缺氧诱导因子-1α对体外模拟CO2气腹下人胃癌细胞凋亡的影响

Effects of hypoxia-inducible factor-1α on human gastric cancer cell apoptosis under in vitro CO2 pneumoperitoneum environment

目的 通过体外模拟CO2气腹环境,构建沉默缺氧诱导因子-1α（HIF-1α）的RNAi表达载体,探讨HIF-1α对CO2气腹环境下人胃癌细胞MKN-45凋亡的影响及机制.方法 利用密闭培养箱模拟CO2气腹,气腹机维持培养箱内压力分别为0、5、10、15 mm Hg（1 mm Hg=0.133 kPa）,采用RT-PCR方法和Western blot方法观察沉默HIF-1α前后MKN-45细胞HIF-1α mRNA和蛋白表达变化.免疫组化技术观察沉默HIF-1α前后细胞bcl-2/bax表达变化.Annexin V-FITC/PI舣标染色、流式细胞仪检测沉默HIF-1α前后细胞凋亡比例变化.结果 沉默HIF-1α前15 mm Hg组细胞HIF-1α mRNA和蛋白表达（1.48±0.22和1.34±0.09）以及10 nnn Hg组细胞蛋门表达（1.25±0.10）均显著高于对照组（0.55±0.17和0.83±0.04）（P＜0.05）.0、5、10 mm Hg组细胞HIF-1α mRNA和0、5 mm Hg蛋白表达与对照组比较差异无统计学意义（P＞0.05）.沉默HIF-1α前15 mm Hg组细胞bcl-2/bax比值（0.78±0.05）较对照组（1.43±0.15）明显降低（P＜0.05）,细胞凋亡比例（11.70±0.12）较对照组（0.22±0.07）显著增加（P＜0.01）.而在沉默HIF-1α后15 mm Hg组细胞HIF-1α mRNA表达（0.52±0.11）和蛋白表达（0.92±0.02）、bcl-2/bax比值（1.57±0.04）、细胞凋亡比例（0.45±0.11）与对照组比较均无显著差异（P＞0.05）.结论 在CO2气腹环境压力为0、5、10 mmHg CO2时MKN-45细胞凋亡比例与对照组比较无显著差异,压力为15 mm Hg时CO2气腹可促进胃癌细胞凋亡,HIF-1α可能为促进细胞凋亡的重要因子.

Objective To study the effect of hypoxia-inducible factor-1α（HIF-1α）on human gastric cancer cells apoptosis in simulated CO2 pneumoperitoneum environment Methods Applied closed box to simulated CO2 pneumoperitoneum environment under the pressure of 0,5.10 and 15 mm Hg （1 mm Hg=0.133 kPa）.Compared HIF-1α mRNA and protein expression of MKN-45 cells before and after silencing HIF-1α by RT-PCR and Westem blot Study the changes of bcl-2/bax expression in MKN-45 cells before and after silencing HIF-1α by immunohistochemistry.The apoptosis ratio of MKN-45 wag measured by using Annexin V-FITC/PI double labelled staining.Results In 15 mm Hg group.HIF-1α mRNA and protein expression of MKN-45 cells（1.48±0.22.1.34±0.09）and HIF-1α protein expression in 10 mm Hg group（1.25±0.10）were significantly higher than those in control group（0.55±0.17,0.83±0.04） （P＜0.05）.But there was no significant differences among 0,5,10 mm Hg group and control group in HIF-1α mRNA（P＞0.05）;and no obvious difference was found among 0,5 mm Hg group and the control group in HIF-1α protein expression（P＞0.05）.In 15 mm Hg CO2 pressure,bcl-2/bax expression（0.78±0.05）was obviously lower than that in the control group（1.43±0.15）（P＜0.05）and the apoptesis ratio（11.70±0.12）was significantly higher than the control group（0.22±0.07）（P＜0.01）before silencing HIF-1α.But once HIF-1α was silenced,HIF-1α mRNA（0.52±0.11）,HIF-1α protein expression（0.92±0.02）,bcl-2/bax ratio（1.57±0.04）and apoptosis ratio（0.45±0.11）in MKN-45 were not significantly different between 15 mm Hg group and the control group（P＞0.05）.Conclusions The apoptosis ratios of MKN-45 under 0,5,10 mm Hg CO2 pneumoperitoneum are comparable with that in the control group before the silencing of HIF-1α.The apoptosis ratio of MKN-45 is increased under 15 mm Hg CO2 pneumoperitoneum environment and HIF-1α may be one of the important factor to improve the apoptosis of human gastric cancer cell.