热疗通过上调基因PUMA表达诱导胃癌MKN45凋亡

Hyperthermia induces apoptosis of gastric cancer MKN45 by upregulating PUMA gene expression

目的探讨热疗诱导胃癌MKN45细胞凋亡及其对胃癌细胞促凋亡蛋白PUMA表达的影响。方法体外复苏与培养人胃癌MKN45细胞。对照组常温(37℃)下培养,实验组按不同时间分组43℃水浴加热胃癌MKN45后培养24h,采用倒置显微镜和透射电镜观察热疗后胃癌细胞的形态结构变化。四甲基偶氮唑盐比色法(MMT)检测细胞增殖抑制。AO/EB荧光双染色法及Annexin-V/PI双染色法流式检测细胞凋亡。Western blot检测促凋亡蛋白PUMA蛋白表达。结果光镜观察发现热疗后MKN45细胞明显皱缩、变圆及细胞漂浮,3h多数细胞漂浮。电镜下可见热疗后MKN45细胞核仁增多,胞核及胞浆出现大小不等的空泡和凋亡小体。MTT实验提示,热疗可明显抑制MKN45细胞生长(P<0.01)。AO/EB荧光双染色显示,热疗后细胞呈淡黄或橘红色,胞核呈现致密斑状。热疗0.5h、1h、2h和3h后,AO/EB荧光双染色法及Annexin-V/PI双染色法流式检测细胞凋亡率基本一致。热疗0.5h后细胞凋亡率明显增高,1h略低于0.5h,2h达最高峰。Western blot显示,MKN45细胞各热疗时间的PUMA蛋白表达明显升高,热疗0.5h开始升高,热疗2h达最高峰,1h略低于0.5h,3h有所回落(P<0.05)。结论热疗2h诱导胃癌细胞凋亡的效果最佳,并且可通过上调基因PUMA表达诱导胃癌MKN45凋亡。

Objective To investigate the effect of hyperthermia inducing apoptosis of gastric cancer MKN45 cells and its effect on the expression of the protein PUMA in gastric carcinoma cells.Methods Human gastric cancer MKN45 cells were resuscitated and cultured in vitro.Following hyperthermia at 43 oC for 0,0.5,1,2or 3h,human gastric cancer MKN45 cells were further cultured for 24 h,Morphology of MKN45 cells was observed by inverted microscopy and transmission electron microscopy.Apoptosis was evaluated by AO/EB double fluorescent labeling and flow cytometry(Annexin vs PI).The cell proliferation was determined by MTT.PUMA protein production was evaluated by Western blot.Results Inverted and transmission electron microscopy revealed morphological shrinkage,rounded and floated cells,karyopyknosis,peripheral chromosome aggregation under the nuclear membrane and cytoplasm vacuolization in MKN45 cells.MTT assay showed inhibited growth of MKN45 cells.AO/EB examination and Flow cytometry showed that the apoptotic rate was 6.2%,31.7%,26.7%,46.1%,41.8%,and 8.8%,41.7%,33.5%,48.1%,41.5%in MKN45 cells at 0,0.5,1,2and 3hpost hyperthermia respectively.Western blot showed that hyperthermia incearsed the expression of Puma protein.Conclusion The best effect of hyperthermia is 2hours for the apoptosis of gastric cancer cells,and can induce the apoptosis of MKN45 by upregulating PUMA gene expression.