CTGF-整合素β_1受体信号途径在PASMC增殖、迁移和细胞外基质沉积中的作用
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摘要
目的：1.探讨整合素β1受体在CTGF促进PASMC增殖、迁移、细胞骨架变化和细胞外基质分泌中的作用机制。2.初步探讨CTGF-整合素β1受体信号途径在肺动脉高压肺血管重构中的作用。方法：1.分离并培养SD大鼠肺动脉中膜平滑肌细胞,实验使用3～7代细胞；2.加入CTGF和不同浓度整合素β1抗体共同孵育24h、48h、72h、96h和120h,采用WST-1检测整合素β1抗体对CTGF促进PASMC增殖的影响；3.在无刺激因子、CTGF(50ng/ml)和CTGF(50ng/ml)+整合素β1抗体(15mg/L)孵育PASMC 24h,采用Transwell小室检测整合素β1抗体对CTGF促进PASMC迁移的影响；4.在无刺激因子、CTGF (50ng/ml)和CTGF (50ng/ml)+整合素β1抗体(15mg/L)孵育PASMC 12h和24h,采用考马斯亮蓝R250染色和激光共聚焦显微镜观察细胞骨架改变情况；5.在无刺激因子、CTGF (50ng/ml)和CTGF (50ng/ml)+整合素β1抗体(15mg/L)孵育PASMC 72h,采用RT-PCR检测整合素β1抗体对CTGF促进CollagenⅠ-α、CollagenⅢ-α1、Fibronectin-1 mRNA表达的影响；并采用蛋白质印迹检测整合素β1抗体对CTGF促进PASMC中Ⅲ型胶原蛋白的表达及细胞外信号调节激酶1和2 (ERK1/2)的磷酸化水平,并研究整合素β1抗体对ERK1/2信号通路的影响。结果：1.用CTGF (50ng/ml)和整合素β1抗体(0、5、10、15 mg/L)同时孵育PASMC 24h、48h、72h96h和120h, WST-1检测结果表明,整合素β1抗体对CTGF促进PASMC增殖有明显抑制作用(P<0.01),并呈现一定的浓度依赖性,其中整合素β1抗体(15mg/L)在72h抑制率最高。2. Transwell小室检测结果显示,以整合素β1抗体(15 mg/L)封闭PASMC 24h后,CTGF刺激的细胞迁移受到明显抑制(P<0.01)。3.考马斯亮蓝R250染色观察细胞骨架变化结果发现,24h时,各组之间细胞骨架变化不明显；在12h时与对照组比较,CTGF组能短暂改变细胞形态：使用整合素β1抗体后,CTGF所引起的细胞形态变化不明显。同时采用激光共聚焦扫描显微镜观察胞内FAK的变化结果发现,12h时,细胞形态及胞质内绿色荧光变化不明显。而CTGF作用PASMCs 24h可见细胞形态缩小,胞质绿色荧光(FAK)减弱,分散。但整合素β1抗体作用24h后细胞形态与正常组相近。4. RT-PCR检测结果显示,与对照组比较,CTGF显著促进Ⅰ型胶原-α1、Ⅲ型胶原-α1和纤维连接蛋白-1mRNA表达(P<0.05)；使用整合素β1抗体后与CTGF组比较三种基因mRNA表达均减少(P<0.05)。5.蛋白质印迹结果表明,与对照组比较CTGF明显促进Ⅲ型胶原蛋白表达；使用整合素β1抗体后Ⅲ型胶原蛋白表达较CTGF组减少(P<0.05),但较对照组表达增加；且整合素β1(15mg/L)抗体能明显抑制CTGF诱导大鼠PASMCs中ERK1/2磷酸化的水平,p-ERK1/2与ERK1/2比值降低(P<0.05)。结论：1.整合素β1介导了CTGF诱导的PASMC增殖、迁移和细胞骨架变化。2.整合素β1可介导外源性CTGF促进PASMC.Ⅰ型胶原-α1、Ⅲ型胶原-α1、纤维连接蛋白-1 mRNA表达,以及介导CTGF促进Ⅲ型胶原蛋白表达。4.整合素β1介导CTGF诱导的PASMC增殖和迁移可能与ERK1/2信号通路有关。4.整合素β1受体信号可能在CTGF介导的PASMC增殖、迁移、细胞外基质沉积和细胞骨架改变等生物学行为中起重要作用；CTGF-整合素受体信号途径可能参与了PAH肺血管重构。
Objective 1. To explore the mechanisms of integrinβ1 on CTGF-induced proliferation, migration, change of cytoskeleton and extracellular matrix deposition of PASMCs in vitro.2. To investigate the effects of CTGF-integrinβ1 signal pathway on pulmonary vascular remodeling in pulmonary hypertension. Methods 1. Pulmonary artery smooth muscle cells of SD Rats were cultured in vitro and 3-7 passages of cultured PASMCs were used in the experiments.2. The PASMCs were cultured with CTGF and different concentrations of anti-integrinβ1 antibody for 24h,48h,72h,96h and 120h, WST-1 assay was used to detect the effects of anti-integrinβ1 antibody on CTGF-induced proliferation.3. The cultured PASMCs were divided into control group, CTGF group and CTGF+anti-integrinβ1 antibody group.Transwell chambers were used to observe the effects of anti-integrin Pi antibody on CTGF-induced migration at 24h.4. The cytoskeletal rearrangement was observed with coomassie brilliant blue R25o staining and Confocal Lasar Scanning Microscopy(CLSM) at 12h and 24h.5.RT-PCR was used to assay the mRNA expression of CollagenⅠ-α1, CollagenⅢ-α1 and Fibronectin-1 at 72h. In addition, the expression of Collagen III protein and the phosphorylation of ERK1/2 were evaluated by Western blot analysis at 72h. Results 1. PASMCs treated with CTGF (50ng/ml)were exposed to anti-integrinβ1 antibody (0,5,10,15 mg/L) for 24h,48h,72h, 96h and 120h. WST-1 assay showed a pattent of concentration-dependent, and there was significant difference (P<0.01) when the concentration of anti-integrinβ1 antibody meet 15mg/L, and inhibition rate of PASMC proliferation was the highest at 72h.2.The migration of PASMCs treated with anti-integrinβ1 antibody (15mg/L) was inhibited by Transwell chambers assay at 24h.3. Coomassie brilliant blue R250 staining assay indicated that the cytoskeletal rearrangement of PASMCs were not changed significantly between each groups at 24h; compared with the control, CTGF could change cytoskeletal rearrangement of PASMCs briefly at 12h, while anti-integrinβ1 antibody inhibited this phenomenon; Meanwhile, Changes of intracellular FAK was observed by CLSM. The cell morphology and green fluorescence (FAK) of cytoplasmic were not channged significantly between each group at 12h. Compared with the control group, CTGF could reduce cell morphology and decrease green fluorescence of cytoplasmic of PASMCs at 24h, while anti-integrinβ1 antibody inhibited this phenomenon.4. compared with the control groups, RT-PCR assay indicated that the expression of Collagen typeⅠ-α1, Collagen typeⅢ-α1, Fibronectin-1 mRNA of PASMCs treated with CTGF (50ng/ml) were significantly increased at 72h (P<0.01), anti-integrinβ1 antibody inhibited these extracellular matrix genes mRNA expression (P<0.05).5. Compared with the control, Western blot assay indicated that CTGF(50ng/ml) promoted significantly the expression of CollagenⅢprotein, while anti-integrinβ1 antibody inhibited the expression of CollagenⅢprotein, but the CollagenⅢprotein expression were still more than control group. Meanwhile, anti-integrinβ1 antibody also obviously reduced phosphorylation of ERK1/2 induced by CTGF. Conclusions 1. Integrinβ1 mediates proliferation, migration, cytoskeletal rearrangement of PASMCs induced by CTGF.2. CTGF could promote the expression of CollagenⅠ-α1, CollagenⅢ-α1, Fibronectin-1 mRNA and protein of CollagenⅢ.3. Integrinβ1 may mediate the expression of CollagenⅠ-α1, CollagenⅢ-α1, Fibronectin-1 mRNA and CollagenⅢprotein in PASMCs induced by CTGF, which may be related to the phosphorylation of ERK1/2 signal pathway.4.The CTGF-integrinβ1 signal pathway may play a key role in proliferation, migration, cytoskeletal rearrangement and extracellular matrix deposition of PASMCs, and this signal pathway may be a new target to interven the pulmonary vascular remodeling in PAH.
Objective 1. To explore the mechanisms of integrinβ1 on CTGF-induced proliferation, migration, change of cytoskeleton and extracellular matrix deposition of PASMCs in vitro.2. To investigate the effects of CTGF-integrinβ1 signal pathway on pulmonary vascular remodeling in pulmonary hypertension. Methods 1. Pulmonary artery smooth muscle cells of SD Rats were cultured in vitro and 3-7 passages of cultured PASMCs were used in the experiments.2. The PASMCs were cultured with CTGF and different concentrations of anti-integrinβ1 antibody for 24h,48h,72h,96h and 120h, WST-1 assay was used to detect the effects of anti-integrinβ1 antibody on CTGF-induced proliferation.3. The cultured PASMCs were divided into control group, CTGF group and CTGF+anti-integrinβ1 antibody group.Transwell chambers were used to observe the effects of anti-integrin Pi antibody on CTGF-induced migration at 24h.4. The cytoskeletal rearrangement was observed with coomassie brilliant blue R25o staining and Confocal Lasar Scanning Microscopy(CLSM) at 12h and 24h.5.RT-PCR was used to assay the mRNA expression of CollagenⅠ-α1, CollagenⅢ-α1 and Fibronectin-1 at 72h. In addition, the expression of Collagen III protein and the phosphorylation of ERK1/2 were evaluated by Western blot analysis at 72h. Results 1. PASMCs treated with CTGF (50ng/ml)were exposed to anti-integrinβ1 antibody (0,5,10,15 mg/L) for 24h,48h,72h, 96h and 120h. WST-1 assay showed a pattent of concentration-dependent, and there was significant difference (P<0.01) when the concentration of anti-integrinβ1 antibody meet 15mg/L, and inhibition rate of PASMC proliferation was the highest at 72h.2.The migration of PASMCs treated with anti-integrinβ1 antibody (15mg/L) was inhibited by Transwell chambers assay at 24h.3. Coomassie brilliant blue R250 staining assay indicated that the cytoskeletal rearrangement of PASMCs were not changed significantly between each groups at 24h; compared with the control, CTGF could change cytoskeletal rearrangement of PASMCs briefly at 12h, while anti-integrinβ1 antibody inhibited this phenomenon; Meanwhile, Changes of intracellular FAK was observed by CLSM. The cell morphology and green fluorescence (FAK) of cytoplasmic were not channged significantly between each group at 12h. Compared with the control group, CTGF could reduce cell morphology and decrease green fluorescence of cytoplasmic of PASMCs at 24h, while anti-integrinβ1 antibody inhibited this phenomenon.4. compared with the control groups, RT-PCR assay indicated that the expression of Collagen typeⅠ-α1, Collagen typeⅢ-α1, Fibronectin-1 mRNA of PASMCs treated with CTGF (50ng/ml) were significantly increased at 72h (P<0.01), anti-integrinβ1 antibody inhibited these extracellular matrix genes mRNA expression (P<0.05).5. Compared with the control, Western blot assay indicated that CTGF(50ng/ml) promoted significantly the expression of CollagenⅢprotein, while anti-integrinβ1 antibody inhibited the expression of CollagenⅢprotein, but the CollagenⅢprotein expression were still more than control group. Meanwhile, anti-integrinβ1 antibody also obviously reduced phosphorylation of ERK1/2 induced by CTGF. Conclusions 1. Integrinβ1 mediates proliferation, migration, cytoskeletal rearrangement of PASMCs induced by CTGF.2. CTGF could promote the expression of CollagenⅠ-α1, CollagenⅢ-α1, Fibronectin-1 mRNA and protein of CollagenⅢ.3. Integrinβ1 may mediate the expression of CollagenⅠ-α1, CollagenⅢ-α1, Fibronectin-1 mRNA and CollagenⅢprotein in PASMCs induced by CTGF, which may be related to the phosphorylation of ERK1/2 signal pathway.4.The CTGF-integrinβ1 signal pathway may play a key role in proliferation, migration, cytoskeletal rearrangement and extracellular matrix deposition of PASMCs, and this signal pathway may be a new target to interven the pulmonary vascular remodeling in PAH.
引文
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