PROLONGED SURVIVAL OF RAT LIVER ALLOGRAFTS TRANSFECTED WITH ADENOVIRUS VECTORS CONTAINNING FAS-LIGAND AND CRM A GENES

目的。Fas配体(Fas-Ligand，FasL)在角膜、睾丸等免疫优势部位逃避宿主免疫反应中起重要作用。细胞因子反应修饰物A(CrmA)是牛痘病毒的一种基因产物，被认为在异基因移植中可以阻断Fas/FasL和穿孔素/颗粒酶介导的凋亡通路。在本研究中，我们发现FasL和CrmA基因是延长大鼠移植肝存活的有效基因。方法：研究方法。以Cre为载体的重组腺病毒载体在供体肝脏中表达FasL和CrmA，免疫染色证实其表达，体外检测其诱导和抑制细胞凋亡的功能。用DA(RT-L)和Lewis(RT-L；L)大鼠组合，用FasL和/或CrmA基因转移的移植物进行原位肝移植。结果。成功构建了表达FasL(AxCALNLFasL)和CrmA(AxCALNLCrmA)的重组腺病毒载体，并通过免疫染色检测了它们在细胞中的表达。表达FasL的COS-7细胞可诱导Jurkat细胞发生凋亡；表达CrmA基因可显著降低Hep2细胞的凋亡率。将转FasL和/或CrmA基因的DA肝移植到Lewis大鼠体内，受体存活时间显著延长。结论。在此基础上，我们得出结论：在供体肝脏中有适当水平的FasL表达可诱导受体活化的T细胞发生凋亡，从而调节移植肝的免疫反应。此外，CrmA是一种有效的基因产物，可通过调节转基因移植物中的细胞凋亡活性来延长受体的存活时间。

Purpose. Fas-ligand (FasL) plays an important role in immune privileged sites including cornea and testis, to escape from host immune response. Cytokine response modifier A (CrmA), a gene product of cowpox virus, is considered to block Fas/FasL and perforin/granzyme mediated apoptotic pathways in allogeneic transplantation. We investigated in the present study, that FasL and CrmA are the effective genes to prolong survival of rat liver allografts. Methods. FasL and CrmA were expressed in donor liver using adenovirus vector with a Cre-mediated gene delivery system The gene expressions were confirmed by immune staining and their functions were examined with in vitro assay for induction and inhibition of the apoptosis. Using DA (RT-l<a>) to Lewis (RT-l^<l>) rat combination, orthotopic liver transplantation was performed with FasL and/or CrmA gene-transfered grafts. Results. The recombinant adenoviral vectors expressing FasL (AxCALNLFasL) and CrmA (AxCALNLCrmA) under a Cre-mediated switching system were successfully generated and their gene expressions were detected in the transfected cells by immune staining. FasL expressed on the COS-7 cells were induced apoptosis in the Jurkat cell ; CrmA gene-expression dramatically reduced apoptosis in Hep2 cells. The FasL and/or CrmA-transfected DA livers were transplanted into Lewis rats, resulting in a significant prolongation of recipient survival time. Conclusions. Based on these observations, we concluded that an appropriate expression level of FasL in donor liver could act to induce apoptosis to recipient activated T cells, which resulted in the regulation of immune response to the liver allograft. In addition, CrmA is an effective gene-product to prolong recipient survival by the apoptosis modulation activity in the gene-transferred graft.