血管紧张素Ⅱ诱导HEK293细胞增殖中Ca^(2+)的变化及意义

Change and significance of Ca^(2+) concentration in proliferation of HEK293 cells induced by angiotensin Ⅱ

目的:建立血管紧张素Ⅱ(AngⅡ)诱导人胚肾细胞(HEK293)增殖模型,检测增殖效果与Ca2+变化及TR-PC6的表达,并观察氯沙坦(Losartan)的干预作用。方法:用不同浓度AngII(10-8 mol/L、10-7 mol/L、10-6 mol/L、10-5mol/L)诱导细胞,在不同时间(0 h、6 h、12 h、24 h、48 h),采用MTT法及台盼蓝法检测细胞增殖及活性,确定最适浓度及时间构建增殖模型。予Losartan干预24 h后用激光扫描共聚焦显微镜测Ca2+变化,RT-PCR、Western Blot检测TRPC6表达情况。结果:AngII10-6mol/L诱导HEK293 24h后细胞增殖及Ca2+升高明显,Losartan干预后细胞增殖及Ca2+明显下降。TRPC6表达(+/-),胞膜、胞质定位不清。结论:AngⅡ诱导HEK293细胞增殖与Ca2+升高有关,具有一定的时间和剂量依赖性。Losartan抑制细胞增殖及Ca2+表达下降可能与AT1R作用相关。HEK293细胞自身无明显表达TRPC6,可利用此表达系统进一步研究外源性TRPC6与Ca2+变化的关系。

Objective： To establish proliferation models of human embryonic kidney 293 （HEK293） cells by angiotensin Ⅱ, detect the effect of proliferation, Ca2＋ concentration change, and expression of TRPC6, and observe the intervention effect of Losartan. Methods： Different concentrations of angiotensin Ⅱ（10-8 mol/L, 10-7 mol/L, 10 -6 mol/L, and 10 -5tool/L） were used to induce HEK293 cells, MTT method and trypan blue staining were used to detect proliferation and activity of HEK293 cells at 0, 6, 12, 24, and 48 hours to confirm the optimal concentration and opportunity to construct proliferation model. After intervention with Losartan for 24 hours, eonfocal laser scanning microscope was used to detect the change of Ca2 ＋ concentration, RT - PCR and Western blot were used to detect the expression of TRPC6. Results： After inducing HEK293 ceils with 10 -6 mol/L angiotensin Ⅱ for 24 hours, cell proliferation and increase of Ca2 ＋ concentration increased obviously, after intervention with Losartan, cell proliferation and increase of Ca2 ＋ concentration decreased significantly. The expression of TRPC6 was too weak to detect in cytomembrane or cytoplasm. Conclusion ： Proliferation of HEK293 cells induced by angiotensin Ⅱ was correlated with increase of Ca2 ＋ concentration, showing a certain time - and dose - dependent manner. Inhibition of cell proliferation and decrease of Ca2＋concentration after intervention of Losartan was correlated with AT1R. TRPC6 didnt express in HEK293 cells, which can be used to further research the relationship between exogenous TRPC6 and change of Ca2 ~ concentration.