蛋白激酶C对低氧诱导小鼠肺动脉平滑肌细胞增殖及蛋白表达的影响  

Effects of typical PKC subtypes on the proliferation of mouse pulmonary artery smooth muscle cells and the expression of ERK1/2 and Akt induced by hypoxia

作　　者：施熠炜[1] 秦小江[2] 曾超[3] 张新日[1] Shi Yiwei;Qin Xiaojiang;Zeng Chao;Zhang Xinri(Department of Respiratory and Critical Care Medicine,Shanxi Medical University Affiliated First Hospital,Taiyuan 030012,China;School of Public Health,Shanxi Medical University,Taiyuan 030001,China;Department of Pediatrics,Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西医科大学第一医院呼吸与危重症监护室,太原030012 [2]山西医科大学公共卫生学院,太原030001 [3]山西医科大学儿科医学系,太原030001

出　　处：《中华结核和呼吸杂志》2022年第5期460-467,共8页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：国家自然科学基金(81803282);山西省高等学校创新基金项目(2020L0193);山西医科大学博士启动基金(03201520);山西省归国留学基金(2020-087,2020-075,2020-167)。

摘　　要：目的探讨经典型蛋白激酶C(PKC)对低氧诱导的小鼠远端肺动脉平滑肌细胞(PASMC)增殖及细胞外信号调节激酶1/2(ERK1/2)、丝氨酸苏氨酸蛋白激酶(Akt)表达的影响。方法利用Dynal MPC-1磁分离器分离含铁粉的远端肺小动脉,原代培养并鉴定肺动脉平滑肌细胞。应用PKC激动剂(PMA)、PKCα抑制剂(safingol)、PKCβⅠ抑制剂(Go6976)、PKCβⅡ抑制剂(LY333531)分别干预细胞。在常氧和低氧条件下,采用免疫印迹法观察cPKC亚型蛋白表达变化以及ERK1/2、Akt磷酸化水平变化。应用PKCα、PKCβ的慢病毒载体,通过病毒转染技术特异性敲减目标基因活性,采用免疫印迹法观察低氧诱导小鼠PASMC中cPKC亚型蛋白表达变化,以及ERK1/2、Akt磷酸化水平变化。结果采用Brdu法,检测到用safingol、Go6976、LY333531分别抑制cPKCα、βⅠ和βⅡ时,低氧诱导的PASMC增殖能力受到明显抑制,与低氧对照组相比,Brdu阳性细胞率分别为(7.35±0.26)%vs(11.28±0.43)%,(3.76±0.25)%vs(7.98±0.28)%和(4.12±0.46)%vs(7.78±0.53)%;且PMA在常氧条件下可显著促进PASMC的增殖能力,与常氧对照组相比,Brdu阳性细胞率分别为(9.65±0.47)%vs(6.34±0.52)%,(9.34±0.38)%vs(5.42±0.21)%和(7.78±0.53)%vs(4.12±0.46)%;此外经过PKCα、PKCβ的慢病毒载体转染细胞后,也发现低氧转染组的PASMC增殖能力较空载对照组显著下降,Brdu阳性细胞率分别为(3.58±0.54)%vs(5.97±0.63)%。用safingol、Go6976、LY333531分别抑制cPKCα、βⅠ和βⅡ表达后,低氧诱导的PASMC中ERK1/2、Akt的磷酸化水平较对照组显著降低,使用safingol之后,ERK1/2、Akt的磷酸化水平分别为:0.56±0.07 vs 1.08±0.13和0.49±0.04 vs 0.97±0.08,使用Go6976之后,ERK1/2、Akt的磷酸化水平分别为:0.41±0.09 vs 0.79±0.10和0.48±0.09 vs 0.82±0.16,使用LY333531之后,ERK1/2、Akt的磷酸化水平分别为:0.42±0.03 vs 0.87±0.06和0.34±0.07 vs 0.78±0.05;PMA可使常氧条件下PASMC中ERK1/2、Akt的磷酸化水平增高,分�Objective To study the effects of specific isoforms of classic protein kinase C(cPKCs)on hypoxia-induced proliferation and the expression of ERK1/2 and Akt using drug intervention or virus transfection in vitro.Methods Dynal MPC-1 magnetic particle concentrator was used to separate iron-containing pulmonary arterioles fragments,and the pulmonary artery smooth muscle cells(PASMCs)were primary cultured and identified.The cells were intervened by PKC agonist(PMA),PKCαinhibitor(safingol),PKCβⅠinhibitor(Go6976)and PKCβⅡinhibitor(LY333531)respectively,and the changes in protein expressions of cPKCs,and the phosphorylation levels of ERK1/2 and Akt were observed by immunoblotting under the condition of normal oxygen or hypoxia.The lentiviral vectors of PKCαand PKCβwere used to specifically knock-down the activity of target genes by virus transfection techniques,and Western blotting was used to observe the protein expressions of cPKCs,and the phosphorylation levels of ERK1/2 and Akt in hypoxia-induced PASMCs in mice.Results With Brdu method,the proliferation of PASMCs induced by hypoxia was significantly inhibited by safingol,Go6976 and LY333531 by inhibiting cPKCα,βⅠandβⅡrespectively.Compared with the hypoxic control group,the rates of Brdu positive cells were(7.35±0.26)%vs(11.28±0.43)%,(3.76±0.25)%vs(7.98±0.28)%and(4.12±0.46)%vs(7.78±0.53)%.We also observed that PMA could significantly promote the proliferation of PASMCs under normoxic condition.Compared with the normoxia control group,the Brdu-positive cell rates were(9.65±0.47)%vs(6.34±0.52)%,(9.34±0.38)%vs(5.42±0.21)%and(7.78±0.53)%vs(4.12±0.46)%.In addition,after transfection with PKCαor PKCβlentiviral vector,the proliferation of PASMCs was significantly lower in hypoxia transfection group than in the control group.The rates of Brdu positive cells were(3.58±0.54)%vs(5.97±0.63)%,respectively.Using Western blotting,we also observed that after being inhibited by safingol,Go6976 and LY333531 respectively,the phosphorylation levels of ERK1

关 键 词：高血压 肺性 增殖 低氧 蛋白激酶C 

分 类 号：R544.1[医药卫生—心血管疾病]