Specific transplantation tolerance refers to the state that the recipient's immune system does not produce immune response to allogenic or heterogeneous donor antigens for a long time, but can produce normal immune response to other antigens without using immunosuppressive drugs. At present, it is considered that inducing immune tolerance involves four mechanisms: immune clearance, immune disability, immune suppression and immune neglect. Since Petersen et al. discovered in 1999 that hepatic oval cells can be derived from bone marrow, identifying hepatic stem cells from bone marrow cells has become a hot spot in recent years. At present, studies have confirmed that multiple subsets of hematopoietic stem cells isolated and purified from bone marrow can differentiate into liver stem cells under certain microenvironment or cytokine induction [B]. It has been found that many cell subsets in bone marrow have the potential to differentiate into hepatocytes. Such as KTLS cells (c-kithighlowlin-SCA-1+) [h], β2-m-Thy- 1+ cells [12], CD44-CD45-HLA-c-kit- pluripotent adult precursor cells (T-cells). These cells all involve many different surface markers, which may represent different development stages or different branches of bone marrow stem cells. These cytokines must exist in the microenvironment of cells, including signal molecules involved in adhesion process in extracellular matrix and cytokines [B] [E] involved in normal cell development, differentiation and maturation. Petersen et al. [L], Theise et al. [M, N] and Alison et al. [O] have successively pointed out that bone marrow stem cells or hematopoietic stem cells can be transformed into hepatic oval cells or even mature hepatocytes and bile duct cells in mouse liver, and proved that this phenomenon also exists in human body. High concentration of hepatocyte growth factor (HGF) induced rat bone marrow cells in vitro, and the expression of albumin and alpha-fetoprotein was detected by RT-PCR. Immunocytochemistry also confirmed that the induced cells had the characteristic expression of AFP, albumin, CK8/ 18 and other liver precursor cells. β2m-Thy- 1+ cells were isolated from human or rat bone marrow cells by magnetic strain cell screening method, which can express the characteristics of hepatocytes [P]. These bone marrow-derived liver stem cells (BDHSC) were quickly integrated into the liver plate after intrahepatic transplantation and differentiated into mature hepatocytes. Adding bile serum during in vitro culture can promote its differentiation into hepatocytes. Cai Yunfeng and others successfully isolated multiple subsets of bone marrow stem cells by immunomagnetic beads method, and proved that β2 microglobulin negative (β2 m-) cells in rat bone marrow were polygonal cells after induction in vitro, and albumin, AFP, CK8/ 1 8 were positive, while other stem cell types did not show similar changes. It is suggested that this subgroup of bone marrow stem cells has the ability to differentiate into liver stem cells. According to the method they introduced, we successfully isolated liver stem cells with the order of 1.5× 105. The purity is 95%, and it can reach the order of 2× 106 after 7 days of culture. Immunohistochemical staining showed that it had specific markers of hepatocytes, bile duct epithelial cells and vascular endothelial cells, so it was speculated that it might differentiate into the above three cells (see the basis of research work). These results indicate that there are stem cells in bone marrow cells that can differentiate into hepatocytes.
A big problem of liver gene therapy is that mature hepatocytes modified by genes are not easy to be expanded and passaged in vitro. Liver stem cells have strong proliferation ability, and even after gene modification, it is still possible to be passaged, which opens up a new way to overcome the main problems in gene therapy at present. As a carrier, liver stem cells have the characteristics of one-time intervention and permanent treatment, and immortalized liver stem cells do not cause cancer [F].
Based on the above results, we assume that liver stem cells can target and colonize around the central vein in the portal area of the liver, and can differentiate and supplement the biological characteristics of damaged or aging vascular endothelial cells, bile duct epithelial cells and hepatocytes when necessary. Using adeno-associated virus as a vector, DcR3 gene was transferred into liver stem cells isolated and purified from the bone marrow of male inbred Wistar rats, and the liver of female inbred Wistar rats was transplanted into female inbred Lewis rats. The liver stem cells transfected with DcR3 gene were injected into the transplanted liver through portal vein. With the colonization of liver stem cells, DcR3 gene is mainly expressed in portal area, and if necessary, with the further differentiation of liver stem cells, it is partially expressed in vascular endothelial cells, bile duct epithelial cells and liver cells, thus inhibiting the occurrence of rejection in the initial (primary) and attack (secondary) stages. The strong immunosuppression of DcR3 gene is limited to the liver, thus inducing liver-specific transplantation tolerance.
refer to
1. Tolerance level of peripheral T cells. Transplantation immunology. 2002, 10(2-3): 109- 1 14
2. Adams DH Goddard. A new method of immunosuppression in liver transplantation. Chinese Journal of Gastroenterology, 2002, 17(2)
3.Petersen BE, Bowen WC, Patrene KD, et al. regard bone markers as a potential source of hepatic oval cells. Science.1999,284 (5417):1168-70
4. Liver stem cells: from bone marrow cells to hepatocytes. Biochemical and biophysical research newsletter 200 1, 28 1(l):l-5
5. Similarity of early histological changes in rat liver induced by ethionine, 2- acetylaminofluorene and 3- methyl -4- dimethylaminoazobenzene. Cancer research 1955, 16: 142-8
6. Expression of stem cell factor and its receptor c-kit during liver regeneration in adult rats. Laboratory investment1994,70 (4): 511-6
7. Rat hepatic oval cells express hematopoietic stem cell marker Thy- 1. Liver disease. 1998，27(2):433-45
8. Expression of stem cell factor receptor c-kit in normal and diseased children's livers: Identification of human hepatic progenitor cells? Liver disease. 1999，30(l): 1 12-7
9. Cells co-expressing CD34 hematopoietic stem cells and CAM 5.2 whole-cell keratin markers were isolated from human fetal liver. Journal of Hepatol1998,29 (3): 450-4
10. The purified hematopoietic stem cells can differentiate into hepatocytes in vivo. National medical center. 2000,6( 1 1): 1229-34
1 1. Gao z, mcallister VC, general manager of Williams. Regeneration of liver endothelial cells by bone marrow-derived cells. The Lancet.2001,357(9260):932-933.
12. Isolation, identification and transplantation of hepatocyte stem cells from bone marrow. Biochemical and biophysical research newsletter 200 1, 288(l): 156-64
13. Daley AK, Japanese CP, Donaldson Point. Immunoregulatory gene polymorphism: towards individualized immunosuppressive therapy? Journal of pharmacogenomics. 2002, 2( 1): 13-23
14. Pitty RM, Master Ltd., Laurenda, et al. Genome amplification of Fas ligand decoy receptor in lung cancer and colon cancer. Nature. 1998,396(67 12): 699-703
A. crosby Ha, Gastroenterology, 200 1, 120:534-544.
Yao Peng, Zhan Yiqun, Xu Wangxiang, et al. Effect of cell growth factor on rat liver stem cells cultured in vitro. Chinese Journal of Hepatology, 2003,11(1): 33 ~ 36.
C. Zhao Chunhua (hsc3)
D. Mr. Alison River
Suzuki A. Journal of Cell Boiling by Cloning and Identification, 2002,156 (1):173.
Alan JE, immortal. Journal of China Academy of Sciences, 2002,99 (6), 3696.
G Theise ND。 The canal. Liver disease,1999,30 (6):1425-1433
Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. National medical center. 2000 1 1 month; 6( 1 1): 1229-34.
Pluripotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest。 May 2002; 109( 10): 129 1-302.
J Danet GH, Luongo JL, Butler G, et al. C 1qRp defines a new human stem cell population with hematopoietic and liver potential. Journal of the National Academy of Sciences August 6, 2002; 99 (16):10441-5. EPUB July 24, 2002.
Why stem cells? Science. February 25, 2000; 287(5457): 1439-4 1.
Length PetersenBE, BowenWC, PatreneKD, et al. Bone Marrow Rowa Potential Sources of HepaticoVallls. Science,1999,284:11681170.
Meter (short for meter) TheiseND, BadveS, SaxenaR, etc. Bone marrow clearance induced by microwave radiation of bone marrow cells. Liver disease, 2000, 3 1∶235 240.
Nouns (short for noun) TheiseND, NimmakayaluM, GardnerR, etc. Chinese Journal of Hepatology, 2000, 32:1165438.
Page (abbreviation of page) AvitalI, InderbitzinD, AokiT, etal. Isolation, identification and transformation of bone marrow-derived hepatocellular carcinoma, 200 1, 288∶ 156 164.
2. Research contents, research objectives and key problems to be solved. (This part focuses on the content.)
2. 1 research content
2. 1. 1 Construct adeno-associated virus vector carrying DcR3 gene, transfect it into AAV293 cells, and collect AAV virus particles for later use.
2. 1.2 bone marrow was extracted from femur and tibia of male inbred Wistar rats, liver stem cells were isolated, and the adeno-associated virus particles were transfected to identify their expression.
2. 1.3 The liver of female inbred Wistar rats was implanted into female inbred Lewis rats, and the transfected liver stem cells of male inbred Wistar rats were injected into the liver through portal vein.
2. 1.4 According to the Y chromosome and the green fluorescent protein (GFP) marker of AAV virus vector, the colonization and distribution of transgenic liver stem cells in donor liver were determined.
2. 1.5 Detect the expression of DcR3 gene transferred into liver stem cells in donor liver.
2. 1.6 Observe the occurrence of graft rejection after liver transplantation.
2.2 Research objectives: This project intends to construct adeno-associated virus carrying DcR3 gene and transfect it into liver stem cells derived from bone marrow of male inbred Wistar rats; The liver of female inbred Wistar rats was implanted into female inbred Lewis rats, and the liver stem cells transfected with DcR3 gene were injected into the transplanted liver through portal vein. DcR3 gene is expressed in donor liver for a long time, thus inducing liver-specific transplantation tolerance. To investigate the colonization and distribution of (1) transgenic liver stem cells in donor liver. (The expression of DcR3 gene in donor liver after transforming into hepatic stem cells. (3) The distribution of transgenic liver stem cells in donor liver and the relationship between their gene expression and transplantation tolerance.
2.3 Key problems to be solved
2.3. 1 Efficiency of adeno-associated virus transfection of liver stem cells: Gene transfection of stem cells is difficult, which is one of the key links in this project. Members of this project (deleted) successfully infected neural stem cells with adenovirus on 200 1, the success rate was about 80% ~ 90%, and they still had the characteristics of stem cells after 12 generations of subculture. The transfection efficiency of adeno-associated virus is higher than that of adenovirus, and the transfected cell types are more extensive than adenovirus. Therefore, our research group has the ability to complete the transfection of liver stem cells by adeno-associated virus.
2.3.2 Colonization quantity of transgenic liver stem cells in portal area of donor liver: The colonization quantity of liver stem cells in portal area is related to the degree of liver injury. The more serious the injury, the more liver stem cells will settle in the portal area, and vice versa. Hepatic ischemia-reperfusion is a kind of injury to the liver during liver transplantation. In order to further increase the number of liver stem cells in portal area, a leaf of rat liver can be removed during cold preservation, and then liver transplantation can be completed.
3. Put forward the research scheme and feasibility analysis. (including the description of relevant methods, technical routes, experimental means and key technologies)
3. 1 research scheme
3. 1. 1 The DcR3 gene was cloned according to the method introduced by Pitti RM et al.
3. 1.2 construct adeno-associated virus vector carrying DcR3 gene, infect AAV-293 cells with it for in vitro expression identification, and collect AAV virus particles for later use.
3. 1.3 Bone marrow of femur and tibia of male inbred Wistar rats was taken, and liver stem cells were isolated, purified and identified according to the method introduced by Itzhak Avital.
Liver stem cells were transfected with 3. 1.4 adeno-associated virus vector and directly infected.
3. 1.5 in vitro test: Liver stem cells expressing DcR3 gene inhibit lymphocyte apoptosis induced by FasL; Liver stem cells expressing DcR3 gene inhibit FasL-induced lymphocyte chemotaxis
3. 1.6 animal experiment: rat liver transplantation was performed by cuff method, and the liver stem cells transfected with DcR3 gene were injected into the liver through portal vein after transplantation; Blood and liver samples were taken on the 3rd, 7th, 65th, 438+04, 26th, 438+0 and 28th day after operation, and the liver function was detected by routine biochemical methods, the expression of DcR3 gene in the liver was detected by Northern blot and Western blot, the infiltration of CD4+ and CD8+ lymphocytes in the donor liver was detected by immunohistochemistry, and the apoptosis in the donor liver was detected by TUNEL method.
3.2 Technical route
3.3 Feasibility analysis
3.3. 1 Theoretically, liver stem cells derived from bone marrow are the precursor cells of oval cells and basophilic small hepatocytes in the portal area of the liver. It has been proved that the Helin tubules in portal area of liver transplanted with hepatic stem cells injected through portal vein can differentiate into vascular endothelial cells, bile duct epithelial cells and hepatocytes. Hepatic hilum is the first infiltration area of CTL cells in liver transplantation rejection, and vascular endothelial cells, bile duct epithelial cells and hepatocytes are the target cells attacked by CTL cells. Therefore, it is feasible in theory that liver stem cells bring the transfected immunosuppressive gene into the liver and express it continuously in vascular endothelial cells, bile duct epithelial cells and liver cells, thus inducing transplantation tolerance specific to the liver.
3.3.2 The technologies involved in this study are all mature immunological technologies; Our institute has the necessary equipment and conditions to carry out this research.
4. Features and innovations of this project.
In this study, the targeting of liver stem cells in the liver is used as the carrier of immunosuppressive molecules, so that the role of non-specific immunosuppressive molecules is limited to the liver, which overcomes the shortcoming of too large range of immunosuppressive molecules, thus inducing liver-specific transplant tolerance.
5 annual research plan and expected research results. (including important academic exchange activities to be organized, international cooperation and exchange plans, etc.). )
Annual research plan
The DcR3 gene was cloned from October 2005 to September 2005, and the adeno-associated virus vector carrying DCR3 gene was constructed for later use.
From 2005. 10/0, 2005 to June, 2006, liver stem cells from the bone marrow of male inbred rats were isolated, purified and identified, and transfected with adeno-associated virus. Expression and identification of DcR3 gene in vitro: functional experiment of transfected DcR3 gene in vitro.
From July 2006 to June 2007, the animal model of liver transplantation was completed, and transfected liver stem cells were injected into the liver through portal vein. Liver samples were taken regularly, and the colonization and distribution of transgenic liver stem cells in donor liver were determined according to Y chromosome marker staining and GFP. Detecting the expression of the gene transferred into liver stem cells in the liver; The occurrence of transplant rejection.
From July, 2007 to June, 2007, and from April, 2007 to February, 2007, I supplemented the experimental omissions, sorted out the experimental data, statistically processed the experimental data, and wrote a paper.
Expected research results
1. It is proved that liver stem cells transfected with DcR3 gene can be continuously expressed in donor portal area and some vascular endothelial cells, bile duct epithelial cells and liver cells, and induce long-term immune tolerance in liver transplantation.
2. Publish 2-3 papers in foreign academic journals, 6-8 papers in domestic core journals, and exchange them at domestic academic conferences.
(2) Research basis and working conditions
1. Work basis (research work accumulation and research results related to this project)
The results came out from the end of February to the beginning of March.
2 working conditions (including the existing experimental conditions, the shortcomings of experimental conditions and solutions, including the planning and implementation of state key laboratories and departmental open laboratories. )
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3. Resume of the applicant (including the academic qualifications and research resumes of the applicant and the main members of the project team, the recently published catalogue of major works related to this project, the academic awards won and the tasks undertaken in this project. )
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(III) Description of fund application (required to be carefully filled out in accordance with the Measures for Fund Management of the National Natural Science Foundation of China), and the direct relevance and necessity of project research should be explained item by item for the procurement of fixed assets and equipment with funds of more than 50,000 yuan. )
(4) List of other attachments (attached materials shall be submitted together with the paper application form after being copied) (attached list submitted together with the paper application form, such as the recommendation letter of the applicant with intermediate technical title or the recommendation letter of the in-service graduate student applying for the project tutor, etc.). ).)