缺氧下HIF-1α对胰腺癌细胞“干性”的影响及机制

Study on the Mechanism of Hypoxia Induced Factor-1α on Pancreatic Cancer "Stemness"

目的:通过构建人胰腺癌PANC-1、As PC细胞缺氧模型、检测缺氧对胰腺癌细胞"干性"及生物学恶性行为的影响。方法:在缺氧培养箱中构建人胰腺癌PANC-1、As PC细胞缺氧模型,通过Western Blot检测胰腺癌细胞中缺氧诱导因子(HIF-la)表达情况;通过悬浮培养成球实验检测肿瘤细胞成球生长能力,流式细胞术检测胰腺癌细胞表面CD133阳性率,PCR和Western blot实验检测常氧和缺氧培养下干性相关基因(BMI-1、OCT4A、Nanog、SOX2)在mRNA以及蛋白水平的表达差异,免疫荧光观察CD133的表达量以及其定位变化。结果:随着缺氧时间的延长,HIF-1a、CD133表达逐渐上升,在16 h时HIF-1a表达处于高峰;缺氧时间超过16 h后,随着HIF-1a表达降低,CD133表达也降低,同时缺氧胰腺癌细胞悬浮成球能力增强,CD133+细胞比例增多,干性标记物mRNA表达上调,差异具有统计学意义(P<0.05);缺氧培养下胰腺癌表面标记CD133阳性率显著上调,差异具有统计学意义(P<0.05);在缺氧条件下,下调HIF-1α后,BMI-1、OCT4A、Nanog、SOX2表达显著下调,差异具有统计学意义(P<0.05)。结论:缺氧可诱导胰腺癌细胞"干性"维持或增强,在缺氧条件下HIF-lα对胰腺癌细胞"干性"调节有着重要作用。

Objective： To investigate the effect of hypoxia on cellular function of pancreatic cancer cells by constructing human pancreatic cancer cells PANC-1,As PC cell hypoxia model,and detecting the effect of hypoxia on pancreatic cancer ＂ stemness＂ and biology malignant behavior. Methods： Constructing hypoxic model of human pancreatic cancer cells PANC-1 and As PC cell hypoxia model in hypoxia incubator,detecting the expression of HIF-1α by Western Blot. Suspension cultured spheroid experiment detected tumor cell spheroid development capability,Flow Cytometry detected pancreatic cancer cell surface CD133 positive rate; PCR and Western blot test detected expression differences of mRNA and protein level of normoxia and hypoxia cultured stemness related genes（ BMI-1,OCT4 A,Nanog,SOX2）; immunofluorescence observed CD133 expression and its location changes. Results：With the hypoxia time prolonged,expression of HIF-1α and CD133 gradually increased; at 16 h,HIF-1α expression reached peak; after being in hypoxia condition over 16 h,with the decreased HIF-1αexpression,CD133 expression decreased at the same time while hypoxia pancreatic cancer cell suspension spheroid capability enhanced,CD133＋cell proportion increased,stemness markers mRNA expression improved,differences were statistically significant（ P〈0. 05）; under hypoxia condition,down-regulated HIF-1α lead to down-regulated expression of MI-1,OCT4 A,Nanog,SOX2,differences were statistically significant（ P〈0. 05）. Conclusion： Hypoxia can maintain or enhance the stemness of pancreatic cancer cell. HIF-1α plays an essential role in the stemness maintainence or enhancement of BCSCs induced by hypoxia.