| Infection with the hepatitis c virus (HCV) is a major public health problem because of its high prevalence and the severity of its complications, liver cirrhosis and hepatocellular carcinoma (HCC), which is one of the main causes of hepatitis c virus. The treatments (chimioembolisation transarterielle, radiofrequency or surgical removal) is associated with an increased risk of tumor recurrence, liver transplantation is the best treatment for HCC and the underlying cirrhosis, however, a liver transplanted is systematically reinfected by HCV, which is associated with a relapse of the disease and to accelerate. New drugs acting directly on viral factors, protease or polymerase or host factors have been or should be the authorisation for placing on the market and have revolutionised the treatment of chronic HCV infection, which significantly improve the cure rate and reduce the side effects of the treatment of historical reference.However, the use of these treatments in the transplant patient is limited because of drug interactions with certain agents, in addition, the eradication of HCV does not reduce the risk of development of HCC. New therapeutic approaches are needed to prevent the reinfection of the liver graft with hepatitis c and to improve the treatment of hepatocellular carcinoma (HCC).There are a number of approaches for the treatment of cancer by adoptive immunotherapy based on the administration of immune response cells, effector cells. However, these approaches are often autologues, meaning using cells from patient, which are often immunocompromised because of its pathology and/or treatment received in the past. Therefore, the lymphocytes are often difficult to amplify and output a batch for a given patient is long and poorly reproducible. The clinical results obtained are often not commensurate with the biological responses observed. In fact, the development of immunotherapeutic approaches is limited by technical and logistical issues related to the production of cell therapy products, specific to each patient. Such a development could be facilitated by the establishment of a bank of products (from different donors of allogeneic patient ’ready to use’ instead of autologous products, which might improve the develop as well as utilization of immunotherapy in the future.[Object]1. Preclinical evaluation of a genetically modified allogeneic cell bank.The allo-reactive can be defined as the ability of the T cells of the immune system of an individual to react against the cells and tissues of an individual. the reactive can provide beneficial effects, including anti-tumor, but also may lead to serious side effects such as the graft versus host disease (gvhd), it is possible to control the side effects by the introduction of specific gene in these cells, a pre conditional toxicity, or "gene suicide, such as the gene of herpes simplex virus thymidine kinase (hsv-tk) or the inducible caspase 9 (icasp9):in the case of side effects, the aSMKCs may then be removed, specifically, by administering a prodrug ganciclovir or AP1903, respectively. Therefore, we intend to establish a "ready-to-use" aSMKCs bank derived from healthy donors, and to compare the therapeutic and preventive effects of HCC and HCV infection.2,Investigate treatment efficiency on HCC:Using cell culture and animal models, we aimed to investigate the efficacy and safety of suicide gene-modified allogeneic killer cells as a cell-based therapy for hepatocellular carcinoma, The apoptosis of immune effector cells mediated by suicide gene, might be quickly mediated by the use of the prodrug of suicide gene, therefore could prevent the GvHD, the main complications of allogeneic transplantation. So far provide a basis of theory and experiment for the treatment of solid tumors by allogeneic transplantation.3,Demonstrate proof-of concept of preventing HCV reinfection in vitro by aSMKCs after liver transplantation:HCV infection has the most serious complication such as cirrhosis and HCC, liver transplantation is the effective procedure for the complications associated with HCV, however, for patients with HCV after liver transplantation, new graft was reinfected by HCV in a short-term, which is a difficult problem to solve. The aSMKCs have been used for a decade in clinical for treating the patients after allo-graft hematopoietic. This study will study the cell secretion function of aSMKCs and its role in anti HCV re infection, and provide effective treatment and prevention methods for the clinical treatment of HCV infection after transplantation. At present, to our knowledge, this is the first research to assess the activity of allogeneic against solid tumors (not hematological malignancies), and anti-viral therapy.[Method]1. The production of retroviral supernatent and extraction of PBMC, after activation, transduction of PMNC by using retroviral spernatent which containing kind of suicide gene--HSV-tk or iCaspase9, and amplification for 2 weeks, then characterized the subset of aSMKCs by the FACS, and immunomagnetic positive sorting the SMKCs with different antibody, followed by reserving aSMKCs as a "ready to use " bank.2. Comparing the mortality and success ration among approaches of intra-spleenic, intrahepatic and intra-portal vein in the mice of SCID, Balb/c and Nu, analyzing the orthotopic model of HCC based on MRI associating with BLI.3. PBMC cells were transfected by aSMKCs and control group HeLa, Huh7 cells were cultured in vitro, aSMKCs cells were cultured in different concentration and time. To observe the effect of on aSMKCs cells against above HCC cells to investigate the cytotoxity of SMKCs, meanwhile, observe the apoptosis of SMKCs when administration the prodrug of suicide gene in vitro and in vivo.4, the anti tumor effect of aSMKCs in subcutaneous and in situ HCC nude mice model, and the preventive effect of suicide gene on the potential of graft versus host reaction were observed, and the anti HCC effect of aSMKCs was also studied.5, using the JFH1 Delta E1E2-luc transfection huh7.5.1 cells established the HCV replicon system, at the same time, the use and HCVc c Jc-1 to establish HCV positive cell culture system.6, in vitro study of aSMKCs on HCV replication system and cell culture system for the inhibition of HCV virus production, the use of monoclonal antibodies to detect the inhibitory effect of the mechanism, and to observe the effect of immune inhibitor.[Result]1.By using of MP71-T34FT plasmid and SFG.iCasp9.2A.ACD19 plasmid, we successful produced HSV-tk/CD34 and iCasp9/CD19 retroviral supernatant and successful transduced PBMC with different retroviral supernatant, after activation as well as culture in vitro, immunomagnetic positive screen can improve the purity of effector cells aSMKCs, before and after the election of aSMKCs proportion respectively 9.1+0.8% and 89.4+1.2%, which T cells, NK cells and NKT cells transfected respectively 65.5+/-1.8 and 7.2+/-1.1,11.2+/-1.0. After transfection was 77.3+/-3.2,11.1+/-1.6 and 5.3+/-2.0 (mean+standard deviation). It indicated that through different management for production of aSMKCs, we could successful establish a "ready-to-use " aSMKCs bank for the following procedure.2. Testing the sensibility of suicide gene’s prodrug to aSMKCs:aSMKCs and were used to compare the sensitivity to CID (1μg/ml) to CD19/iCasp9+aSMKCs and (1 μg/ml)GCV to HSV-tk/CD34+, the sensibility of suicide gene’s prodrug was 92.5% and 95%, respectively, while PBMC had no obviously effect neither GCV nor CID.3. Comparing the different methods in the field of establish HCC in situ models:the mortality of HCC, IS and IPT in nude mice was 16%,6% and 3%, p<0.05, and IS was 25%,3% and 2%, p<0.01, IH was 3% and 2%, p>0.05, and IH. Through the analysis of MRI and BLI, the HCC, IS and IH in situ HCC model were 52% vs 19% vs 32%, p<0.05.4. Testing the anti-tumor ability in vitro of aSMKCs against HCC:aSMKCs has obvious anti HCC effect in vitro, and the transduction of suicide gene has no effect on aSMKCs cytotoxicity. The anti HCC effect of aSMKCs is non MHC-I restrict, in which the main effector cells are NKT cells and NK cells. Importantly, immunosupressant did not show inhibition effect on aSMKCs cytotoxicity against HCC, while their could be eliminated quickly by using the suicide gene’s prodrug therefore preventing the major complication of allreactivity GvHD.5. In vivo study showed that the aSMKCs cells transfected with the suicide gene had significantly hepatic homing property, SMKCs showed significant anti tumor ability on HCC in situ mice model. The pathological tissue confirmed that the main effector cells were NKT and NK cells, and the anti HCC effect was not affected by the Immunosuppressant. Suicide gene in vivo rapid eliminated aSMKCs cells expressing the suicide gene, thus safe and effective prevent GvHD.6. Using HCV replication system and HCVcc system shown that aSMKCs cells in vitro had obviously anti-HCV effect, the main effector cells were NKT and NK cells, the anti-HCV effect was implemented by EFN-γ.[Conclusion]1. By using kind of retroviral vector, we could establish the cell-banks from different heath donors, in which the cells stably expressing suicide gene, aSMKCs could provide a powerful cell banks in order to clinical using to exert anti-tumor effect against solid tumor including HCC as well as HCV re-infection.2. aSMKCs in vitro has obvious anti-tumor effect, compared with the control group, the suicide gene transduction has no significant effect on the cytotoxicity of allogeneic immune cells;3. The best way to establish the model of orthotopic liver cancer in nude mice by the way of injection of intrahepatic when comparing with other methods such as IP and IPT;4. aSMKCs has an obviously anti HCC effect in vivo, and this function is IL-2-dependent, the anti tumor effect was not influenced by immunosupressant, while the main effector cells of aSMKCs was NK and NKT cells.5. aSMKCs in vitro has a significant anti HCV effect, the role of the play is also IL-2 dependent, and through cytokine of IFN secreted by NK and NKT cell, also, these antiviral effect did not influenced by immunosupressant.6. The suicide gene’s prodrug could induce apoptosis of aSMKC which expressing stably suicide gene, comparing with HSV-tk-GCV system, iCasp/9-CID system presented a more faster apoptosis effect in several minutes, while both of two system could effectively prevent the occurrence of graft versus host response. |
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