不同浓度氯化钴对原代脑微血管内皮细胞增殖与缺氧诱导因子HIF-1α表达的研究

Effects of different concentrations of cobalt chloride on proliferation of primary brain microvascular endothelial cells and expression of hypoxia-inducible factor HIF-1α

目的研究不同浓度的氯化钴(CoCl_2)对原代脑微血管内皮细胞(BMECs)细胞增殖和缺氧诱导因子-1α(HIF-1α)表达的影响。方法利用不同浓度的(0、50、100、200、400、800μmol/L)CoCl_2建立原代脑微血管内皮细胞缺氧模型。通过MTT法检测CoCl_2对原代脑微血管内皮细胞的体外生长增殖活性的影响。定量PCR(RT-PCR)、蛋白免疫印迹法检测BMECs中HIF-1α的mRNA及蛋白表达情况。结果 MTT显示CoCl_2对BMECs细胞增殖抑制作用表现出浓度依赖性抑制作用。免疫印迹法显示HIF-1α的蛋白表达水平在(50、100、400、800)μmol/L,HIF-1α在细胞中的表达与CoCl_2浓度0μmol/L没有差异改变(P> 0.05),浓度为200μmol/L时,HIF-1α蛋白表达与对照组CoCl_2浓度为0μmol/L相比有显著差异(P <0.01)。RT-PCR显示BMECs细胞HIF-1α的mRNA表达随CoCl_2浓度升高无明显变化(P>0.05)。结论 CoCl_2可抑制细胞的增殖,同时CoCl_2化学缺氧模型可较好的模拟缺氧引起的BMECs细胞HIF-1α上调;通过上调HIF-1α可增加对脑微血管内皮细胞的损害,为下一步耐缺氧脑损伤药物实验奠定基础。

Objective To investigate the proliferative effect as well as variation of hypoxia inducible factor-1α(HIF-1α)expression on primary brain microvascular endothelial cells(BMECs) under induced hypoxia by different concentration of Cobalt chloride(CoCl2). Method Hypoxic cell models were established by CoCl, with different concentration(0、50、100、200、400、800 μ mol/L).MTT assay was used to detect the proliferative of BMECs. Quantitative reverse transcription PCR(RT-PCR) was used to detect the expression levels of HIF-1α mRNA. Western blot was adopted to detect the levels of HIF-1α protein. Result The inhibition on the proliferation capabilities of BMECs cells exhibited a concentration-dependent. In the hypoxic microenvironment(0、50、100、400 and 800μ mol/L of CoCl2),the HIF-la protein of BMECs did not significantly differ from that in the control group(0μ mol/L of CoCl2)(P >0.05).At200μ mol/L of CoCl2, There were significant differences between 200μ mol/L and 0μ mol/L(P<0.01). RT-PCR showed that the expression of HIF-1α mRNA did not have significant change along with elevated concentration CoCl2(P>0.05). Conclusion CoCl2 can inhibit the proliferation of BMECs cell under induced hypoxia at different concentration, Simultaneously, CoCl2-induced BMECs cell model could simulate the up-regulated expression of HIF-1α. Upregulating HIF-1α can increase cerebral microvascular endothelial cell damage and lay a foundation for the hypoxia brain injury model in the next step.