微囊化腺病毒介导的Bcl-2基因转染对缺血再灌注损伤肠黏膜的保护作用  

Microencapsulated adenovirus-mediated gene transfer of Bcl-2 protects rat intestinal mucosa after ischemia-reperfusion injury

作　　者：伊远学[1,2] 刘晓平[1] 李宝金[1] 张超[1] 冷希圣[1] 刘筑 罗效梅 杨瑞兵 

机构地区：[1]北京大学香港科技大学医学中心,北京大学深圳医院肝胆腔镜外科,广东深圳518036 [2]武警重庆总队医院检验科,重庆400061

出　　处：《第三军医大学学报》2010年第14期1516-1519,共4页Journal of Third Military Medical University

基　　金：广东省自然科学基金博士启动项目(06300934);广东省医学科研基金(A2007595)~~

摘　　要：目的研究微囊化重组腺病毒AdCMV/Bcl-2转染对缺血再灌注损伤肠黏膜的保护作用及其机制。方法利用气体雾化技术将携带Bcl-2基因的复制缺陷型重组腺病毒AdCMV/Bcl-2微囊化,检测其在人工胃、肠液中的融出率;将等量的重组腺病毒通过静脉注射、腹腔注射与口服微囊3种转染途径转染大鼠,应用RT-PCR和Western blot方法检测目的基因Bcl-2在小肠中的表达,并检测各组大鼠伤后血浆内毒素、二胺氧化酶(DAO)、D-乳酸含量和肠上皮细胞凋亡数量的变化。结果制备的微囊在人工胃液中仅有少量崩解,而在肠液中40min崩解融出率即达到(57.2±3.9)%,肠溶性良好;AdCMV/Bcl-2转染后48h大鼠肠黏膜Bcl-2基因的表达显著增强,其中,口服微囊组Bcl-2表达最强;AdCMV/Bcl-2转染使大鼠肠上皮细胞凋亡减少,伤后血浆D-乳酸、DAO及内毒素水平显著降低(P<0.01)。结论复制缺陷型重组腺病毒微囊化可保护其免受胃液损伤,并在肠道中被释放吸收。AdCMV/Bcl-2转染提高肠黏膜组织Bcl-2的表达在缺血再灌注损伤过程中可显著保护肠黏膜屏障功能。Objective To investigate the protective effects of microencapsulated adenovirus-mediated gene transfer of Bcl-2 on intestinal mucosa in rats after ischemia-reperfusion injury and underlying mechanism. Methods Alginate microcapsules containing the recombinant adenovirus AdCMV/Bcl-2 was prepared by using air atomization technique. Its acid resistance and dissolution in intestinal fluid were tested in artificial gastric juice and intestinal fluid. After the microencapsulated adenovirus （5×109 PFU） were injected by venous and intraperitoneal injection,and oral taking into the rats in 48 h before ligating superior mesenteric artery for 1 h followed by reperfusing for 6 h （ischemia-reperfusion injury）,the expression of Bcl-2 in intestinal mucosa was detected by RT-PCR and Western blot analysis. The protective role of Bcl-2 in the intestinal mucosa in rats was analyzed by measuring plasma levels of endotoxin,D-lactic acid,and DAO. Results The recombinant adenovirus AdCMV/Bcl-2 contained in microcapsules was still survivable in vitro and also could transfect and proliferate in 293 cells.The prepared microcapsule was almost insoluble in the simulated gastric juice but soluble in the simulated intestinal fluid in which the solution ratio arrived to 57.2% at 40 min. The AdCMV/Bcl-2 transfection in vivo successfully induced Bcl-2 over-expression in the intestinal mucosa,which protected the intestinal mucosa against ischemia and reperfusion injury. The AdCMV/Bcl-2 transfection in vivo attenuated intestinal epithelial cell apoptosis and structural mucosal damages,and decreased plasma endotoxin,DAO and D-lactic acid content following ischemia and reperfusion. Conclusion The AdCMV/Bcl-2 transfection in vivo successfully induces Bcl-2 over-expression in the intestinal mucosa,and protects the intestinal mucosa against the ischemia and reperfusion injury through attenuating intestinal epithelial cell apoptosis and decreasing plasma contents of endotoxin,D-lactic acid,and DAO. The microencapsulated adenovirus taken orally c

关 键 词：小肠 缺血再灌注损伤 基因转染 Bcl-2 微囊 腺病毒载体 

分 类 号：R364.12[医药卫生—病理学] R574[医药卫生—基础医学]