CAJ | 학술논문

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72 h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(PI)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、瘤质最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.
목적 통과선상관병독(AAV)개도적RNA간우(RNAi)억제폐선암세포A549혈관생장소(ANG)표체,관찰기대암세포생장화성류능력적영향.방법 구건H1계동자구동적표체침대ANG적소간우RNA(siRNA)중조선상관병독(AAV-shANG),전염A549세포,동시이정상A549세포이급전염AAV-Null적A549세포작위대조,관찰중조선상관병독개도적RNAi억제ANG표체대A549세포생장、치류、종류세포증식、조망급종류미혈관밀도적영향.용t검험분석각조간차별,소유수거균용x±s표시.결과 체외실험결과표명중조선상관병독AAV-shANG성공구건,중조선상관병독전염A549세포72 h후ANG단백표체수평명현저우A549세포조급AAV-Null조;전기인A549세포세포주기분석결과제시,정상A549세포、AAV-Null전염세포화AAV-shANG전염세포적증식지수(PI)분별위0.32±0.29、0.35±0.38화0.31±0.43,차이불명현.체내실험결과표명,AAV-shANG전염세포조흉선결함소서적성류체적、류질최균명현저우량대조조.각조미혈관밀도분별위9.4±1.5、9.8±2.1화5.7±1.9,제시AAV-shANG전염세포조여정상A549세포화AAV-Null전염세포조유명현차이.각조조망세포적백분솔분별위(7.7±3.1)%、(8.5±5.4)%화(17.1±8.6)%.AAV-shANG전염세포조조망세포적백분솔명현고우정상A549세포조화AAV-Null전염세포조,각조세포증식핵항원(PCNA)양성표체솔분별위(84.8±9.7)%、(85.8±9.8)%、(70.4±10.1)%,AAV-shANG전염세포조PCNA표체솔저우량대조조.결론 AVV-siRNA표체능현저억제종류세포ANG표체급종류세포증식,촉진종류세포조망,억제종류생장.
Objective To study the inhibition of angiogenin (ANG) expression in human lung squamous cancer cell strain-A549 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 microvessel-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 A.549 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 AAV-mediated expression of siRNA could reduce the erpression of ANG in cancer cells, significantly enough to inhibit cell proliferation, promote cell apoptosis and inhibit tumor growth.