CN117814181A

CN117814181A - Construction and application of a mouse model of liver fibrosis based on human hepatic stellate cell lines

Info

Publication number: CN117814181A
Application number: CN202311777915.9A
Authority: CN
Inventors: 庄立琨; 林中华; 赵真真; 辛永宁; 陆祎; 马学峰
Original assignee: Qingdao Municipal Hospital
Current assignee: Qingdao Municipal Hospital
Priority date: 2023-12-22
Filing date: 2023-12-22
Publication date: 2024-04-05
Anticipated expiration: 2043-12-22
Also published as: CN117814181B

Abstract

The invention belongs to the technical field of animal model construction, and provides a construction method and application of a hepatic fibrosis mouse model based on a human hepatic stellate cell strain, aiming at the problem of hepatic fibrosis animal model caused by lack of human hepatic stellate cells. The invention takes an immunodeficiency mouse as an animal model, uses luciferase and LX-2 marked by red fluorescent protein, and uses the MCT to be injected intraperitoneally to prepare a liver injury model of the mouse, and LX-II is injected into the spleen after liver injury2, transplanting the humanized LX-2 cells into NSG mice, and then injecting CCl into the intraperitoneal of the mice ₄ . By this method, the implantation, distribution, proliferation and hepatic fibrosis of transplanted LX-2 in the liver were successfully studied. The model is suitable for in vivo experiments of therapeutic methods such as biological therapy and drug therapy of targeted hepatic stellate cells or fibroblasts.

Description

Construction method and application of hepatic fibrosis mouse model based on human hepatic stellate cell strain

Technical Field

The invention belongs to the technical field of animal model construction, and particularly relates to a method for constructing a liver fibrosis mouse model.

Background

Liver fibrosis is characterized by excessive accumulation of fibrous connective tissue in the liver, resulting in impaired liver function. Hepatic stellate cells (hepatic stellate cells, HSCs) are considered to be major contributors to liver fibrosis. Following liver injury, HSCs migrate to the site of injury and become live, producing excessive extracellular matrix (extracellular matrix, ECM) components, ultimately leading to liver fibrosis.

Current in vivo studies in mice directed to liver fibrosis have relied primarily on exogenously added carbon tetrachloride (CCl ₄ ) Chemical inducers such as Dimethylnitrosamine (DMNA), D-galactosamine (DGA) and Diethylnitrosamine (DEN) are used for constructing liver fibrosis models of mice. By CCl ₄ The induced liver fibrosis model is simple and easy, but has higher death rate and CCl ₄ The liver fibrosis state caused by isochemical induction is difficult to simulate the occurrence and development states of fibrosis diseases, and the mouse fibrosis model is difficult to simulate the liver fibrosis disease progress of human beings due to the difference of species of mice and human beings. LX-2 is a primary human hepatic stellate cell strain capable of serial passage by SV40 large T antigen treatment. Xu et al established a serial-passaged LX-2 cell line and provided a source of stably proliferating human hepatic stellate cells (Xu L, hui AY, albanis E, arthur MJ, O' Byrne SM, blancer WS, mukherjee P, friedman SL, eng FJ.Human hepatic stellate cell lines, LX-1and LX-2:new tools for analysis of hepatic fibrosis.Gut.2005Jan;54 (1): 142-51). LX-2 retains key characteristics of liver astrocyte cytokine signal, nerve gene expression, retinol metabolism and fibrosis, and is very suitable for useCulture-based studies of human liver fibrosis were performed in combination. Because of the difficulty in the field planting of humanized cells in other animal models, the survival rate is low, and the current research on humanized liver fibrosis models is mainly in vitro models, for example, patent document CN109337860a discloses a method for constructing a liver fibrosis 3D model in vitro, single cells of hepatic parenchyma cells, single cells of hepatic stellate cells and single cells of cumic cells are inoculated on a 3D culture medium for co-culture, and a liver fibrosis inducer is added into the obtained 3D co-cultured cells for induction to obtain the liver fibrosis 3D model. Although such in vitro models can be constructed based on humanized cells, in vitro models cannot mimic complex conditions in an organism, for example, changes in the levels of fluids in an organism, changes in metabolites and in vivo environments can result in changes in the state of cell viability.

Although it has been found that an immunodeficient animal model is more suitable for human cell transplantation, the inventors have found during the course of the present invention that the selection of an immunodeficient animal model alone does not actually result in the desired engraftment rate and survival rate, and that human hepatic stellate cells are present in an immunodeficient mouse,

thus, there is a need to construct a humanized liver fibrosis mouse model for better scientific research.

Disclosure of Invention

Aiming at the problems that a mouse with liver fibers of a murine origin cannot fully meet scientific research requirements and a liver fibrosis animal model caused by human hepatic stellate cells is lacking at present, the invention provides a construction method of the liver fibrosis mouse model based on a human hepatic stellate cell strain. The invention takes an immunodeficiency mouse as an animal model, uses LX-2 marked by Luciferase (LUC) and Red Fluorescent Protein (RFP) to inject MCT intraperitoneally to prepare a liver injury model of the mouse, and uses LX-2 injected from spleen after liver injury to transplant human LX-2 cells into NSG mice, and then performs intraperitoneally injection CCl of the mice ₄ By this method, the implantation, distribution, proliferation and hepatic fibrosis of transplanted LX-2 in the liver were successfully studied.

The invention is realized by the following technical scheme:

a method for constructing a hepatic fibrosis mouse model based on a human hepatic stellate cell strain comprises the following steps:

(1) Constructing LX-2-LUC-RFP cells, screening puromycin-resistant cells, monoclonalizing the cells, and screening monoclonal cells FFLuc-RFP LX-2 with good cell state and high fluorescence intensity;

(2) Taking an immunodeficiency mouse as an animal model, injecting 180-220mg/kg of Monocrotaline (MCT) into the abdominal cavity within 16-24 hours before cell transplantation, preparing an injection by dissolving the monocrotaline in a medically available solvent, and injecting an equivalent solvent into a control group;

(3) Transplanting FFLuc-RFP LX-2 cells into NSG mice by spleen injection;

(4) Starting on day 10 after cell transplantation, intraperitoneal injections of 0.18-0.22mL/kg body weight CCl were given twice a week ₄ ，CCl ₄ The corn oil was mixed with the control to prepare an injection for 6 weeks, and the control was injected with an equal amount of corn oil.

Preferably the mice are heavy immunodeficient mice, more preferably NSG mice, which lack mature T, B and NK cells.

Preferably by intraperitoneal injection of MCT at 200mg/kg body weight. Preferably, the solvent that dissolves MCT is phosphate buffer.

Preferably, 0.2mL/kg body weight CCl is administered intraperitoneally ₄ . Preferably CCl ₄ Mixing with corn oil according to the following ratio of 1: (6-15) by volume ratio, more preferably 1:10 volume ratio.

The LX-2-LUC-RFP cells are constructed by a method comprising the steps of:

(1) Constructing a plasmid pCDH-CMV-fLuc-EF1-turboRFP-T2A-Puro containing a target gene for expressing a luciferase reporter gene and a red fluorescent protein;

(2) Respectively carrying out high-purity endotoxin-free extraction on the three plasmid vectors, transfecting 293T cells in a growing period together, collecting virus supernatant and concentrating;

(3) And adding viruses into the LX-2 cells for incubation to obtain the LX-2-LUC-RFP cells.

Further, the 293T cells were replaced with complete medium 6 hours after co-transfection, cell supernatants containing lentiviral-enriched particles were collected at 24 and 48 hours of culture, and virus supernatants were concentrated by ultracentrifugation.

Further, LX-2 cells were plated on 24 well plates 18-24 hours prior to lentivirus transfection, the culture just prepared was replaced with the original medium the next day, 5 μg/mL of transfection reagent was added, and virus suspension was added followed by incubation at 37 ℃ for 24 hours, after which fresh medium was replaced with virus containing medium.

Pathological sections are made of NSG mouse livers at different time points after the injection of the monocrotaline, and the damage condition of liver tissues after the MCT injection is observed.

Performing living imaging of the mice at different time points after the spleen of the mice is injected with FFLuc-RFP LX-2, and observing the proliferation condition of LX-2 cells; after the mice were sacrificed, pathological sections of liver tissues were prepared, and the distribution of LX-2 cells in liver tissues was observed.

In mouse CCl ₄ Performing in-vivo imaging of mice at different time points after injection, and observing the distribution of LX-2 cells in liver tissues; after the mice were sacrificed, pathological sections of liver tissue were prepared, and liver fibrosis was observed.

The liver fibrosis animal model obtained by the method for constructing the liver fibrosis mouse model based on the human hepatic stellate cell strain is applied to research of liver fibrosis mechanism or drug effect evaluation or pharmacological experiment of drugs.

Preferably, the application is in vivo experiments of human origin of biotherapy or drug therapy targeting hepatic stellate cells or fibroblasts.

The invention has the following beneficial effects:

according to the invention, NSG mice are used for experiments, and an external stimulus is combined to construct a humanized liver fibrosis mouse model, so that the engraftment rate and the survival rate of human cells are improved, and the human liver fibrosis process is better simulated.

According to the invention, MCT is injected intraperitoneally to prepare a liver injury model of a mouse, after liver injury, LX-2 injected by spleen is easy to colonize in liver, and the colonization quantity of LX-2 in the liver can be obviously increased.

The invention can be stably planted in the liver of a mouse after the LX-2 cell strain is injected through the spleen and is positioned in CCl ₄ Is rapidly proliferated under the stimulation of (a).

The model is suitable for in vivo experiments of therapeutic methods such as biological therapy and drug therapy of targeted hepatic stellate cells or fibroblasts.

Compared with the model constructed by using primary human hepatic stellate cells or hepatic fibroblasts, the invention has small difficulty and is suitable for popularization and application in laboratories.

Drawings

FIG. 1 is a plasmid map of example expression of Luciferase and RFP;

FIG. 2 is a fluorescence image (fluorescence microscope, 200X) of a monoclonal cell line of example FFLuc-RFP LX-2;

FIG. 3 is a flow chart showing the procedure of the procedure for the colonization and proliferation of example LX-2 in the liver of NSG mice;

FIG. 4 is a graph of HE staining of the liver injury model of the example (left graph, liver HE staining of normal NSG mice; right graph, liver HE staining of liver injured NSG mice, 100X);

FIG. 5 is a photograph of a living animal of example LX-2 cells in liver colonization (left: not colonized; middle: no MCT; right: MCT);

FIG. 6 shows the results of frozen sections of liver after liver colonization of example LX-2 (fluorescence microscope, 200X);

FIG. 7 is a schematic diagram of an embodiment CCl ₄ After injection, LX-2 cells were imaged in vivo in animals with liver proliferation (left panel, 3 weeks; right panel, 6 weeks);

FIG. 8 is a schematic diagram of an embodiment CCl ₄ 3D imaging of proliferated FFLuc-RFP LX-2 colonization in liver 6 weeks after injection;

FIG. 9 is a schematic diagram of an embodiment CCl ₄ Distribution of FFLuc-RFP LX-2 cells in liver tissue after 6 weeks of injection (left panel, non-transplanted liver tissue; right panel, transplanted liver tissue, arrow FFLuc-RFP LX-2, 100×);

FIG. 10 is a schematic diagram of an embodiment CCl ₄ Sirius scarlet staining of mouse liver 6 weeks after injectionColor (Sirius red), HE staining, anti-human collagen type I staining (left side not injected CCl) ₄ A group; right side injection CCl ₄ Group, 100×).

Detailed Description

The invention will be described in further detail with reference to specific embodiments and drawings.

The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.

Example 1: selection of animal models

And 6-8 weeks old NSG mice are selected to construct a human model. In order to ensure the health status of mice, the required feeding goods should be autoclaved and the feeding environment should reach SPF level, which is very advantageous for feeding immunodeficient mice. A high fat low protein feed can be used, while the NSG mice feed should contain more than or equal to 18% protein and more than or equal to 6% fat, and be sterilized by Co-60 irradiation.

2. Construction of LX-2-LUC-RFP cells

(1) Plasmid construction

Construction of plasmid pCDH-CMV-fLuc-EF1-turboRFP-T2A-Puro expressing Luciferase reporter gene and red fluorescent protein RFP, plasmid map is shown in FIG. 1.

(2) Virus package

Lentiviral plasmids and their auxiliary packaging original vector plasmids were prepared, and three plasmid vectors were highly purified, respectively, to ensure endotoxin-free extraction. Then, 293T cells were co-transfected and replaced with complete medium 6 hours after transfection. Cell supernatants containing enriched lentiviral particles were collected at 24 and 48 hours of incubation, respectively. Finally, the virus supernatant is concentrated by ultracentrifugation.

(3) Viral transfection plasmid

1X 10 will be taken 18-24 hours prior to lentivirus transfection ⁵ The LX-2 cells were plated on 24-well plates to ensure that each well was transfected withLX-2 cell number was about 1X 10 ⁵ . The next day, the freshly prepared medium (1.5 mL) was replaced with the original medium, 5. Mu.g/mL polybrene was added, and the appropriate amount of virus suspension was added, followed by incubation at 37 ℃. After a continuous incubation time of 24 hours, the virus-containing medium was replaced with fresh medium.

(4) Screening of monoclonal cell lines

As the plasmid contains puromycin resistance gene, a proper amount of puromycin is added for screening to obtain drug-resistant LX-2 cells, and the cells are named as FFLuc-RFP LX-2, so that the subsequent FFLuc-RFP LX-2 cell pool with high brightness is formed. FFLuc-RFP LX-2 cell pool with good cell state is selected, cells are subjected to suspension treatment by PBS, and counted by a cell counter. 300 cells were taken, mixed well with 30mL of medium, and then added to 3 96-well plates, 100uL per well. The plates were placed in a cell incubator for cultivation. On the third day of culture, 100uL of fluid was supplied per well. After 10 days of culture, the growth and fluorescence of the cells were observed by using a fluorescence microscope. And selecting a monoclonal cell strain with good cell state and strongest fluorescence intensity for amplification. For cells with poor cell state and poor agglomeration effect, the proliferation effect of the cells may be negatively affected and even the proliferation failure may occur when the cells are stimulated by carbon tetrachloride. In addition, when selecting cell lines, preference should be given to monoclonal cell lines which have a strong fluorescent intensity and which are convenient to track using live imaging of small animals and staining of pathological sections with antibodies. Such a choice will help to improve the accuracy and reliability of the experiment.

3. Experimental procedure for LX-2 colonization and proliferation in the liver of NSG mice

(1) And establishing a liver injury model. 200mg/kg of Monocrotaline (MCT) was dissolved in Phosphate Buffered Saline (PBS) at pH 7.4 by intraperitoneal injection during 16-24 hours prior to cell transplantation. The control group was injected with an equal dose of PBS.

(2) Using 2X 10 ⁵ The FFLuc-RFP LX-2 cells were suspended in 200uL of PBS for use. Subsequently, mice were anesthetized with tribromoethanol and usedIodophors disinfect the skin of mice. During the procedure, the back skin of the mice was gently lifted using sterilized forceps and an incision of about 1cm was made along the skin using sterilized scissors. Next, the endothelial tissue at the incision was grasped using another pair of sterilized forceps, and a 1cm incision was cut along the endothelium again using scissors so as to fully expose the spleen. Then, 200uL of the suspended FFLuc-RFP LX-2 cells were aspirated using a 1mL injection needle, and injected from the spleen site of the mouse. Immediately after injection, the sterile cotton ball was used to stop bleeding by compression. Subsequently, a suturing operation is performed using a sterile suture needle line, which first sutures the endothelium and then the skin. The mice can be injected with an appropriate amount of antibiotics, as desired. Finally, the mice were placed on a warm and sterilized pad and waited for awakening.

(3) To induce toxin-mediated liver fibrosis, 0.2mL/kg CCl was injected intraperitoneally twice a week, starting on day 10 after cell transplantation ₄ And corn oil 1:10 mixture for 6 weeks. The control group received only corn oil.

(4) Six weeks later, in vivo imaging and pathology biopsy were performed to evaluate model effects.

4. LX-2 colonization in NSG mouse liver

(1) Liver injury model

NSG mouse liver sections were prepared by hematoxylin-eosin staining (HE) following MCT injection. As can be clearly seen from the slice images of figure 4, the liver tissue was significantly damaged after MCT injection.

(2) Liver field planting condition

To examine the status of LX-2 colonization in liver cells, mice spleen was imaged in vivo (using PerkinElmer IVIS Spectrum) in small animals on day 10 of FFLuc-RFP LX-2 injection, as shown in FIG. 5. The results showed that FFLuc-RFP LX-2 was transferred from the spleen to the liver and that MCT-injected mice had more liver colonization with FFLuc-RFP LX-2 than without MCT injection.

After the mice were sacrificed, their tissues were subjected to frozen section observation, see fig. 6. The results showed that FFLuc-RFP LX-2 cells transferred from the spleen to the liver.

5. Proliferation of LX-2 in the liver of NSG mice and liver fibrosis

Mouse CCl ₄ Animals were imaged in vivo at weeks 3 and 6 after injection, as shown in fig. 7. The results showed that LX-2 cells proliferated in the liver. In the process of CCl ₄ 3 weeks after intraperitoneal injection, the bioluminescence intensity of the liver part of the mouse reached a peak value of 3×10 ⁵ And (3) radius. As the injection time was prolonged, the peak of bioluminescence intensity increased to 1.33X10 by 6 weeks ⁶ And (3) radius. This result is derived from the fact that FFLuc-RFP LX-2 has a bioluminescence gene, and thus the bioluminescence intensity can reflect the proliferation status of FFLuc-RFP LX-2. Experimental data shows that in CCl ₄ Under stimulation, FFLuc-RFP LX-2 proliferated significantly.

CCl ₄ At the 6 th week after injection, observing the coronal plane, sagittal plane and cross section of the mouse, wherein the region with higher bioluminescence intensity is mainly distributed at the liver part of the mouse; further construction of three-dimensional images, the results were more pronounced, showing that the higher bioluminescence intensity remained concentrated in the mouse liver region, as shown in fig. 8.

After the mice were sacrificed, liver tissues were taken and observed for anti-RFP immunohistochemical staining, and the results are shown in fig. 9. Compared to the control group, the experimental group exhibited numerous areas of tan-colored clusters, as indicated by the arrows on the right in fig. 9, which were FFLuc-RFP LX-2 stained with anti-RFP antibodies, characterized by regular distribution along fibrous tissue, but not within hepatic lobules.

HE staining was then performed, and liver fiber-related Sirius red (Sirius red) and anti-human collagen type I were stained, and the results are shown in FIG. 10. HE staining experiments showed that, via CCl ₄ After injection treatment, mice had developed liver fibrosis. The fibrosis-related staining, such as Sirius red staining and anti-human collagen type I staining, was positive compared to the control, with a clear distribution of fibrous tissue.

The above examples are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the claims of the present invention without departing from the spirit of the present invention.

Claims

1. A method for constructing a liver fibrosis mouse model based on a human hepatic stellate cell line, characterized in that it comprises the following steps:

(1) Construct LX-2-LUC-RFP cells, screen for puromycin-resistant cells, clone the cells and screen for monoclonal cells with good cell status and high fluorescence intensity FFLuc-RFP LX-2;

(2) Using immunodeficient mice as an animal model, 180-220 mg/kg body weight of monocrotaline was intraperitoneally injected within 16-24 hours before cell transplantation. Monocrotaline was dissolved in a medically acceptable solvent to prepare an injection, and the control group was injected with an equal amount of solvent;

(3) Transplantation of FFLuc-RFP LX-2 cells into mice by intrasplenic injection;

(4) Starting from the 10th day after cell transplantation, 0.18-0.22 mL/kg body weight of CCl ₄ was injected intraperitoneally twice a week. CCl ₄ was mixed with corn oil to prepare an injection. This lasted for 6 weeks. The control group was injected with an equal amount of corn oil.

2. The construction method according to claim 1, wherein the immunodeficient mice are NSG mice.

3. Construction The construction method of claim 1, characterized in that the LX-2-LUC-RFP cell is constructed by a method comprising the following steps:

(1) Construct the plasmid pCDH-CMV-fLuc-EF1-turboRFP-T2A-Puro containing the target gene expressing the luciferase reporter gene and red fluorescent protein;

(2) The three plasmid vectors were extracted to high purity and without endotoxin, and co-transfected into growing 293T cells. The viral supernatant was collected and concentrated;

(3) Add the virus to LX-2 cells for incubation to obtain LX-2-LUC-RFP cells.

4. The construction method of claim 3, characterized in that the 293T cells are co-transfected and replaced with complete culture medium 6 hours later, and the cell supernatant containing enriched lentiviral particles is collected at 24 and 48 hours of culture, respectively, and the viral supernatant is concentrated by ultracentrifugation.

5. The construction method of claim 3, characterized in that 18-24 hours before lentiviral transfection, LX-2 cells are plated on a 24-well plate, the next day the culture medium is replaced with the original culture medium, the transfection reagent is added, and the virus suspension is added, followed by incubation at 37°C. After continuous culture for 24 hours, the culture medium containing the virus is replaced with a fresh culture medium.

6. The method of constructing claim 1, characterized in that pathological sections are made on the liver of mice at different time points after injection of monocrotaline to observe the damage of liver tissue after MCT injection.

7. The construction method of claim 1, characterized in that live imaging of mice is performed at different time points after FFLuc-RFP LX-2 is injected into the spleen of mice to observe the proliferation of LX-2 cells; after the mice are killed, liver tissue pathological sections are made to observe the distribution of LX-2 cells in the liver tissue.

8. The method of constructing claim 1, characterized in that in vivo imaging of mice is performed at different time points after CCl ₄ injection in mice to observe the distribution of LX-2 cells in liver tissue; after killing the mice, liver tissue pathological sections are made to observe liver fibrosis.

9. Use of the liver fibrosis animal model obtained by the method for constructing a liver fibrosis mouse model based on human hepatic stellate cells according to any one of claims 1 to 8 in studying the mechanism of liver fibrosis or in drug efficacy evaluation or pharmacological experiments.

10. The use according to claim 9, characterized in that the use is a human in vivo experiment of biological therapy or drug therapy targeting hepatic stellate cells or fibroblasts.