低氧对小鼠肺动脉平滑肌细胞增殖及经典型蛋白激酶C表达的影响

Effects of hypoxia on proliferation of pulmonary artery smooth muscle cells and expression of classical protein kinase C in mice

目的原代培养小鼠远端肺动脉平滑肌细胞(PASMC),探讨低氧对PASMC增殖及经典型蛋白激酶C(cPKC)各亚型表达的影响。方法原代培养小鼠远端PASMC,实验用4~8代传代细胞。采用免疫印迹、免疫荧光技术检测PASMC中cPKC各亚型的表达情况。将PASMC分为常氧组和低氧组,分别置于普通培养箱(5%CO_(2))和低氧培养箱(3%O_(2)、5%CO_(2))中培养。常氧和低氧培养24 h、48 h、72 h后,Brdu法检测两组细胞增殖能力变化,免疫印迹技术、流式细胞术检测PASMC中cPKC各亚型蛋白的表达变化。结果免疫印迹、免疫荧光技术检测显示小鼠PASMC存在cPKCα、cPKCβⅠ和cPKCβⅡ的表达,而无cPKCγ表达。免疫荧光检测显示,与常氧组相比,低氧24 h、48 h、72 h后低氧组PASMC的Brdu阳性细胞与DAPI阳性细胞比值均显著增高(均P<0.05),其中低氧72 h时最明显。免疫印迹技术、流式细胞术检测结果显示,与常氧组比较,低氧24 h、48 h、72 h后低氧组PASMC中cPKCα、cPKCβⅠ、cPKCβⅡ蛋白表达均增高(均P<0.05)。结论低氧可能通过调控cPKCα、βⅠ和βⅡ信号通路诱导小鼠远端PASMC异常增殖。

Objective To perform a primary culture of mouse distal pulmonary artery smooth muscle cells(PASMCs),and to investigate the effect of hypoxia on PASMCs proliferation and expression of classical protein kinase C(cPKC)isoforms.Methods Primary culture of mouse distal PASMCs was performd.PASMCs of 4 to 8 passage were used in the experiment.Western blotting and immunofluorescence staining were used to examine the expression of cPKC isoforms in PASMCs.The PASMCs were then divided into normoxic group and hypoxic group,which were cultured in usual incubator(5%CO_(2))and hypoxic incubator(3%O_(2),5%CO_(2)),respectively.After 24 h,48 h,and 72 h culture in normoxia and hypoxia,Brdu method was used to examine the changes in cell proliferation in the two groups;Western blotting and flow cytometry were used to examine the changes in expression of cPKC isoform proteins in PASMCs.Results Western blotting and immunofluorescence staining showed that mouse PASMCs expressed cPKCα,cPKCβⅠand cPKCβⅡ,but not cPKCγ.Immunofluorescence staining showed that,compared with the normoxia group,the ratio of Brdu-positive cells to DAPI-positive cells in PASMCs in the hypoxic group increased significantly at 24 h,48 h,and 72 h of hypoxia(all P<0.05),and was most significant at 72 h.Western blotting and flow cytometry showed that the expression of cPKCα,cPKCβⅠ,and cPKCβⅡproteins in PASMCs of hypoxic group increased at 24 h,48 h,and 72 h of hypoxia compared with the normoxic group(all P<0.05).Conclusion Hypoxia may induce abnormal proliferation of mouse distal PASMCs by regulating cPKCα,βⅠandβⅡsignaling pathways.