二苯乙烯苷抑制肺动脉平滑肌细胞增殖的机制研究

2,3,5,4-Tetrahydroxyl Diphenylethylene-2-o-glucoside Blocked the Proliferation of Pulmonary Artery Smooth Muscle Cells Induced by Platelet-Derived Growth Factor-BB

目的 建立PDGF-BB诱导的原代大鼠肺动脉平滑肌细胞（pulmonary vascular smooth muscle cells,PASMCs）增殖的细胞模型,并探讨二苯乙烯苷（2,3,5,4-tetrahydroxyl diphenylethylene-2-o-glucoside,TSG）对PASMCs增殖的影响及其机制,为肺血管重构防治寻找新药物.方法 采用酶消化法分离培养大鼠原代PASMCs,通过20ng/ml PDGF-BB诱导PASMCs增殖建立细胞模型,采用1 ～ 100μmol/L TSG干预PDGF-BB诱导的PASMCs增殖,通过CCK-8检测PASMCs增殖及TSG的细胞毒作用,BRDU检测DNA的合成,流式细胞仪分析细胞周期,实时定量PCR（RT-PCR）检测CyclinD1、CyclinE、CDK2/4/6 mRNA的表达,Western blot法检测总的和磷酸化AKT/GSK3β的表达.结果 CCK-8检测结果表明TSG抑制PDGF-BB诱导的PASMCs增殖及DNA合成具有浓度依赖性,并且实验浓度的TSG对PASMCs无明显毒性不良反应;流式细胞仪分析结果表明TSG能够阻滞细胞周期于G0/G1～S期,RT-PCR结果表明TSG能够抑制CyclinD1、CyclinE、CDK2/4/6 mRNA的表达;Westernblot法检测结果表明TSG能够抑制AKT/GSK3β活化.结论 TSG通过阻滞细胞周期于G0/G1～S抑制PDGF-BB诱导的PASMCs增殖.TSG抑制PASMCs增殖与抑制AKT/GSK3β信号通路的活化有关.

Objective To establish the platelet - derived growth factor - BB （PDGF - BB） - induced model of cell proliferation in rat pulmonary artery smooth muscle cells（PASMCs） , and investigate the effects of ：2,3,5,4 - tetrahydroxyl diphenylethylene - 2 - o - glu- coside（TSG）on the proliferation of PASMCs induced by PDGF- BB, as to search for new drugs for the treatment of pulmonary vascular remodeling. Methods Enzymatic digestion was used to separate the PASMCs and use PDGF - BB to induced the proliferation of PASMCs, and the effect of TSG on the proliferation of PASMCs was observed, After PDGF - BB treatment for 24 h, the proliferation was deteetedby CCK - 8, cell cycleby, flow cytometry at the mRNA expression of Cyelin D1, CyclinE and CDK2/4/6 by RFPCR. After PDGF -BB treatment for 15 min, the total and phosphorylated forms AKT, proto - synthase kinase 313 （GSK313） was observed by Western Blot. Results CCK - 8 test results showed that TSG blocked PDGF - BB - induced proliferation of PASMCs ; flow cytometry analysis indicated that TSG arrested the cell cycle in G0/G1 to S phase. RT - PCR results demonstrated that TSG inhibited the mRNA expression of CyclinD1 , CyclinE and CDK2/4/6. Western Blot indicated that TSG suppressed the activation of AKT/GSK313 signaling pathway. Conclusions TSG blocked the proliferation of PASMCs induced by PDGF - BB through arrestting the cell cycle in G0/G1 phase. Further study indicates that TSG suppress PDGF - BB - induced PASMCs proliferation is associated with an inhibition of AKT/GSK313 signal path- way.