微环境乏氧上调胰腺癌细胞HIF-1α表达并诱导凋亡

目的 探讨CoCl2诱导缺氧对体外培养的胰腺癌PC-2细胞缺氧诱导因子-1(HIF-1α)表达变化及诱导凋亡作用.方法 以50、100、150、200μmol/L不同浓度CoCl2处理PC-2细胞模拟肿瘤体内乏氧模型,MTT法检测不同浓度、不同时间CoCl2处理后的PC-2细胞生长情况;透射电镜下观察乏氧微环境作用下的PC-2细胞超微结构的改变;免疫细胞化学和RT-PCR方法 检测乏氧微环境对PC-2细胞HIF-1α基因表达的影响;AnnexinV-FITC/PI双标记染色,流式细胞术检测肿瘤细胞凋亡情况.结果 不同浓度CoCl2对胰腺癌PC-2细胞生长曲线有不同程度的改变,对数生长期和平台期提前,对数期缩短.随着CoCl2浓度增加,曲线越趋于低平.透射电镜下可见线粒体肿胀,染色质边集等凋亡早期表现,少见凋亡小体.免疫细胞化学和RT-PCR结果 显示,化学缺氧剂COCl2模拟的乏氧微环境可引起胰腺癌PC-2细胞HIF-1α表达的上调;流式细胞仪检测结果 显示,正常对照组、100、150、200 μmol/L CoCl2不同浓度组PC-2细胞凋亡率分别为10.77%、34.32%、40.17%和52.30%.结论 CoCl2对胰腺癌PC-2细胞具有一定的凋亡诱导作用,同时也缩短了细胞正常的生长期,细胞形态发生相应改变,可能与上调HIF-1α的表达有关.

Hypoxia simulated by cobalt chloride can up-regulate expression of hypoxia inducible factor-1α and induce apoptosis in pancreatic cancer PC-2 cells

Objective To investigate the variation of hypoxia inducible factor-1α (HIF-1α) ex-pression and inducing apoptosis effect of pancreatic cancer PC-2 cells by cobalt chloride (CoCl2) simu-lated hypoxia. Methods MTT assay was used to examine the proliferation of PC-2 cells treated by dif-ferent concentrations of CoCl2 for 24-96 h. The cellular morphology of PC-2 cells was observed by light inverted microscope and transmission electron microscope (EM). The expression of HIF-1α was determined by semi-quantitive RT-PCR and immunohistochemieal SP methods. Flow cytometry was performed to analyze the apoptosis of PC-2 cells by staining with annexinV-FITC/PI. Results The in-hibition of proliferation of PC-2 cells in vitro was observed in time-dependent but no dose-dependent manner. Log phase growth and platform phase advanced. The cellular growth period became shorter and cellular morphology was changed correspondingly. Chondrosomes were swelling,chromatin accu-mulated under the nuclear membrane and apoptosis bodies were also seen by EM. CoCl2 simulated hy-poxia could effectively up-regulate the expression of HIF-1α both at the Mrna and protein levels. The expression of HIF-1α was inhibited in a dose-and time-dependent manner. The apoptotic rate of control group and 100,150,200 μmol/L CoCl2 groups were 10.77%,34.32%,40. 17% and 52.30%, respec-tively. Conclusion Hypoxia simulated by CoCl2 can effectively induce apoptosis in PC-2 cells and cellu-lar growth period becomes shorter. The cellular morphology is changed correspondingly. The mecha-nism might be related to over-expression of HIF-1α.