重组腺相关病毒介导的RNA干扰抑制血管生长素表达对肺腺癌细胞成瘤能力的影响  

Recombinant adeno-associated virus mediated RNA interference of angiogenin expression inhibits cell growth of human lung adenocarcinoma

作　　者：李白翎[1] 张冠鑫[1] 侯霄雷[2] 谈梦伟[1] 袁扬[1] 刘晓红[1] 龚德军[1] 黄盛东[1] 

机构地区：[1]第二军医大学附属长海医院胸心外科,上海200433 [2]上海中医药大学附属岳阳中西医结合医院重症监护室

出　　处：《中华结核和呼吸杂志》2009年第3期188-192,共5页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：国家自然科学基金资助项目（30471718）;上海市科委重点项目基金资助项目（04JC14006）

摘　　要：目的通过腺相关病毒（AAV）介导的RNA干扰（RNAi）抑制肺腺癌细胞A549血管生长素（ANG）表达，观察其对癌细胞生长和成瘤能力的影响。方法构建H1启动子驱动的表达针对ANG的小干扰RNA（siRNA）重组腺相关病毒（AAV-shANG），转染A549细胞，同时以正常A549细胞以及转染AAV—Null的A549细胞作为对照，观察重组腺相关病毒介导的RNAi抑制ANG表达对A549细胞生长、致瘤、肿瘤细胞增殖、凋亡及肿瘤微血管密度的影响。用t检验分析各组间差别，所有数据均用x^-±s表示。结果体外实验结果表明重组腺相关病毒AAV—shANG成功构建，重组腺相关病毒转染A549细胞72h后ANG蛋白表达水平明显低于A549细胞组及AAV—Null组；转基因A549细胞细胞周期分析结果提示，正常A549细胞、AAV—Null转染细胞和AAV—shANG转染细胞的增殖指数（P1）分别为0．32±0．29、0．35±0．38和0．31±0．43，差异不明显。体内实验结果表明，AAV—shANG转染细胞组胸腺缺陷小鼠的成瘤体积、瘤质量均明显低于两对照组。各组微血管密度分别为9．4±1．5、9．8±2．1和5．7±1．9，提示AAV—shANG转染细胞组与正常A549细胞和AAV—Null转染细胞组有明显差异。各组凋亡细胞的百分率分别为（7．7±3．1）％、（8．5±5．4）％和（17．1±8．6）％。AAV—shANG转染细胞组凋亡细胞的百分率明显高于正常A549细胞组和AAV—Null转染细胞组，各组细胞增殖核抗原（PCNA）阳性表达率分别为（84.8±9．7）％、（85．8±9．8）％、（70．4±10．1）％，AAV—shANG转染细胞组PCNA表达率低于两对照组。结论AVV—siRNA表达能显著抑制肿瘤细胞ANG表达及肿瘤细胞增殖，促进肿瘤细胞凋亡，抑制肿瘤生长。Objective To study the inhibition of angiogenin （ANG） expression in human lung squamous cancer cell strain-AS49 through adeno-associated virus （AAV） -mediated RNA-interference, and therefore to observe its effect on the growth of cancer cells and tumor formation. Methods Recombinant AAV expressing H1-promoter-induced small-interference- RNA（siRNA） targeting ANG （AAV-shANG） was constructed, and then transfected into A549 cells. A549 cells and cells transfected with AAV-Null were used as the control groups. The effects of the reduced expression of ANG by RNAi from AAV-shANG on the growth, formation, reproduction, apoptosis, and mierovessel-density of the carcinoma were observed. Results In vitro experiment showed that AAV-shANG was constructed successfully, There was an significant decrease in the expression of ANG protein 72 h after transfection, compared with the normal A459 cells and AAV-Null cells （P 〈 0. 01 ）. Cell cycle analysis showed that the proliferation index（PI） of normal A549 cells, AAV-Null cells and AAVshANG cells were 0. 32 ± 0. 29,0. 35± 0. 38 and 0. 31 ± 0. 43, respectively. There was no statistic difference in the PIs among the 3 groups （ P 〉 0. 05 ）. In vivo experiment using thymus-defect mice showed that, there was an remarkable reduction in the mass and volume of tumors in AAV-shANG transfected group, compared to the control groups. Microvessel-density was 9.4 ± 1.5, 9. 8 ± 2. 1 and 5.7 ± 1.9, respectively in the 3 groups, a statistic difference among the AAV-shANG-transfected group, the normal A549 group and the AAV-Null transfected group. The percentages of apoptotic cells in each group were （7. 7 ± 3.1 ）% , （8. 5 ± 5.4 ）% , （17. 1 ± 8.6 ）% , respectively, the experimental group being higher than those of the control groups. Positive rates of PCNA were （ 84. 8 ± 9. 7 ） % , （ 85.8 ± 9. 8）% ,and （70.4 ± 10. 1）%, respectively, the AAV-shANG transfected cancer cells showing a lower PCNA index than the control groups. Conclusion

关 键 词：腺病毒科 腺癌 血管生长素 

分 类 号：R686[医药卫生—骨科学]