雷帕霉素通过mTORC1/p70S6K通路调控大鼠系膜细胞增殖及细胞周期的机制研究  

Mechanisms of rapamycin on regulating rat mesangial cell proliferation and cell cycle through mTORC1/p70S6K pathways

作　　者：常思佳 王艳红[1] 郭海秀[1] 周晓霜 冀贺 田继华[1] CHANG Si-jia;WANG Yan-hong;GUO Hai-xiu;ZHOU Xiao-shuang;JI He;TIAN Ji-hua(Shanxi Medical University,Taiyuan 030001,China;Department of Nephrology,the Affiliated People's Hospital of Shanxi Medical University,Taiyuan 030012,China)

机构地区：[1]山西医科大学,山西太原030001 [2]山西医科大学附属人民医院肾内科,山西太原030012

出　　处：《药学学报》2020年第2期235-240,共6页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金青年科学基金项目(81500529,81500518);山西医科大学博士启动基金(03201403)

摘　　要：为了探究雷帕霉素对大鼠系膜细胞增殖、细胞周期的影响及其作用机制,将大鼠系膜细胞(HBZY-1)分为以下6组:对照组(control)、血小板衍生生长因子(platelet derived growth factor,PDGF)20 ng·mL^-1组、PDGF+雷帕霉素1、10、100和1000 nmol·L^-1组;MTT法和流式细胞术检测雷帕霉素(1、10、100、1000 nmol·L^-1)作用后对细胞增殖和周期的影响;Western blot法检测周期蛋白D1(cyclin D1)、周期蛋白E(cyclin E)、周期素依赖性蛋白激酶2(cyclin-dependent kinase 2,CDK2)、周期素依赖性蛋白激酶4(cyclin-dependent kinase 4,CDK4)、p70S6K/p-p70S6K和p27的蛋白表达;Real-time-PCR法检测p27 mRNA。结果显示,雷帕霉素能显著抑制PDGF刺激系膜细胞(glomerular mesangial cells,MCs)引起的增殖,且呈剂量及时间依赖性,但随着浓度增加(1~1000 nmol·L^-1),时间依赖性逐渐减弱;雷帕霉素可以将细胞周期阻滞在G0/G1期;对照组cyclin D1、cyclin E及CDK2、CDK4表达较低,PDGF组各蛋白表达均上调,与对照组比较差异有统计学意义(P<0.05),但雷帕霉素干预组与PDGF组比较,cyclin D1、cyclin E及CDK2、CDK4的表达均未见明显改变;雷帕霉素可以明显抑制p70S6K磷酸化,上调p27蛋白及mRNA的表达。上述结果表明,雷帕霉素具有抑制系膜细胞增殖的作用,该作用是通过mTORC1/p70S6K通路调控p27mRNA转录,使其蛋白表达增加,导致cyclin-CDK的活性降低,将细胞周期阻断在G0/G1期实现的。To explore the affect and mechanisms of rapamycin on mesangial cell proliferation and cell cycle,rat mesangial cells(HBZY-1)were cultured and divided into the six groups:normal;normal with platelet derived growth factor(PDGF)20 ng·mL^-1;PDGF+rapamycin 1,10,100,1000 nmol·L^-1.The cell proliferation was measured by MTT in 24 and 48 h;flow cytometry was used to detect the cell cycle phase.Western blot was performed to determine cyclin D1,cyclin E,cyclin-dependent kinase 2(CDK2),cyclin-dependent kinase 4(CDK4),p27,p70 S6 K/p-p70 S6 K protein expression.The p27 mRNA was detect by Real-time PCR.The results showed that rapamycin significantly suppressed PDGF induced glomerular mesangial cells(MCs)proliferation in a dose and time-dependent manner,but with the dose increased(1 to 1000 nmol·L^-1),the time dependence gradually weakened.Rapamycin inhibited mesangial cell proliferation and arrested the cell cycle in the G0/G1 phase.PDGF at 20 ng·mL^-1 significantly increased the expression of cyclin D1,cyclin E and CDK2,CDK4(P<0.05),but rapamycin did not affect the expression of cyclin D1,cyclin E and CDK2,CDK4.Rapamycin can significantly inhibited p70 S6 K phosphorylation,up-regulated the expression of p27 protein and mRNA.Collectively,rapamycin has the effect of inhibiting the glomerular mesangial cells proliferation of mesangial cells by regulating the transcription of p27 mRNA,increasing its protein expression through the mTORC1/p70 S6 K pathway,resulting in decreased activity of cyclin-CDK,and blocking cell cycle in G0/G1 phase.

关 键 词：雷帕霉素 P27 系膜细胞 mTORC1/p70S6K 

分 类 号：R965[医药卫生—药理学]