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目的探讨新型内皮细胞抑制素血管内皮细胞生长抑制因子151融合基因(hENDOVEGI151)治疗胃癌的作用机制。方法应用重复感染系数(MOI=100)的重组腺病毒AdhENDOVEGI151转染胃癌SGC7901、MKN28细胞和血管内皮ECV304细胞4h后,继续培养6d,噻唑蓝(MTT)比色法检测第1至6天3种细胞的存活率;流式细胞仪(FCM)丙化碘锭(PI)单染色法检测转染后48h胃癌和内皮细胞凋亡的情况;应用DNA片断化试验分析转染后12、24、36、48h时ECV304凋亡情况;应用逆转录聚合酶链反应(RTPCR)和蛋白印迹法(Westernblot)检测融合基因转染对SGC7901表达促血管生成因子VEGF165的影响。结果AdhENDOVEGI151治疗强烈抑制ECV304增殖,72h抑制率为55.18%,144h抑制率89.86%;FCM检测出现明显凋亡峰,凋亡细胞约占(20.70±5.83)%,并且出现G1期阻滞(65.41±2.38)%和S期明显减少(21.81±1.52)%,与AdLacZ组和对照组比较差异有统计学意义(P<0.01);转染组细胞DNA出现典型的梯形条带,尤以转染后24～36h最为明显,AdLacZ组及对照组DNA无裂解。AdhENDOVEGI151转染对胃癌细胞无直接毒性作用,但明显下调胃癌细胞VEGF165的表达水平。结论AdhENDOVEGI151治疗一方面强烈抑制内皮细胞增殖,诱导凋亡;另一方面抑制胃癌细胞表达VEGF165,多角度联合抑制肿瘤新生血管形成,使肿瘤细胞因缺血而发生大量凋亡。
Objective To investigate the mechanism of the novel endothelial cell inhibitor vascular endothelial growth inhibitor 151 fusion gene (hENDOVEGI151) in the treatment of gastric cancer. METHODS: The recombinant adenovirus AdhENDOVEGI151 with repeated infection coefficient (MOI=100) was transfected into gastric cancer SGC7901, MKN28 cells and vascular endothelial ECV304 cells for 4 h, and cultured for 6 days. MTT colorimetry was used to detect the first 3 days from 1st to 6th days. Cell viability; Flow cytometry (FCM) iodide iodide (PI) single staining method was used to detect the apoptosis of gastric cancer and endothelial cells 48 h after transfection; DNA fragmentation assay was used to analyze 12, 24, and 36 after transfection. At 48h, the apoptosis of ECV304 was detected. The effect of transfection of fusion gene on expression of angiogenic factor VEGF165 in SGC7901 was detected by reverse transcription polymerase chain reaction (RTPCR) and Western blot. Results AdhENDOVEGI151 strongly inhibited the proliferation of ECV304, the inhibition rate was 55.18% at 72h and the inhibition rate was 89.86% at 144h. Apoptotic peak was detected by FCM, apoptotic cells accounted for approximately (20.70±5.83)%, and G1 arrest occurred (65.41±). 2.38)% and S phase decreased significantly (21.81±1.52)%, and there was significant difference between AdLacZ group and control group (P<0.01). Typical DNA ladders appeared in transfected cells, especially after transfection. From 24 to 36h, the DNA was not lysed in the AdLacZ group and the control group. AdhENDOVEGI151 transfection had no direct toxic effect on gastric cancer cells, but significantly down-regulated the expression of VEGF165 in gastric cancer cells. Conclusion On the one hand, AdhENDOVEGI151 treatment strongly inhibits the proliferation of endothelial cells and induces apoptosis. On the other hand, it inhibits the expression of VEGF165 in gastric cancer cells and inhibits the formation of tumor neovascularization through multi-angles, resulting in massive apoptosis of tumor cells due to ischemia.