重组腺病毒载体Ad-CRT/MAGE-A3抑制肺腺癌细胞A549侵袭的体外研究

Inhibition of transfected Ad-CRT/MAGE-A3 on A549 cell in vitro

目的:探讨同时携带CRT基因及MAGE-A3基因的重组腺病毒载体转染人肺腺癌细胞A549后基因的表达及其对体外增殖、侵袭、凋亡及血管形成能力的影响。方法:Ad-CRT/MAGE-A3转染A549细胞,转染48h后以Western blotting法检测CRT及MAGE-A3蛋白的表达;MTT法检测Ad-CRT/MAGE-A3对A549细胞增殖的影响;Transwell方法检测Ad-CRT/MAGE-A3对A549细胞侵袭的影响;AnnexinⅤ-FITC/PI双染法检测Ad-CRT/MAGE-A3对A549细胞凋亡的影响;小管形成实验检测Ad-CRT/MAGE-A3对血管形成的影响。结果:转染48h,Western blotting结果显示转染Ad-CRT/MAGE-A3的A549细胞能够表达CRT及MAGE-A3蛋白;MTT检测结果显示Ad-CRT/MAGE-A3能够明显抑制A549细胞的增殖;Tran-swell检测结果显示Ad-CRT/MAGE-A3能够明显抑制A549细胞的侵袭能力;AnnexinⅤ-FITC/PI双染法检测结果显示Ad-CRT/MAGE-A3能够诱导A549细胞凋亡;小管形成实验结果显示Ad-CRT/MAGE-A3能够明显抑制血管形成。结论:Ad-CRT/MAGE-A3能够明显抑制A549的增殖及侵袭能力,诱导其凋亡,并能明显抑制血管内皮细胞血管生成,其作用机制可能与CRT及MAGE-A3蛋白同时表达后相互作用有关,提示Ad-CRT/MAGE-A3可以作为潜在的肺癌基因治疗的新方法。

Objective： To study the expression of CRT and MAGE - A3 and inhibition of Ad - CRT/MAGE - A3 on the invasion, apoptosis and anti - angiogenesis of A549. Methods：Adenovirus expression vector for CRT/MAGE - A3 （Ad -CRT/MAGE -A3 ） was transfected into A549. Forty -eight hours after transfection, Western blotting assay was adopted to identify the expression of CRT and MAGE - A3 ; the effect of growth suppressing was quantified by MTT assay ; anti - invasion was observed by transwell assay ; apoptosis was assayed with Annexin V - FITC/PI doub- led stained flow cytometry; tube formation assay was used to detect the effects of Ad - CRT/MAGE - A3 on human umbilical vein endothelial cells （ HUVEC ） angiogenesis. Results ： Forty - eight hours after transfeetion, expression of CRT and MAGE -A3 were identified by Western blotting; by MTT assay,Ad-CRT/MAGE-A3 inhibited cell pro- liferation significantly; by transwell assay ,Ad- CRT/MAGE-A3 inhibited cell invasion significantly; Annexin V - FITC/PI doubled stained flow cytometry figured that Ad - CRT/MAGE - A3 induced apoptosis on A549 ; by tube for- mation assay, Ad -CRT/MAGE -A3 exhibited efficient inhibitory effects on angiogenesis of HUVEC. Conclusion：Ad - CRT/MAGE - A3 can inhibit the proliferation and invasion of A549 and induce the apoptosis of A549. It showed the anti - angiogenesis effect on HUVEC. The co - expression of CRT and MAGE - A3 may possess its certain inhibi-tory action on A549. Ad - CRT/MAGE - A3 may hold great promise as a novel gene - therapy for lung carcinoma.