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

转染含有 FAS 配体和 CRM A 基因的腺病毒载体的大鼠同种异体肝脏移植物的存活时间延长

基本信息

批准号：
12671171
负责人：
LI Xiao-Kang
金额：
$ 2.18万
依托单位：
National Children's Medical Research Center
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671171/
关键词：
Adenovirus Fas-ligand CrmA organ transplantation Cre loxP apoptosis gene therapy liver HepG2

项目摘要

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.

目的。 FAS-Ligand（FASL）在包括角膜和睾丸在内的免疫特权部位中起着重要作用，以摆脱宿主免疫反应。 Cypox病毒的基因产物A（CRMA）被认为可以阻止同种异体移植中的FAS/FASL和穿孔/颗粒酶介导的凋亡途径。我们在本研究中研究了FASL和CRMA是延长大鼠肝移植物生存的有效基因。方法。使用腺病毒载体和CRE介导的基因递送系统在供体肝脏中表达FASL和CRMA。通过免疫染色证实了基因表达，并通过体外测定法检查了其功能，以诱导和抑制凋亡。使用DA（RT-L <a>）与Lewis（RT-L^<l>）大鼠组合，使用FASL和/或CRMA基因转移的移植物进行原位肝移植。结果。在CRE介导的开关系统下表达FASL（AxcalnlFASL）和CRMA（Axcalnlcrma）的重组腺病毒载体成功地产生，并通过免疫染色在转染的细胞中检测到其基因表达。在cos-7细胞上表达的FASL在Jurkat细胞中诱导了凋亡。 CRMA基因表达大大降低了HEP2细胞的凋亡。将FASL和/或CRMA转染的DA肝移植到刘易斯大鼠中，从而显着延长受体存活时间。结论。基于这些观察结果，我们得出的结论是，供体肝脏中FASL的适当表达水平可以作用于对受体激活的T细胞的细胞凋亡，从而调节对同种异体移植的免疫反应。此外，CRMA是一种有效的基因产物，可通过基因转移的移植物中的凋亡调节活性延长受体存活。