大鼠肝移植中脂质体介导IL-10基因转染供肝的实验研究

Liposome Mediated Allograft Transduction of IL-10 in Rat Liver Transplantation

目的 观察供肝转染 IL- 10后基因表达情况。方法 应用改良“二袖套法”行 Lewis到 BN大鼠肝移植11例,然后分为对照组3例; 空载体转染组4例,术中供肝冷保存期门静脉注射 Lipofectamine 2000 pCR3.1 空载体质粒复合物,保存45 min后行肝移植; 重组 IL- 10(rIL- 10)转染组 4 例,术中供肝冷保存期门静脉注射 Lipo fectamine 2000 pCR3.1 rIL- 10复合物,保存45 min后行肝移植。术后第 6 天处死全部大鼠,取血清检测 IL-- 10 水平,另取肝组织行免疫组化染色和RT PCR检测肝细胞 IL -10 表达水平。结果 rIL- 10 转染组血清 IL -10 水平明显升高,肝上下腔静脉 IL 10 水平可达( 639. 27±67. 11 ) pg/ml,是肝下下腔静脉 IL 10 水平的近 1. 4 倍(P=0.024); 免疫组化染色结果示肝细胞胞桨呈棕黄色或深棕色,而其他两组肝细胞着色轻微或不明显。肝组织RT -PCR显示,IL- 10 mRNA的表达在 rIL -10转染组明显处于高水平(P=0.000)。结论 脂质体介导,体外冷保存期经门静脉途径进行供肝转染 IL- 10,可以使 IL -10在肝脏获得较高水平的表达。

Objective To investigate the effect of genetic modulation of the hepatic graft with IL 10 during liver preservation in rat liver transplantation. Methods Eleven cases of orthotopic liver transplantation were performed in Lewis to BN rats according to the cuff’s technique. All rats were divided into 3 groups,which were control group( n =3), Lipo group( n =4) and Lipo rIL 10 group( n =4). Lipofectamine 2000 pCR3.1 complex and Lipofectamine 2000 pCR3.1 rIL 10 complex were respectively injected into portal vein and kept for 45 minutes to transfect grafts during cold preservation in vitro . All rats were killed on postoperative day 6. Serum samples were collected for decting IL 10 by means ELISA. Transgene expression of rIL 10 was assessed by means of RT PCR and immunohistochemistry. Results In Lipo rIL 10 group, levels of IL 10 from suprahepatic vena cava were significant higher than those from infrahepatic vena cava ( P =0.024), transgene expression of rIL 10 in Lipo rIL 10 were higher than those of control group and Lipo group assessed by means of RT PCR and immunohistochemistry. Conclusion During cold preservation in vitro through portal vein injection to donor liver, liposome mediated gene transfection can successfully achieve local gene expression.