氯化钴作用下大鼠肝癌细胞株RH35的HIF-1α与VEGF表达及侵袭性影响的研究

Effect of CoCl_2 on expression of HIF-1α and VEGF,and tumor invasion in rat RH35 hepatoma cells

目的研究不同浓度的氯化钴(CoCl2)对大鼠肝癌细胞株(RH35)的缺氧诱导因子-1α(HIF-1α)及血管内皮生长因子(VEGF)表达的影响,及对RH35增殖及侵袭能力影响。方法利用不同浓度的(0、50、100、150、200μmol/L)CoCl2建立RH35缺氧模型。通过Cell Counting Kit-8(CCK-8)检测CoCl2对RH35的体外生长增殖活性的影响。定量PCR(Q-PCR)、蛋白免疫印迹法及ELISA检测RH35中HIF-1α及VEGF的mRNA及蛋白表达情况。体外侵袭实验(Transwell)检测细胞的体外侵袭迁移力。结果 CCK-8显示在CoCl2浓度为50、100、150μmol/L时,RH35细胞体外生长增殖活性与0μmol/L无差异(P>0.05),在200μmol/L时,RH35增殖活性降为(62.90±1.57)%,与0μmol/L时比较差异有统计学意义(P﹤0.01)。Q-PCR显示RH35细胞HIF-1α及VEGF的mRNA表达随CoCl2浓度升高而增加,并且HIF-1α与VEGF呈显著正相关(r=0.980,P=0.020)。蛋白免疫印迹法及ELISA显示HIF-1α及VEGF的蛋白表达均随CoCl2浓度升高而增加。Transwell显示RH35随CoCl2浓度增高,侵袭能力增加。结论 CoCl2化学缺氧模型可较好的模拟缺氧引起的肝癌细胞HIF-1α上调,使VEGF表达增加,并且两者具有相关性;通过上调HIF-1α可增加肝癌细胞的侵袭能力,为下一步实验奠定基础。

Objective To investigate the effect of different concentration of Cobalt chloride （CoCl2 ） on the expression levels of hypoxia inducible factor-1α（HIF-1α） and vascular endothelial growth factor （VEGF）in rat RH35 hepatoma cell line,and the influence of HIF-1αon the tumor invasion. Methods Hy-poxic cell models were established by CoCl2 with different concentration （0,50,100,150 and 200 μmol/L）. Cell counting kit-8 （CCK-8）was used to detect the proliferation of RH35. Quantitative polymerase chain reac-tion （Q-PCR）was employed to detect the expression levels of HIF-1αand VEGF mRNA. Western blot was adopted to detect the levels of HIF-1αprotein and enzyme-linked immunosorbent assay （ELISA）was conduc-ted to detect the level of VEGF protein. Invasion assay in vitro （Transwell）was applied to evaluate the inva-sion and migration of RH35. Results In the hypoxic microenvironment （50,100 and 150μmol/L of CoCl2 ）, the proliferation of RH35 did not significantly differ from that in the control group （0 μmol/L of CoCl2 ）. At 200 μmol/L of CoCl2,the proliferation of RH35 decreased by （62. 90 ±1. 57）%. Q-PCR revealed that the expression of HIF-1αand VEGF increased along with elevated concentration of CoCl2 and HIF-1αwas positive-ly correlated with VEGF （r=0. 980,P=0. 020）. Western blot and ELISA demonstrated that the expression levels of HIF-1αand VEGF proteins were increased along with the rising concentration of CoCl2. Transwell method showed that the invasion and migration of RH35 were enhanced over the rising concentration of CoCl2. Conclusions CoCl2-induced RH35 cell model could simulate the up-regulated expression of HIF-1αby hy-poxia in hepatocellular carcinoma and then enhance the expression of VEGF. And it also could improve the in-vasion and migration of RH35 by up-regulating the expression of HIF-1α,offering the basis for subsequent in-vestigations related to HIF-1αin hepatocellular carcinoma.