内皮细胞抑制素-血管内皮细胞生长抑制因子151融合基因治疗胃癌的作用及其机制

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Ad hENDOVEGI151转染胃癌SGC-7901、MKN-28细胞和血管内皮ECV-304细胞4 h后,继续培养6 d,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48 h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、48 h时ECV-304凋亡情况;应用逆转录-聚合酶链反应(RT-PCR)和蛋白印迹法(Western blot)检测融合基因转染对SGC-7901表达促血管生成因子VEGF165的影响.结果AdhENDO-VEGI151治疗强烈抑制ECV-304增殖,72 h抑制率为55.18%,144 h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Ad LacZ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24～36 h最为明显,Ad LacZ组及对照组DNA无裂解.Ad hENDO-VEGI151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Ad hENDO-VEGI151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.

Inhibitory mechanisms of endostatin-vascular endothelial growth inhibitor151 chimeric gene therapy on gastric carcinoma neovascularization

Objective To study the inhibitory mechanisms of endostatin-vascular endothelial growth inhibitor_ 151 (hENDO-VEGI_ 151) chimeric gene therapy on gastric carcinoma neovascularization. Methods Recombinant adenoviruses Ad hENDO-VEGI_ 151 was used to treat human vascular endothelial cell line ECV304,human gastric carcinoma cell line SGC-7901 and MKN-28.Fusion protein expression of Ad hENDO-VEGI_ 151 in vitro was identified by Western blot.MTT assay was used to determine the SGC-7901,MKN-28 and ECV-304 cells growth rate.Apoptosis was analyzed by FCM and detection of DNA fragmentation.RT-PCR and Western blot were employed to investigate VEGF_ 165 expression in gastric cancer cells. Results Western blot showed that the molecular weight of fusion protein was about 41kD after infection of ECV-304,SGC-7901 and MKN-28 cells with supernatant of Ad hENDO-VEGI_ 151.No changes in the growth rate and DNA content of SGC-7901 were found.However,Ad hENDO-VEGI_ 151 showed a specific inhibition on the proliferation of ECV-304 cells,and the inhibition rate reached 55.18% in 72 h and 89.86% in 144 h,respectively.A sub-G_1 peak was detected in Ad hENDO-VEGI_ 151 treated ECV-304,and percentage of apoptosis cells was 1.51% of Ad LacZ treated, 1.83% of controls and 20.7% of Ad hENDO-VEGI_ 151 treated respectively,with cells growth arrest in G_1 phrase of the cell cycle.DNA of Ad hENDO-VEGI_ 151 treated ECV-304 showed a typical DNA ladder at 24-36 h after infecting.An obvious down-regulation of VEGF_ 165 expression in cancer cells was identified by RT-PCR and Western blot. Conclusion The fusion gene therapy can directly affect endothelial cell functions by inhibiting the proliferation and inducing apoptosis;On the other hand,it can decrease VEGF_ 165 production by tumor cells or endothelial cells and stromal cells.Our findings strongly suggest that the fusion combines two potent anti-angiogenic genes to increase the suppression of tumor angiogenesis.Fusion protein,operating through different mechanisms,may result in synergistic effects.