人IL-10逆转录病毒载体的构建及对大鼠原位肝移植存活期的影响

Construction of human IL-10 retrovirus vectors and their effects on survival of rats after orthotopic liver transplantation

目的 探讨人白细胞介素 10 (hIL 10 )逆转录病毒载体的构建及对大鼠原位肝移植存活期的影响。方法 将hIL 10克隆基因片断 ,经双酶切后定向插入到逆转录病毒载体 (MSCV)中 ,用脂质体法转染PT6 7包装细胞 ,以G418筛选阳性克隆。将Wistar对SD大鼠原位肝移植模型分三组 ,组Ⅰ为空白对照组 ,组Ⅱ为MSCV空载体灌洗组 ,组Ⅲ为MSCV hIL 10灌洗组。观察大鼠存活期及肝脏功能变化。结果 hIL 10基因片断插入到MSCV载体中经转染包装后得到 2× 10 7CFU ml的分泌hIL 10的病毒悬液 ,hIL 10分泌量为 12 2 0ng 10 6 细胞 2 4h。组Ⅲ与组Ⅰ、组Ⅱ比 ,大鼠存活期明显延长 (P <0 0 1) ,肝脏功能无明显差异。结论 获得MSCV hIL 10重组质粒 ,获得高滴度的分泌hIL 10的病毒悬液 ,hIL 10的表达可延长大鼠原位肝移植的存活期。

Objective To study the construction of human IL-10 retrovirus vectors and their effects on survival of rats after orthotopic liver transplantation. Methods Human IL-10 gene was inserted into MSCV vectors with double digestion. Then the recombinant vectors were transfected into PT67 package cells by lipofectin method and were screened by G418 selection medium. After the orthotopic liver transplantation between Wistar rats and SD rats was conducted, all the rats were divided into 3 groups: group Ⅰ was the control, rats in group Ⅱ were perfused with empty MSCV and those in group Ⅲ with MSCV-hIL-10. Then the rat survival and liver function were observed. Results Human IL-10 gene has been inserted into MSCV vectors and the titration was up to 2×107 CFU/ml in condensed virus solution. HIL-10 secreting concentration was 1 220 ng/106 cell/24 h. The rat survival was significantly longer in group Ⅲ than in other 2 groups (P<0.01). There was no marked difference in liver function among the 3 groups. Conclusions The retrovirus recombinant vectors containing hIL-10 gene have been constructed and the condensed virus solution with high titration acquired. HIL-10 can prolong the survival of rats after orthotopic liver transplantation.