CN1566330A - Activated HLA semi-coincident allogene mixed hematopoietic stem cell and method for making same - Google Patents
Activated HLA semi-coincident allogene mixed hematopoietic stem cell and method for making same Download PDFInfo
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Abstract
The invention provides an activated HLA semi-coincident allogene mixed hematopoietic stem cell and method for making same which comprises, collecting outer blood of cancer patients and their relatives, combining the blood and charging the stem cells into specific culture medium for culturing.
Description
Technical field
The present invention relates to a kind of stem cell and preparation method thereof, specifically, the invention provides a kind of activatory HLA half-matched recessive allele Combination hemopoietic stem cell and preparation method thereof, this stem cell derives from parent, stem cell micro chimerism with among its offspring has antitumor action.After feeding back to this cell in the body, can improve immunizing power, thus the at present positive popular SARS virus of kill tumor and resistance.
Background technology
Achievement in research shows, can utilize the stem cell in various sources, treats aging, wound, heredity and metabolic disease, has good potential applicability in clinical practice.Stem cell is cultivated and directional induction by body outer clone, can become: 1) suitable Transplanted cells material is used for the repairing damage organ; 2) stem cell is used for therapeutic transgene as transgene carrier; 3) utilize the human chronic disease of cellular replacement therapy such as bone marrow stem cell or cord blood stem cell transplantation treatment leukemia etc.The foundation of stem cell line must be satisfied two conditions: duplicate in the cell cycle and can obtain a large amount of daughter cells, promptly have the immortalization characteristic; The essential pedigree heredity phenotype feature that keeps initiating cell of clone.The problem that research at present at first will solve is the stem cell line that how to obtain can be used for clinical application, and can be at amplification in vitro.
External existing multiple technologies route is set up stem cell line: 1) set up clone from the ancester cell of neurocele and neural crest derivation.2) isolated cell system from the spontaneous generation tumour cell, as by Augusti-Tocco and Sato, the mouse neuroblastoma (mouse neuroblastomaC1300) that Schubert etc. (1969) set up, the human brain cortical tumor has also been set up the clone (Ronnett etc., 1990) with neuron behavior.3) utilize X-rays or chemical inducer inducing tumor cell, set up and separate neuronal cell line, as the PC12 clone that is widely used is to induce foundation (Greene and Tischler, 1976) by X-rays.4) carry the special transfection neural precursor of proto-oncogene (neuralprecusor) with reversal of viral carrier or adenovirus carrier and set up cell clone.5) foundation is carried and is contained the hybridization of neural tumor clone, sets up immortalization neuronal cell line (Greene etc., 1975; Choi etc., 1991).7) employing nervus centralis serum-free culture of former generation, Urogastron (EGF) stimulates, being immortalized cell clone (Reynolds and Weiss, 1992; Ray etc., 1993).8) utilize embryo stem cell for directional to induce and set up neural stem cell system, induce neuron cell with Retinoic acid as Gottlieb (University of Washington).9) current research is dynamically paid close attention to the karyomit(e) order short dna tumor-necrosis factor glycoproteins relevant with the regulating cell life-span, i.e. telomere at present.A few cell behind the division growth, is rebuild telomere by Telomerase each time, keeps cell immortalityization.Although above-mentioned technology can obtain stem cell, but some technology relates to the relevant ethics problem of embryonic stem cell, some clone and cancerous tumor cell are not easily distinguishable, the stem cell that utilizes virus vector transfection goal gene to obtain has clinical safety in utilization problem etc., and people expect that a technology can obtain clinical applicable stem cell line.
At home, the research in stem cell field is subjected to the attention of a plurality of subjects gradually.Domestic research has related to the research of the human tissue engineering of skin, cartilage and nervous system injury.Many cases with leukemia patients' life has been saved in the transplanting of hemopoietic stem cell.The situation of present domestic research is taught for doctor Cheng Guoxiang, Sheng Huizhen with the Shanghai City transgenic research institute and the Shanghai City No2. University of Medical Sciences and professor Cao Yilin is the technological line that utilizes human embryo stem cell of representative, the human specific histoorgan of differentiation, as skin, cartilage heart, liver etc., but there is ethics problem in this method.BeiJing ZhongKe department system is that clone's national treasure giant panda of representative is national key subjects with professor Chen Dayuan.Kunming system of the Chinese Academy of Sciences is main characteristic with the primates clone.The Ministry of Agriculture of Xibei Agriculture Science-Technology Univ. open laboratory is main direction of studying with livestock industry.The research is all being actively developed in many laboratories strong or local characteristic, the whole nation at present.
Verified, the stem cell of little transplanting carries out killing the leukemia cell (referring to Champlin.R etc., Reinventing bone marrow transplantation:reducing toxicity usingnonmyeloablative, preparative regimens and induction of graft-versus-malignancy, Curr Opin Oncol, 1999,11:87-91).Artlertt.CM etc. report, pregnancy for many years back parent and its offspring's cell be still chimeric (referring to Artlertt.CM etc., Identification of fetalDNA and cells in skin lesions from women with systemic sclerosis, N Engl JMed, 1998,338:1186-91), therefore, the cell that parent may be able to tolerate its offspring also allows the cell existence of injecting and produces a kind of transplanting with antitumous effect.
Summary of the invention
So purpose of the present invention just is to provide a kind of activatory HLA half-matched recessive allele Combination hemopoietic stem cell, this stem cell derives from donor, and the lymphocyte micro chimerism with in its acceptor has antitumor action.Acceptor wherein can be daughter, son, and donor is that father, mother or donor are daughter, son, and acceptor is father, mother.
Another object of the present invention provides a kind of method that obtains above-mentioned stem cell, this method comprises the peripheral blood of gathering cancer patients and family members thereof, for blood group identical patient and family members, before gathering peripheral hematopoietic stem cells patient and family members thereof are closed blood, the peripheral hematopoietic stem cells that collects is poured in the specific substratum cultivated; If patient and its family members' blood group incompatibility earlier are separated into mononuclearcell respectively with the peripheral blood that collects, and then with substratum co-cultivation same as described above.Family members wherein comprise son and daughter.
The peripheral hematopoietic stem cells of cultivating is collected, and washing feeds back and gives the patient, feeds back the back and finds that patient's appetite increases, and sleep improves, and physical situation index improves, and indivedual pathology have oedema, further are clearly better after the treatment.This show feed back to this cell in the body after, can improve immunizing power, thus kill tumor, therefore, can predict this stem cell has certain effect at present positive popular SARS (severe acute respiratory syndrome, the acute respiratory distress syndrome) virus of resistance also tool.
Used substratum called after X-VIV020 (CAMBREX) in the above-mentioned cultural method contains the lipid acid, the cholesterol of 1.5ug/ml, the glyceryl ester of 5-20ug/ml, the gamma-interferon of 100-5000U/ml, the Transferrins,iron complexes of 300ug/ml of IL-2,0.1-10ug/ml of Dulbecco minimum medium (IMDM), the 100-5000U/ml of Iscove improvement.Buffered soln is sodium bicarbonate or HEPES, and the pH value of substratum is 7.0.
Description of drawings:
Fig. 1: HLA somatotype PCR electrophorogram, the positive products size is about 500bp.
Fig. 2: the cell colony that obtains with the X-VIV020 culture medium culturing.
Embodiment
The present invention is further described with the following examples, but scope of the present invention is not provided constraints.
Embodiment 1: the substratum of culturing stem cells: X-VIV020 (CAMBREX)
In IMDM, add linolic acid 8ug/ml, cholesterol 1.5ug/ml, triglyceride 15ug/ml, Tween80 5ug/ml, gamma-interferon 1000U/ml, Transferrins,iron complexes 300ug/ml, 2600ug/mlHEPES damping fluid (pH 7.0), 2500ug/ml sodium bicarbonate.Before using substratum, in substratum, add the IL-2 of 1000U/ml.
Embodiment 2: the source of stem cell
Source of human stem cell is in a cancer patients's peripheral blood, and this patient is an Asia women of 52 years old, suffers from serious minute voltinism epithelium thymic carcinoma.She is seriously short of breath and have the venous lumen syndromes in late period, carries out CT scan on computers and finds a earth mediastinal tumor, bag heart piece, increasing upper left and right midbrain encephalomere tumor mass, right liver piece.Her total Karnofsky score is 30-40, and Ca19-9 is 70U/ml (normally being 35).Her weight loss 60Kg, the life of expection is very short.Its daughter is normal.
Embodiment 3: separation of stem cell and cultivation
Handle daughter with granulocyte scavenger cell clone's stimulating factor (GM-CSF) and from daughter's patient peripheral blood, take out blood sample after 5 days, if this patient and its daughter's blood group is harmonious, then close blood, divide mononuclearcell in the peripheral blood sample behind the clutch blood with the U.S.'s CS-3000plus of Baxter company blood cell separator.After the peripheral hematopoietic stem cells collection stem cell in the collecting bag is poured in the 50ml centrifuge tube, washes 3 times with physiological saline, simultaneously IL-2 is joined among the substratum X-VIV020 (CAMBREX), concentration is 5 * 10
3/ ml is total to 1000ml, the stem cell of washing is joined put into 5%CO in the substratum
2Cultivated in the incubator 3 hours, during every half an hour the incubator bottle is shaken once, prevent that it is adherent.
If patient and its daughter's blood group incompatibility, then gather peripheral blood cells washing after, use Ficoll lymphocyte separation medium 1800rpm respectively, the 18min isolated lymphocytes is collected mononuclearcell, after physiological saline washing 2 times, co-cultivation as stated above.Cell colony figure referring to Fig. 1.
Embodiment 4: the evaluation of the stem cell that is obtained
1. the DNA extraction of the stem cell that is obtained
Get the above-mentioned stem cell of 3ml, the centrifugal 5min of 1500rpm abandons supernatant.Get the 20ul protein lysate and add in the 1.5ml centrifuge tube, add the centrifugation of 200ul peripheral blood, add the AL damping fluid of 200ul again, concussion 15s.Hatch 10min for 56 ℃, of short duration centrifugal, add the 200ul dehydrated alcohol, change sample over to QLAampspin column, the centrifugal 1min of 12000rpm adds 500ulAW1, the washing of AW2 damping fluid 2 times successively, with the DNA stripping on the filter membrane, collect in another centrifuge tube standby with 50ulmili-Q water.
2.DNA quality examination
With the DNA detection OD260 of said extracted, the absorbancy of OD280, sample OD260/OD280＞1.8, and calculate DNA concentration.
1.PCR detect the HLA somatotype
PCR system: PCR mix 7.1ul
Taq enzyme 0.1ul
DNA(75-125ng/ul) 1ul
The PCR condition:
The circulation step temperature (℃) time (Sec)
1 1 96 60
5 1 96 25
2 70 50
3 72 45
21 1 96 25
2 65 50
3 72 45
4 1 96 25
2 55 60
3 72 120
Keep 14
2. electrophoresis
Configuration 2%TBE sepharose, 120V electrophoresis 15min observes result of determination, positive products 500bp (see figure 1).
What adopt in this experiment is " sequence specific primers " amplification technique, is called for short SSP (sequence spesificprimer).From patient's peripheral blood karyocyte, extract DNA and carry out the evaluation and the screening of HLA type.The HLA that it is based on dna sequence dna joins the type test kit, and the method for using pcr amplification is carried out the somatotype of HLA-A2 site low resolution gene, the primer of design be used to the to increase allelotrope of the international council of WHO definition.Adopt this method to detect 41 patient's peripheral bloods altogether in this experiment, wherein 19 be called the HLA-A2 positives, positive rate is 46.34%.
Embodiment 5: the treatment tumor promotion of cultivating the HLA half-matched recessive allele Combination hemopoietic stem cell of gained
The stem cell that cultivation is obtained feeds back to the patient, feeds back after 3 days, and patient's cough weakens, and appetite increases, and its Karnofsky value is increased to 70, and late period, ground venous lumen syndromes improved.By the 25th day, the Karnofsky value was increased to 90, and CT scan finds that upper left and right midbrain encephalomere tumor mass significantly reduces, and Ca19-9 is reduced to 32U/ml.By the 210th day, her weight increase 66Kg, gone up normal, great-hearted life again excessively.Treat after 1 year, CT scan shows the completely dissolve of upper left and right midbrain encephalomere tumor mass, and the liver piece also disappears.
Claims (7)
1, a kind of method that obtains activatory half-matched HLA recessive allele Combination hemopoietic stem cell, this method comprises the peripheral blood of gathering cancer patients and family members thereof, for blood group identical patient and family members, gather before the peripheral hematopoietic stem cells and patient and family members thereof to be carried out HLA close blood, the peripheral hematopoietic stem cells that collects is cultivated in specific substratum; If patient and its family members' blood group incompatibility earlier are separated into mononuclearcell respectively with the peripheral blood that collects, and then with substratum co-cultivation same as described above.
2, the cancer that the process of claim 1 wherein comprises malignant thymoma, digestive tube knurl, respiratory tract knurl, urologic neoplasms and ovarian cancer and mammary cancer.
3, the family members that the process of claim 1 wherein comprise father, mother, son and/or daughter.
4, the method for claim 1, substratum wherein is X-VIV015 (CAMBREX) substratum, the lipid acid, the cholesterol of 1.5ug/ml, the glyceryl ester of 5-20ug/ml, the gamma-interferon of 100-5000U/ml, the Transferrins,iron complexes of 300ug/ml of IL-2,0.1-10ug/ml that contains Dulbecco minimum medium (IMDM), the 100-5000U/ml of Iscove improvement, buffered soln is sodium bicarbonate or HEPES, and the pH value is 7.0.
5, with the half-matched recessive allele Combination hemopoietic stem cell that method obtained of claim 1.
6, the stem cell of claim 5 is used to prepare the purposes of the medicine for the treatment of tumor disease.
7, the stem cell of claim 5 is used to prepare the purposes of the medicine for the treatment of SARS.
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| Application Number | Priority Date | Filing Date | Title |
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| CNA031478735A CN1566330A (en) | 2003-06-27 | 2003-06-27 | Activated HLA semi-coincident allogene mixed hematopoietic stem cell and method for making same |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101085983A (en) * | 2006-06-06 | 2007-12-12 | 刘永详 | Autologous stem cell, target cell transformed therefrom and its uses |
| JP2023521671A (en) * | 2020-04-03 | 2023-05-25 | ユニバーシティー オブ フロリダ リサーチ ファンデーション， インク． | Stem cell immunomodulatory therapy for COVID-19 infection |
-
2003
- 2003-06-27 CN CNA031478735A patent/CN1566330A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101085983A (en) * | 2006-06-06 | 2007-12-12 | 刘永详 | Autologous stem cell, target cell transformed therefrom and its uses |
| JP2023521671A (en) * | 2020-04-03 | 2023-05-25 | ユニバーシティー オブ フロリダ リサーチ ファンデーション， インク． | Stem cell immunomodulatory therapy for COVID-19 infection |
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