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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 a model of proliferation of human embryonic kidney (HEK293) cells induced by angiotensin Ⅱ (Ang II) and to detect the proliferation, the changes of Ca2 + and the expression of TR-PC6 and to observe the intervention of Losartan. Methods: The cells were induced with different concentrations of AngII (10-8 mol / L, 10-7 mol / L, 10-6 mol / L, 10-5 mol / L) 24 h, 48 h). MTT assay and trypan blue assay were used to detect cell proliferation and activity, and the optimal concentration and time were established to build a proliferation model. Twenty-four hours after intervention with Losartan, the changes of Ca2 + were detected by laser scanning confocal microscopy. The expression of TRPC6 was detected by RT-PCR and Western Blot. Results: The proliferation and Ca2 + of HEK293 cells induced by AngII10-6mol / L for 24 hours were obviously increased. The cell proliferation and Ca2 + decreased obviously after Losartan intervention. TRPC6 expression (+/-), membrane, cytoplasm localization unclear. CONCLUSION: The proliferation of HEK293 cells induced by AngⅡ is related to the increase of Ca2 +, which is time-and dose-dependent. Losartan inhibition of cell proliferation and decreased expression of Ca2 may be related to the role of AT1R. HEK293 cells did not express TRPC6 itself, and this expression system could be used to further study the relationship between exogenous TRPC6 and Ca2 + changes.