人白细胞介素-10和Bcl-2基因重组腺病毒经冠状动脉转染大鼠心肌的可行性  

Intracoronary adenovirus-mediated human IL-10 and Bcl-2 gene transfer to myocardium in rats

作　　者：杨昭云[1] 徐军美 姜金玉[1] 郭亮[1] 廖用琼[1] 钱其军 

机构地区：[1]中南大学湘雅二医院麻醉科,长沙市410011 [2]上海新霁生物科技有限公司

出　　处：《中华麻醉学杂志》2006年第11期1001-1004,共4页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金资助项目(30371377)

摘　　要：目的研究人白细胞介素-10(IL-10)、Bcl-2基因重组腺病毒经冠状动脉转染大鼠心肌的可行性。方法以细胞内同源重组法构建复制缺陷型重组腺病毒Ad-EGFP、Ad-hIL-10、Ad-hBcl-2,在293细胞内分别扩增,应用氯化铯密度梯度离心法纯化病毒。105只雄性SD大鼠,随机分为5组: Ad-EGFP组(Ⅰ组,n=5)、PBS对照组(Ⅱ组,n=25)、Ad-hBcl-2组(Ⅲ组,n=25)、Ad．hIL-10组(Ⅳ组,n =25)、Ad-hIL-10+Ad-hBcl-2组(Ⅴ组,n=25)。采用左心室腔注射,同时阻断主动脉、肺动脉10 s的方法来实施冠状动脉转染。Ⅰ组于转染后3 d处死大鼠,取心、肝、肺、肾、脑,作快速冰冻切片,光镜下观察绿色荧光蛋白(EGFP)表达情况。其余各组于转染后1、3、7、14、28 d各随机处死5只大鼠,用ELISA法测定静脉血浆及心肌组织IL-10、Bcl-2表达水平;取心、肝、肺、肾组织,光镜下观察炎性反应;测定血常规、肝功能指标、肾功能指标及心肌酶。结果Ad-EGFP、Ad-hIL-10、Ad-hBcl-2经氯化铯纯化滴度分别为1．99×109、2．10×1010、1．70×1010 pfu／ml。Ⅰ组于转染后3 d心肌组织可见大量EGFP表达,其它器官未检测到EGFP的表达。Ⅲ组-Ⅴ组心肌组织目的基因蛋白含量于转染后3 d达高峰,并持续至转染后14 d,Ⅲ组、Ⅴ组心肌组织Bcl-2峰含量分别为(71．6±17．9)×104、(95．5±25．7)×104 pg/g,Ⅳ组、Ⅴ组心肌组织IL-10峰含量分别为(16．8±3．7)×104、(19．3±3．3)×104 Pg／g,Ⅲ组-Ⅴ组转染后1-14 d心肌组织目的基因蛋白含量均高于Ⅱ组,Ⅴ组转染后3d心肌组织目的基因蛋白含量高于Ⅲ组、Ⅳ组(P<0．05)。Ⅲ组-Ⅴ组血浆IL-10、Bcl-2浓度与Ⅱ组比较差异无统计学意义。光镜下各组心、肝、肺、肾组织未见明显炎症反应。与Ⅱ组比较,Ⅲ组-Ⅴ组血常规、肝功能指标、肾功能指标、心肌酶差异无统计学意义。结论重组腺病毒介导人IL-10、Bcl-2基因经冠状动脉转染大鼠后,心肌能够高效地表Objective To investigate the feasibility of transferring adenovirus-mediated human IL-10 and Bcl-2 gene to myocardium through coronary artery. Methods Recombinant adenovirns Ad-EGFP, Ad-hIL-10 and Ad-hBcl-2 were produced by homologoas recombination in 293 cells, amplified also in 293 cells on a large scale and purified by uhracentrifugation in CsCl step gradient solutions, One hundred and five male SD rats weighing 250-300 g were randomly divided into 5 groups： group Ⅰ Ad-EGFP; groupⅡ PBS control; group Ⅲ Ad-hBcl-2; group Ⅳ Ad-hIL-10 and group Ⅴ Ad-hIL-10 ＋ Ad-hBcl-2. Frozen sections of the heart, liver, lung and kidney were made to examine the expression of EGFP by fluorescence microscopy on day 3 in group Ⅰ. The hIL-10 and hBcl-2 levels were determined by ELISA in plasma and myocardial homogenate in the other 4 groups on day 1, 3, 7, 14 and 28 after vector delivery. Histologic examination of the heart, lung, liver and kidney was made for inflammatory response. Blood samples were obtained for blood routine, liver, kidney and myocardial enzyme tests. Resuits （1） The concentration of recombinant adenovirus Ad-EGFP purified by ultracontrifugation in CsCl step gradient solutions was 1.99 × 10^9 pfu· ml^-1, that of Ad-hIL- 10 was 2.10 × 10^10 pfu· ml^-1 and that of Ad-hBcl-2 1.70 × 10^10 pfu· ml^-1 . （2） EGFP expression was demonstrated by fluorescence microscopy on the frozen section of myocardium on day 3 after transfection and there was no detectable EGFP in other organs. （3） ELISA analysis of myocardial homogenate showed that the expression of hIL-10 and hBcl-2 peaked on the 3rd day in the other 3 groups. The content of IL-10 was （16.8± 3.7） × 10^4 pg·mg^-1 ; that of Bcl-2 was （71.6± 17.9） × 10^4pg·mg^-1 . The expression persistod for 28 days. IL-10 and Bcl-2 gone expression was not detected in PBS control group and plasma. （4） There was no significant difference in blood routine, cardiac ereatase, renal and hepatic function between the experiment groups and Ⅱ

关 键 词：白细胞介素10 基因 BCL-2 腺病毒科 心肌 转染 冠状血管 

分 类 号：R654.2[医药卫生—外科学]