转化生长因子β1抑制肺动脉平滑肌细胞凋亡的机制研究
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篇名: 转化生长因子β1抑制肺动脉平滑肌细胞凋亡的机制研究
TITLE:
摘要: 目的:研究转化生长因子β1(TGF-β1)抑制肺动脉平滑肌细胞(PASMCs)凋亡的可能机制。方法:以无血清培养基进行饥饿诱导(SD),MTT法检测0.1、1、5、10 ng/ml的TGF-β1对SD诱导PASMCs细胞存活率的影响。Hoechst 33342染色观察正常对照组、SD组、SD+LY294002[磷脂酰肌醇3-激酶(PI3K)抑制剂]+TGF-β1组、SD+Wortmannin(PI3K抑制剂)+TGF-β1组、SD+TGF-β1组细胞凋亡情况。分别用Western blot法考察正常对照组、SD组、TGF-β1组、SD+TGF-β1组细胞内蛋白激酶B(Akt)、磷酸化(p)-Akt蛋白的表达,正常对照组、SD组、SD+TGF-β1组、SD+LY294002组、SD+LY294002+TGF-β1组细胞环磷腺苷效应元件结合蛋白(CREB)、p-CREB蛋白表达,正常对照组、SD组、SD+TGF-β1组、SD+Wortmannin组、SD+Wortmannin+TGF-β1组细胞CREB和p-CREB蛋白表达,LY294002、Wortmannin、TGF-β1的加入浓度分别为10 μmol/L、50 nmol/L、10 ng/ml。结果:与SD比较,TGF-β1作用后细胞存活率呈剂量依赖性增加,其中5、10 ng/ml质量浓度下细胞存活率差异有统计学意义(P<0.05)。与正常对照组比较,SD组、SD+LY294002+TGF-β1组、SD+Wortmannin+TGF-β1组细胞凋亡率均增加(P<0.05);与SD组比较,SD+TGF-β1组细胞凋亡率下降(P<0.05)。与正常对照组比较,TGF-β1组细胞内p-Akt蛋白表达增强,p-Akt/Akt增加;与SD组比较,SD+TGF-β1组细胞内p-Akt蛋白表达增强,p-Akt/Akt增加。与正常对照组比较,SD组细胞内CREB蛋白表达减弱;与SD组比较,SD+TGF-β1组细胞内CREB蛋白表达增强;与SD+ TGF-β1组比较,SD+LY294002+TGF-β1组和SD+Wortmannin+TGF-β1组细胞内CREB蛋白表达均减弱,以上差异均有统计学意义(P<0.05)。结论:TGF-β1可能通过激活PI3K/Akt信号通路抑制PASMCs凋亡。
ABSTRACT: OBJECTIVE: To study the potential mechanism of TGF-β1 inhibiting the apoptosis of pulmonary artery smooth muscle cells (PASMCs). METHODS: The serum free medium was used for serum deprivation (SD); MTT assay was used to detect the effects of 0.1, 1, 5, 10 ng/ml TGF-β1 on the survival rate of PASMCs. The apoptosis of PASMCs were observed by Hoechst 33342 staining in normal control group, SD group, SD+LY294002 [PI3K inhibitor]+TGF-β1 group, SD+Wortmannin (PI3K inhibitor)+TGF-β1 group and SD+TGF-β1 group. Western blot technique was applied to investigate the protein expression of Akt and p-Akt in normal control group, SD group, TGF-β1 group and SD+TGF-β1 group; the protein expression of CREB and p-CREB were investigated in normal control group, SD group, SD+TGF-β1 group, SD+LY294002 group and SD+LY294002+TGF-β1 group; the protein expression of CREB and p-CREB were investigated in normal control group, SD group, SD+TGF-β1 group, SD+Wortmannin group and SD+Wortmannin+TGF-β1 group. The concentrations of LY294002, Wortmannin and TGF-β1 were 10     μmol/L, 50 nmol/L, 10 ng/ml, respectively. RESULTS: Compared with SD, survival rate of PASMCs increased as dosage after treated by TGF-β1, with statistical significance at 5, 10 ng/ml (P<0.05). Compared with normal control group, apoptotic rate of PASMCs increased in SD group, SD+LY294002+TGF-β1 group and SD+Wortmannin+TGF-β1 group (P<0.05); compared with SD group, the apoptotic rate of PASMCs decreased in SD+TGF-β1 group (P<0.05). Compared with normal control group, the protein expression of p-Akt and p-Akt/Akt ratio increased in TGF-β1 group; compared with SD group, the protein expression of p-Akt and p-Akt/Akt ratio increased in SD+TGF-β1 group. Compared with normal control group, the protein expression of CREB decreased in SD group; compared with SD group, the protein expression of CREB increased in SD+TGF-β1 group; compared with SD+TGF-β1 group, the protein expression of CREB decreased in SD+LY294002+TGF-β1 group and SD+Wortmannin+TGF-β1 group, with statistical significance (P<0.05). CONCLUSIONS: TGF-β1 can inhibit the apoptosis of PASMCs by activating PI3K/Akt signaling pathway.
期刊: 2016年第27卷第34期
作者: 刘云,朱金权,孙增先
AUTHORS: LIU Yun,ZHU Jinquan,SUN Zengxian
关键字: 肺动脉高压;转化生长因子β1;磷脂酰肌醇3-激酶/蛋白激酶B;凋亡
KEYWORDS: Pulmonary arterial hypertension; TGF-β1; PI3K/Akt; Apoptosis
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