the patient in mice. The acute lymphoblastic leukemia cells with high expression of ERG involved in this application are all transfected with biofluorescence, which can continuously observe the proliferation, invasion and invasion of human leukemia cells in mice through a small animal imaging system in vivo. Tumor processes such as metastasis. The observation time point can be 1 day, 3 days, 5 days, 7 days, 10 days, 14 days, 17 days, 21 days, 24 days, 28 days, etc. after the injection. The growth of leukemia cells from the cell line in the mouse can also be monitored by monitoring the ratio of huCD45 and muCD45 cells in the peripheral blood of the injected mouse. The huCD45 and muCD45 cells can be cells expressing human CD45 molecules and murine CD45 molecules on the cell surface, respectively. In certain embodiments, the method of monitoring the ratio of huCD45 and muCD45 cells in the peripheral blood of mice can be, for example, flow cytometry, antibody staining, or other methods that specifically detect CD45.

The method for preparing a leukemia humanized disease model may further include: a small animal imaging system monitors the fluorescein value of the humanized cells of the mouse humanized leukemia model, and in the in situ model, when the abdomen of the mouse is imaged in the abdominal position. Or bilateral femurs show fluorescence, and the maximum value is greater than 500 (about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, About 1800, about 1900, etc.), the candidate drug is administered to the model; in the ectopic model, the candidate drug can be administered to the model when the inoculation site shows fluorescence.

The method for preparing a humanized disease model of leukemia may further include: when the proportion of huCD45 and muCD45 cells in the peripheral blood of the mouse inoculated by injection is at least about 5%, at least about 10, based on the number of 10,000 cells. %, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60% %, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or more, isolating the femur of the mouse, extracting bone marrow from the femur, Single-cell suspensions were made by pipetting with a Pasteur pipette, centrifuged, bone marrow cells were resuspended in erythrocyte lysate, erythrocytes were removed by centrifugation again, and the ratio of huCD45 and muCD45 cells in the bone marrow was determined by flow cytometry.

The method for preparing a humanized disease model of leukemia may also include: freezing and storing the above-obtained stock solution in liquid nitrogen for later use. The liquid nitrogen can generally provide a temperature not higher than about -196°C.

In certain embodiments, the method for preparing a humanized disease model of leukemia may further comprise: identifying the clinical relevance of the disease model. In the present application, identifying the clinical relevance of the disease model may include: i) administering a drug candidate to the humanized disease model of leukemia; ii) detecting the persistence of complete disease remission in the model following administration of the drug candidate time; iii) comparing the duration of complete remission of the disease detected in ii) in the model with the duration of complete remission of the disease in the patient from which the model was derived after treatment with the drug candidate and iv) determine the clinical relevance of the model according to the comparison results of iii). In certain embodiments, the disease model is identified.

The clinical relevance may include monitoring the fluorescein value of the humanized cells of the mouse humanized leukemia model. In the in situ model, when the abdomen or bilateral femurs of the mouse are imaged in the abdominal position, fluorescence appears, and the maximum value is greater than 500 ( about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, etc.), applying candidates to the model Drugs; candidate drugs can be administered to the ectopic model by the appearance of fluorescence at the inoculation site.

The candidate drug is selected from one or more of the following group: EIP, a small molecule polypeptide drug that inhibits ERG, and an antitumor drug cytarabine, vincristine, tranexamic acid, dexamethasone, doxorubicin, levothyroxine Asparagine, topotecan, clofarabine, carfilzomib, temsirolimus, dasatinib, bortezomib, SAR3419, all-trans retinoic acid, SAHA, sunitinib, cyclophosphamide and retinoids. A single drug or a combination of a small molecule polypeptide drug that can inhibit the expression of ERG protein and a classic anti-leukemia drug can be selected. For example, the drug candidate may include a combination of small molecule polypeptide EIP and cytarabine, and the drug is administered to the model in the following manner: administered by intraperitoneal injection, and the amount of each administration is: the EIP group is administered according to 10 mg/ The dose of kg is administered 5 times a week for 2-4 consecutive weeks; cytarabine is administered at a dose of 250 mg/kg, twice a week for 2 consecutive weeks; combined administration In the group, the two drugs were co-administered on a group of mice according to their respective doses. For example, the drug candidate may comprise a combination of small molecule polypeptide EIP and SAHA, and the drug is administered to the model in the following manner: by intraperitoneal injection, each administration amount is: EIP group at a dose of 10 mg/kg Administration, 5 times a week, continuous administration for 2-4 weeks; SAHA is administered at a dose of 100 mg/kg, 5 times a week, continuous administration for 2-4 weeks; the combined administration group has two The drugs were administered in combination to a group of mice at their respective doses. For example, the drug candidate may include doxorubicin administered alone, and the doxorubicin administered to the model by intravenous injection at 0.5-3 mg/kg once a week and Continue administration for 4-6 weeks. For example, the drug candidate may include L-asparagine, and the L-asparagine is administered to the model by intraperitoneal injection in an amount of 500-2000 KU/kg once daily and Continue administration for 4-6 weeks. In certain embodiments, the drug candidate comprises topotecan, and the topotecan is administered to the model by intraperitoneal injection in an amount of 0.1-5 mg/kg, administered daily Once administered for 4-8 weeks, with two weeks of dosing followed by one week off. In certain embodiments, the drug candidate comprises temsirolimus, and the temsirolimus is administered to the model by intraperitoneal injection in an amount of 5-50 mg/kg per administration , once daily for 1-3 weeks. In certain embodiments, the drug candidate comprises dasatinib, and the dasatinib is administered to the model by oral administration in an amount of 5-50 mg/kg daily Administer once and continue for 4-6 weeks. In certain embodiments, the drug candidate comprises bortezomib, and the bortezomib is administered to the model by intraperitoneal injection in an amount of 0.1-20 mg/kg, administered weekly 2 times for 4-8 weeks. In certain embodiments, the drug candidate comprises SAR3419, and the SAR3419 is administered to the model by intraperitoneal injection in an amount of 0.5-50 mg/kg, administered once a week and continuously Administer for 4-8 weeks. In certain embodiments, the drug candidate comprises all-trans retinoic acid, and the all-trans retinoic acid is administered to the model by intraperitoneal injection in an amount of 0.5-10 mg/kg per administration , administered once a week and continued for 4-6 weeks. In certain embodiments, in certain embodiments, the drug candidate comprises cyclophosphamide, and the cyclophosphamide is administered to the model by intraperitoneal injection in an amount of 30- 300 mg/kg administered once daily for 4-6 weeks. In certain embodiments, the drug candidate comprises retinoid, and the retinoid is administered to the model by intraperitoneal injection in an amount of 20-200 mg/kg per administration , once daily for 4-6 weeks.

In the process of identifying the clinical relevance of the disease model, after the drug candidate is administered, the small animal imaging system monitors the change in the fluorescence value of humanized leukemia cells within 2-4 weeks of administration, and the collected fluorescence value increases slowly Or the bone marrow and peripheral blood of the stopped group of mice, the proportion of human (hu) and murine (mu) CD45 ⁺ cells was analyzed by flow cytometry, when the huCD45 ⁺ cells accounted for the peripheral blood cells of the model. The drug candidate is judged to be effective when the ratio remains below 1% for at least 1 week.

In certain embodiments, identifying the clinical relevance of the disease model further comprises performing a genomic analysis (eg, genome-wide gene expression analysis) of the cells in the model and correlating the results of the genomic analysis with acute lymphoblastic leukemia The results of the genomic analysis of the patients were compared. For example, the clinical relevance of the disease model can be determined by comparing the results (eg, how well the gene expression profiles match). In certain embodiments, the genes analyzed include, but are not limited to, eg, ERG, GDF15, CCL3, CCL4, MEK, ERK, c-MYC, and the like.

Leukemia humanized disease model

In another aspect, the present application provides a leukemia humanized disease model, which can be prepared by the method for preparing a leukemia humanized disease model described in the present application. The small animal imaging system monitors the fluorescein value of the humanized cells of the mouse humanized leukemia model. In the in situ model, when the abdomen or bilateral femurs of the mouse are imaged in the abdominal position, fluorescence appears, and the maximum value is greater than 500 (approximately 500). 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, etc.).

For example, the leukemia humanized disease model can be prepared by the following method:

a) Acute lymphoblastic leukemia cell lines with high ERG expression were purchased from the cell bank, and after the identification of the cell lines, the cells were expanded and cultured, and the cells were extracted for protein. lymphocytic leukemia cell line. The identified cell line is expanded and cultured, the leukemia cell suspension to be inoculated is collected, and the final density of leukemia cells in the leukemia cell suspension to be inoculated is adjusted to 1× ^{10 7} /ml.

b) Lentiviral transfection to construct the luciferase-labeled cell line described in a), after transfection, use the steady-Gloluciferase assay systemc to measure the bioluminescence value of the cell suspension to determine the success rate of transfection.

c) Packaging and transfection of shRNA ERG recombinant lentivirus b) The acute lymphoblastic leukemia cell line with high expression of ERG, and subsequent identification to obtain an acute lymphoblastic leukemia cell line with silent ERG protein. The successfully transfected cells were sorted by flow cytometry and continued to be cultured; the wild-type cell line was cultured at the same time, and the proliferation rates of the two cells were compared.

d) Judging the success rate of transfection of sorted cells: observe the proportion of cells with GPF green fluorescence under a fluorescence microscope; amplify sorted cells and collect cells to extract cytosol and nuclear proteins, and WB to detect target knockout In addition to the expression of protein ERG, if the expression of ERG protein is lower than 70% of wild-type cells, it can be used for subsequent experiments; the identified leukemia cells are expanded and cultured, the leukemia cell suspension to be inoculated is collected, and the The final density of leukemia cells in the seeded leukemia cell suspension was ^1x107 /ml.

e) Select an appropriate immunodeficiency mouse according to the type of model to be prepared, such as preparing an in situ acute lymphoblastic leukemia mouse model, select a severe combined immunodeficiency model such as NSG, etc.; such as preparing an ectopic acute lymphoblastic leukemia The mouse model is selected from immune-deficient mice such as nude mice or Scid.

For the acute lymphoblastic leukemia model, use the identified wild-type leukemia cells obtained in a) to prepare the leukemia cell suspension to be inoculated; for the study of the effect of ERG protein on the tumor progression such as nesting, proliferation and metastasis during the onset of leukemia , using the shERG leukemia cells obtained from d) to prepare a leukemia cell suspension to be inoculated.

The leukemia cell suspension to be inoculated obtained from a and d) is injected into the immunodeficiency mice of different degrees described in e) to prepare an orthotopic or ectopic model.

The growth of leukemia cells from the patient in the mouse can also be monitored. Human leukemia cells from the cell line are monitored for growth, ie, tumor progression, in the mice. Those skilled in the art can select appropriate methods to monitor the growth of leukemia cells from the patient in mice. The acute lymphoblastic leukemia cells with high expression of ERG involved in this application are all transfected with biofluorescence, which can continuously observe the proliferation, invasion and invasion of human leukemia cells in mice through a small animal imaging system in vivo. Tumor processes such as metastasis. The observation time point can be 1 day, 3 days, 5 days, 7 days, 10 days, 14 days, 17 days, 21 days, 24 days, 28 days, etc. after the injection. The growth of leukemia cells from the cell line in the mouse can also be monitored by monitoring the ratio of huCD45 and muCD45 cells in the peripheral blood of the injected mouse. The huCD45 and muCD45 cells can be cells expressing human CD45 molecules and murine CD45 molecules on the cell surface, respectively. In certain embodiments, the method of monitoring the ratio of huCD45 and muCD45 cells in the peripheral blood of mice can be, for example, flow cytometry, antibody staining, or other methods that specifically detect CD45.

leukemia drug screening method

In another aspect, the present application provides a leukemia drug screening method, the method comprising: obtaining the leukemia humanized disease model.

The leukemia drug screening method described in the present application may also include: a small animal imaging system monitors the fluorescein value of the humanized cells of the mouse humanized leukemia model, and in the in situ model, when the mouse abdomen is imaged in the abdomen or double When the lateral femur shows fluorescence, and the maximum value is greater than 500 (about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800 , about 1900, etc.), and administer the candidate drug to the model; in the ectopic model, the candidate drug can be administered to the model when fluorescence occurs at the inoculation site.

The leukemia drug screening method described in the present application may also include: after the drug candidate is administered, continuing to monitor the fluorescein value of the humanized cells of the mouse humanized leukemia model to evaluate the efficacy of the drug candidate. After the drug candidate is administered, the fluorescein value of the model humanized cells may be decreased, substantially unchanged or increased compared with before the drug candidate, or compared with other groups of drugs, and then it can be judged accordingly. potency of the drug candidate. In certain embodiments, the fluorescein value of the humanized cells of the mouse humanized leukemia model can be monitored by a small animal imaging system. In certain embodiments, when the fluorescein value of the humanized cells of the mouse humanized leukemia model monitored by the small animal imaging system remains lower than the value before administration of the drug or lower than that of the other drug groups, the fluorescence value is collected In the bone marrow and peripheral blood of the slow-growing or stopped group mice, the proportion of human (hu) and murine (mu) CD45 ⁺ cells was analyzed by flow cytometry, when the huCD45 ⁺ cells were in the peripheral blood cells of the model It can be determined when the proportion remains below 1% for at least about 1 week, at least about 1.5 weeks, at least about 2 weeks, at least about 2.5 weeks, at least about 3 weeks, at least about 3.5 weeks or more The drug candidate is potent.

The candidate drug is selected from one or more of the following group: EIP, a small molecule polypeptide drug that inhibits ERG, and an antitumor drug cytarabine, vincristine, tranexamic acid, dexamethasone, doxorubicin, levothyroxine Asparagine, topotecan, clofarabine, carfilzomib, temsirolimus, dasatinib, bortezomib, SAR3419, all-trans retinoic acid, SAHA, sunitinib, cyclophosphamide and retinoids. A single drug or a combination of a small molecule polypeptide drug that can inhibit the expression of ERG protein and a classic anti-leukemia drug can be selected.

For example, the drug candidate may include a combination of small molecule polypeptide EIP and cytarabine, and the drug is administered to the model in the following manner: administered by intraperitoneal injection, and the amount of each administration is: the EIP group is administered according to 10 mg/ The dose of kg is administered 5 times a week for 2-4 consecutive weeks; cytarabine is administered at a dose of 250 mg/kg, twice a week for 2 consecutive weeks; combined administration In the group, the two drugs were co-administered on a group of mice according to their respective doses.

For example, the drug candidate may comprise a combination of small molecule polypeptide EIP and SAHA, and the drug is administered to the model in the following manner: by intraperitoneal injection, each administration amount is: EIP group at a dose of 10 mg/kg Administration, 5 times a week, continuous administration for 2-4 weeks; SAHA is administered at a dose of 100 mg/kg, 5 times a week, continuous administration for 2-4 weeks; the combined administration group has two The drugs were administered in combination to a group of mice at their respective doses.

For example, the drug candidate may include doxorubicin administered alone, and the doxorubicin administered to the model by intravenous injection at 0.5-3 mg/kg once a week and Continue administration for 4-6 weeks.

For example, the drug candidate may include L-asparagine, and the L-asparagine is administered to the model by intraperitoneal injection in an amount of 500-2000 KU/kg once daily and Continue administration for 4-6 weeks.

In certain embodiments, the drug candidate comprises topotecan, and the topotecan is administered to the model by intraperitoneal injection in an amount of 0.1-5 mg/kg, administered daily Once administered for 4-8 weeks, with two weeks of dosing followed by one week off.

In certain embodiments, the drug candidate comprises temsirolimus, and the temsirolimus is administered to the model by intraperitoneal injection in an amount of 5-50 mg/kg per administration , once daily for 1-3 weeks.

In certain embodiments, the drug candidate comprises dasatinib, and the dasatinib is administered to the model by oral administration in an amount of 5-50 mg/kg daily Administer once and continue for 4-6 weeks.

In certain embodiments, the drug candidate comprises bortezomib, and the bortezomib is administered to the model by intraperitoneal injection in an amount of 0.1-20 mg/kg, administered weekly 2 times for 4-8 weeks.

In certain embodiments, the drug candidate comprises SAR3419, and the SAR3419 is administered to the model by intraperitoneal injection in an amount of 0.5-50 mg/kg, administered once a week and continuously Administer for 4-8 weeks.

In certain embodiments, the drug candidate comprises all-trans retinoic acid, and the all-trans retinoic acid is administered to the model by intraperitoneal injection in an amount of 0.5-10 mg/kg per administration , administered once a week and continued for 4-6 weeks.

In certain embodiments, in certain embodiments, the drug candidate comprises cyclophosphamide, and the cyclophosphamide is administered to the model by intraperitoneal injection in an amount of 30- 300 mg/kg administered once daily for 4-6 weeks.

In certain embodiments, the drug candidate comprises retinoid, and the retinoid is administered to the model by intraperitoneal injection in an amount of 20-200 mg/kg per administration , once daily for 4-6 weeks.

Without wishing to be bound by any theory, the following examples are only intended to illustrate the working manner of the apparatus, method and system of the present application, and are not intended to limit the scope of the invention of the present application.

Figure 1 is an example, A indicates that the cell proliferation rate of transfected HAL-01 compared with the corresponding wild-type cells has no statistical difference; B indicates that the cell proliferation rate of the transfected Reh cells compared with the corresponding wild-type cells No statistical difference. Figure 2 is an example, the polypeptide drug significantly inhibited the expression of ERG protein in HAL-01 cell line. Figure 3 shows the results of transfection of shRNA ERG recombinant lentivirus in one example. A indicates that HAL-01 was selected for transfection. Compared with the transfected empty plasmid shcontrol, the shERG cell line after successful transfection showed no statistical difference in the proliferation rate of the two cell lines within 14 days. B shows the process of inoculating the expressed cell line after transfection into the blood system of immunodeficient mice. C indicates that the efficiency of reducing ERG protein in cell lines was confirmed by immunoblotting. Figure 4 is an example of observing tumor processes such as nesting, invasion and metastasis of human cells in mice by non-invasive small animal imaging methods. A indicates that Reh-luc cell line was used to inoculate NSG of immunodeficient mice, and after 7 days, a small animal imaging system was used to monitor the tumor processes such as nesting, invasion and metastasis of human leukemia cells in mice; B indicates that Reh-luc cells were selected The strain was inoculated into NSG of immunodeficient mice, and after 14 days, a small animal imaging system was used to monitor tumor processes such as nesting, invasion and metastasis of human leukemia cells in mice. C: Reh-luc cell line was used to inoculate the NSG of immunodeficient mice, and after 21 days, a small animal imaging system was used to monitor the tumor processes such as nesting, invasion and metastasis of human leukemia cells in the mice. D shows the process of inoculating the expressed cell line after transfection into the blood system of immunodeficient mice. Figure 5 shows an example of flow cytometry analysis of human and murine cells and found that human leukemia cells mainly nest in mouse bone marrow. Figure 6 is an example, inoculation of HAL-01 cell line carrying stable shRNA ERG in NSG mice to delay tumor progression in a leukemia model. A represents the nesting and invasion of human leukemia cells in mice inoculated with HAL-01 cell lines transfected with shERG and shControl, respectively, 3 days, 7 days and 14 days after inoculation. and tumor progression such as metastasis. B shows the survival cycle of NSG mice inoculated with shERG and shControl-transfected HAL-01 cell lines. C represents the expression efficiency of ERG protein in HAL-01 cell line transfected with shERG detected by western blotting. Figure 7 is an example of the effect of combined administration on the tumor progression of the HAL-01 cell line inoculated with the NSG mouse leukemia model. Figure 7 is an example of the effect of combined administration on the tumor progression of the HAL-01 cell line inoculated with the NSG mouse leukemia model. A represents the comparison between the combined administration group and the blank group, and the small animal imaging system was used to detect the levels of human leukemia cells in mice at different administration time points, that is, 7 days, 14 days, 21 days, 25 days, and 28 days after administration. proliferation. B and C represent the growth of tumor tissue between the combined administration group and the blank group. D represents the change of the body weight of the mice in the combined administration group compared with the blank group during the administration period.

Claims (5)

1. a construction method of acute lymphoblastic leukemia mouse model, is characterized in that, comprises the following steps: a) Acute lymphoblastic leukemia cell lines with high ERG expression were purchased from the cell bank, and after the identification of the cell lines, the cells were expanded and cultured, and the cells were extracted for protein. Lymphocytic leukemia cell line, the identified cell line is amplified and cultured, the leukemia cell suspension to be inoculated is collected, and the concentration is adjusted to obtain the cell line; b) lentiviral transfection to construct the luciferase-labeled cell line described in a), after transfection, measure the bioluminescence value of the cell suspension to determine the success rate of transfection; c) Packaging and transfection of shRNA ERG recombinant lentivirus Step b) The obtained acute lymphoblastic leukemia cell line with high expression of ERG, and subsequent identification was performed to obtain an acute lymphoblastic leukemia cell line with silent ERG protein, flow cytometry The successfully transfected cells were sorted by cytometer and continued to be cultured; the wild-type cell line was cultured at the same time, and the proliferation rate of the two cells was compared; d) Determine the success rate of transfection of sorted cells: observe the proportion of cells with GPF green fluorescence under a fluorescence microscope; expand sorted cells and collect cells to extract cytosol and nuclear proteins, and detect target knockout The expression of protein ERG, if the expression of ERG protein is lower than 70% of wild-type cells, can be used for subsequent experiments; the identified leukemia cells are expanded and cultured, the leukemia cell suspension to be inoculated is collected, and the concentration is adjusted to obtain the target cell suspension. liquid; e) Select immunodeficient mice according to the type of model to be prepared, and construct a mouse model of acute lymphoblastic leukemia. 2. the construction method of acute lymphoblastic leukemia mouse model according to claim 1, is characterized in that, in step a), in described cell line, adjust the leukemia cells in the leukemia cell suspension to be inoculated The final density is 0.5xl0 ⁷ ~ 2xl0 ⁷ /ml. 3. the construction method of acute lymphoblastic leukemia mouse model according to claim 1 is characterized in that, in step b), utilize steady-Glo luciferase assay systemc to measure the bioluminescence value of cell suspension. 4. The method for constructing an acute lymphoblastic leukemia mouse model according to claim 1, wherein in step d), the final density of leukemia cells in the leukemia cell suspension to be inoculated is adjusted to be 0.5× ^{10 7} ～2×10 ⁷ /ml to obtain the target cell suspension. 5. the construction method of acute lymphoblastic leukemia mouse model according to claim 1, is characterized in that, constructs acute lymphoblastic leukemia mouse model, specifically comprises: Inoculate the cell line obtained in step a) and the target cell suspension obtained in step d) into the peripheral blood of e) immunodeficiency mice or immunodeficiency mice of different degrees to prepare in situ or ectopic models .

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