The Therapeutic Efficacy Of Lentiviral Vector Mediated Hepatocyte Growth Factor Gene-modified Mesenchymal Stem Cells On Rat Liver Allografts

Bone marrow mesenchymal stem cells(MSC) are adult stem cell of mesodermal origin with abilities of self-renewal and multiple lineage differentiations including osteoblasts,adipocytes, chondrocytes,myoblasts,and early progenitors of neural cells. Researches show that MSC have the immunosuppressive abilities,which inhibit lymphocyte proliferation ,and alleviate allograft rejection. Data have indicated soluble growth factors are relevant to the immunosuppression of MSC, including hepatocyte growth factor(HGF) especially, Which has the functions of promoting cell proliferation, reducing apoptosis,cell protection and immunosuppression. MSC can be purified and cultured easily in vitro,and can take ectogenesis gene easily. In order to study the immune synergistic effect of MSC and HGF,we construct lentiviral vector carrying the human HGF,and then infect the MSC to get hHGF gene-modified MSC(MSC/hHGF). The biological properties and immunoregulatory function in vitro and in vivo of MSC/hHGF were investigated. MSC/hHGF could exhibit the immunosuppression synergetic effect of hHGF and MSC, reduce the acute rejection and prolong the survival time. It can supply a novel strategy and method to induce immune tolerance on liver allograft.PART 1 Modified technique of orthotopic liver transplantation in rats Objective To investigate the surgical technique of establishing a reliable rat model of orthotopic liver transplantation. Methods Syngeneic group SD-SD and allogeneic group SD-Wistar rats liver transplantation were performed in 70 and 60 cases, respectively. 130 cases of orthotopic liver transplantations in rats were performed using modified Kamada's two-cuff technique.Under the sufficient exposure of the porta hepatis,the liver was perfused through the portal vein with cold perfusion without touching the liver. The anastomosis of the suprahepatic vena cave was sutured end- to-end with 8/0 prolene line. Guided by double line, the continuity of portal vein was established by means of cuff method easily. The fluid was supplemented sufficiently after operation to maintain the stabilization of hemodynamics. Results Orthotopic liver transplantations were performed in 130 rats.The time for donor operation was 38.2±2.5 min,the receptor operative and anhepatic time were 45.6±3.5 min and 15.1±2.2 min respectively.The successful operative rate was 93%.The survival rate after one week was 92%. All the data is better than that of traditional method.There were 64 survivals in SD-SD group and 57 in SD-Wistar group after liver transplantation, and the survival time were long-term and 8～20 days(mean 10.5 days) respectively.The liver function recovered well in SD-SD group ,while in SD-Wistar group we found the liver functional failure and acute rejection in pathology in 3-5 days after liver transplantation which lead to death all without any therapy. Conclusions The modified method is proved to be ideal for its advantages of simple operation,short anhepatic phase and high operative successful rate. The SD to Wistar rat combination is a rejection model and can be used to study immune tolerance in liver transplantation.PART 2 Isolation,culture and identification of the bone marrow mesenchymal stem cells(MSC) in vitro and the distribution of MSC in the rat transplantation liver. Objective To isolate,culture and identify the rat bone marrow mesenchymal stem cells(MSC) .To study the distribution of MSC after injection into portal vein in rats of orthotopic liver transplantation. Methods MSC were collected by flushing the femurs and tibias from 3-4-week-old male SD rat under sterile condition. MSC were separated with direct anchoring method,and then they were amplified and cultured.The characters of the cell,such as morphology,cell growth curve,phenotype were demonstrated.MSC were identified using flow cytometry. The abilities to differentiate along adipocytic and osteoblastic were investigated. MSC were labeled by DAPI and were injected into portal vein in rats after orthotopic liver transplantation. The distribution of MSC was observed by fluorescence after frozen section. Results By direct anchoring method, MSC were gained after purification and proliferation,and passaged every five days.The cell population consisted of spindle-shaped cells with significant renewal capacity,stabilities of the biology as they were cultured until 20 passages. Flow-cytometric analysis showed the 3th passage MSC were positive against CD44 and CD90,with a positive rate of 94.81% and 99.53%,while negative against CD34,CD45 and CD11b with a negative rate of 0.04%,1.94% and 1.42%.MSC can be differentiated into adipocyte and osteoblast cell. The positive rate DAPI reached 100%. MSC labeled with blue fluorescence can be founed in livers of rats. Conclusions Direct anchoring method is simple and feasible when used to separate and culture MSC,so it deserves popularizing.Purified MSC with steady biological characters were gained.MSC can successfully reside in the liver of rats.PART 3 Construction of lentiviral vector recombined by hHGF gene and construction of hHGF gene-modified MSCObjective Construct lentiviral vector carrying the human Hepatocyte growth factor gene,and then infect the MSC to get hHGF gene-modified MSC(MSC/hHGF). The biological properties and immunoregulatory function in vitro of MSC/hHGF were investigated. Methods The hHGF gene was obtained with PCR from pcDNA-hHGF plasmid. The recombination lentiviral vector plasmid hHGF was constructed with digestion and gene recombinant,then identified with PCR and sequencing. The three plasmids system of lentiviral vector were co-transfected to 293T cells to produce hHGF gene. The supernatant was collected and concentrated by ultracentrifugation and the titer of lentivirus was measured by qRT-PCR. The MSC were infected by the constructed lentivirus and were identified with flurescent microscope, flow-cytometric analysis ,ELISA analysis and RT-PCR. The immunosuppression functions of MSC and MSC/hHGF were observed by mixed lymphocyte reaction(MLR). Results Lentiviral vector carrying hHGF gene had been constructed successfully. The titer of lentivirus was 1×108TU/ml. The infection efficiency of MSC by hHGF lentiviral was high and reached up to 98% tested by FCM when the best MOI was 10. A great mount of green fluorescence was observed with the flurescent microscope until 28 day after infection. Peak concentration of hHGF secreted by MSC/hHGF reached to 40.5ng/ml on 5 day. The concentration can maintain in a high level until 28 day after infection. MSC/hHGF could express hHGF mRNA. The MLR test showed that MSC/hHGF could exhibit more inhibitory effect(64.42%) on lymphocyte proliferation than MSC(46.34%) in vitro. Conclusions By Lentiviral vector, hHGF gene was intergrated into MSC genome ,and transcribed and expressed correctly. MSC/hHGF could exhibit more inhibitory effect.MSC and hHGF have immunosuppression synergetic effect in virto. PART 4 The immunosuppression of lentiviral vector mediated hHGF gene-modified MSC on rat liver allograftsObjective TO study the immunosuppression effect of recombinant lentiviral vector mediated hHGF gene-modified MSC on rat liver allografts rejection in vivo, and to reveal the mechanism of immune tolerance. Methods In the rat liver allografts model,after harvest, 1ml MSC/hHGF containing 2x106 cells were implanted to acceptor via portal vein immediately in the MSC/hHGF group. As controls,1ml MSC/GFP and 1ml MSC containing 2x106 cells were implanted in MSC/GFP group and MSC group respectively by the same method ,and 1ml physiological saline in control group. Then we observed the survival time,examined the liver function and pathology, measured the level of cytokine including hHGF,IL-2,IL-4,IL-10 and IFN-γby ELISA analysis,examined the level of apoptosis of gragts by Tunel method and the expression level of TNF-α,NF-κB,Bcl-2 and PCNA by immunohistology and RT-PCR. Results RT-PCR demonstrated the transcription of hHGF gene in the grafts. Green fluorescence could been observed with the flurescent microscope.The serum cytokine hHGF reached to 6.2ng/ml. The survival time was repectively >100 days, 23.1±3.9 days,23.4±3.1 days in MSC/hHGF group,MSC/GFP group and MSC group,which was significantly higher than control group(10.5±4.2 days). Both of group MSC/hHGF group and MSC group could reduce the histologic rejection Banff scores and improve liver function. Compared with MSC group, MSC/hHGF group exhibit more inhibitory effect, liver function significantly improved,level of acute rejection alleviated,level of cytokine IL-2 and IFN-γdecreased, level of cytokine IL-4 and IL-10 increased,level of apoptosis reduced,the expression level of Bcl-2 and PCNA increased,but the expression of TNF-αand NF-κB descended. Conclusions These data in vivo demonstrate planting MSC/hHGF via portal to rat liver allograft is efficient and effectively and can induce immune tolerance. Compared with injection of MSC alone, MSC/hHGF treatment could alleviate acute rejection and prolong the survival time more significantly.MSC/hHGF could exhibit the immunosuppression synergetic effect of hHGF and MSC ,which made the Th1 cell toward Th2 cell, suppressed IL-2 and IFN-γ,and hence enhanced the expression of Th2 cytokines such as IL-4 and IL-10,suppressed production of proinflammatory factor such TNF-αand NF-κB,reduced the level of apoptosis,promoted liver regeneration. It can supply a novel strategy and method to induce immune tolerance on liver allograft.