缺氧诱导因子1α对CO2气腹环境下人胃癌细胞凋亡的影响

Effects of HIF-1α on human gastric cancer cell＇s apoptosis in 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、15mmHg。采用荧光定量RT-PCR方法和Western blot方法观察沉默HIF-1α前后MKN-45细胞HIF-1α mRNA和蛋白表达的变化；免疫细胞化学技术观察沉默HIF-1α前后细胞bcl-2／bax表达的变化；Annexin V-FITC／PI双标染色、流式细胞仪检测沉默HIF-1α前后细胞凋亡比例的变化。结果沉默HIF-1α前15mmHg组细胞HIF-1α mRNA和蛋白表达（分别为1．51±0．04、4．44±0．30）均显著高于对照组（0．58±0．06、2．01±0．06）（P〈0．01）。沉默HIF-1α前15mmHg组细胞bcl-2／bax比值（0．77±0．05）较对照组（1．43±0．02）明显降低（P〈0．05），细胞凋亡比例（11．60±2．11）较对照组（0．30±0．02）增加。而在沉默HIF-1α后15mmHg组细胞HIF-1α mRNA（0．46±0．04）和蛋白表达（0．92±0．02）、bcl-2／bax比值（1．61±0．04）、细胞凋亡比例（0．41±0．03）与对照组相比差异均无统计学意义（P〉0．05）。结论在CO2气腹环境压力为0、5、10mmHg CO2时MKN-45细胞凋亡比例与对照组比较无差异，压力为15mmHg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原因之一。

Objective To study the effect and mechanism of human gastric cancer cell＇s apoptosis in simulated CO2 pneumoperitoneum environment by HIF-1α. Methods We used a closed box to simulate CO2 pneumoperitoneum environment, a standard surgical insufflator maintained pressure of the box at 0, 5, 10 and 15 mm Hg respectively. MKN-45 cell＇s HIF-1α mRNA and protein expression were observed before and after silencing cell＇s HIF-1α by real time RT-PCR and Western blot. Cell＇s bcl-2/bax was studied by immunohistochemistry before and after silencing HIF-1α. Cell＇s apoptosis ratio was measured using Annexin V-FITC/PI double labelled staining. Results In 15 mm Hg group, MKN-45 cell＇s HIF-1α mRNA and protein expression were significantly higher than control group （ P 〈 0.01 ） , while the difference was not significant among 0, 5, 10 mm Hg group and control group（P 〉0.05）. In 15 mm Hg CO2 pressure, cell＇s bcl-2/bax was obviously lower than in control group （ P 〈0. 05 ） and apoptosis ratio was more than control group（P〈0.01）. When HIF-1α was silenced, cell＇s bcl-2/bax and apoptosis ratio weren＇t significantly different between 15 mm Hg group and control group （P 〉 0. 05 ）. Conclusions MKN-45 cell＇s apoptosis did not experience any alterations under 0, 5, 10 mm Hg. 15 mm Hg CO2 pneumoperitoneum environment enhanced cell＇s apoptosis probably by way of HIF-1α.