大鼠肺动脉平滑肌细胞Rho激酶对这p-Smad1核迁移的作用及机制
发布时间：2018-10-24 17:26
【摘要】：目的 揭示Rho激酶对p-Smad1核迁移的作用及信号转导途径,探讨它们在肺血管重构中的作用机制。 材料与方法 分离培养大鼠远端肺动脉平滑肌细胞,应用PDGF-BB启动肺动脉平滑肌细胞Rho激酶信号通路,应用BMP-2启动BMP信号通路,并用Rho激酶抑制剂Y-27632、MEK抑制剂-U0126进行干预。培养细胞分成五组：(1)对照组；(2)BMP-2组；(3)BMP-2+PDGF-BB组；(4)BMP-2+PDGF-BB+Y-27632组；(5)BMP-2+PDGF-BB+U0126组。免疫荧光染色标记p-Smad1在细胞核内外的分布并计算p-Smad1核迁移阳性细胞百分数(即核迁移率)。分离平滑肌细胞核蛋白和胞浆蛋白,western blotting分析p-Smad1在细胞内的总量和细胞核内外相对含量的变化。Cell Counting Kit-WST-8试剂盒检测细胞增殖。 结果 BMP-2组p-Smad1在细胞内的总量以及在细胞核中的相对含量和核迁移率明显高于对照组(p0.05); BMP-2+PDGF-BB组p-Smad1的核迁移率和在细胞核中的相对含量明显低于BMP-2组(p0.05),但是细胞内p-Smad1总量无明显改变(p0.05); BMP-2+PDGF-BB+ Y-27632组和BMP-2+PDGF-BB+U0126组细胞内p-Smad1的总量以及在细胞核中的相对含量和核迁移率与BMP-2组基本相似(p0.05)。BMP-2组,OD值明显小于对照组(p0.05); PDGF-BB+BMP-2组,OD值明显大于BMP-2组(p0.05)且大于对照组(p0.05); BMP-2+PDGF-BB+Y-27632组和BMP-2+PDGF-BB+U0126组OD值均小于PDGF-BB+BMP-2组(P0.05)。 结论 在大鼠肺动脉平滑肌细胞,PDGF-BB激活的Rho激酶通过MEK/ERK1/2抑制BMP-2引起的p-Smad1的核迁移,促进平滑肌细胞增殖,进而引起肺血管重构。
[Abstract]:Aim to investigate the effect of Rho kinase on nuclear migration of p-Smad1 and its signal transduction pathway, and to explore the mechanism of its role in pulmonary vascular remodeling. Materials and methods Rat pulmonary artery smooth muscle cells were isolated and cultured. PDGF-BB was used to activate the Rho kinase signaling pathway in pulmonary smooth muscle cells, and BMP-2 was used to initiate the BMP signal pathway. Rho kinase inhibitor Y-27632 was used to intervene with U0126. The cultured cells were divided into five groups: (1) control group; (2) BMP-2 group; (3) BMP-2 PDGF-BB group; (4) BMP-2 PDGF-BB Y-27632 group; (5) BMP-2 PDGF-BB U0126 group. The distribution of p-Smad1 in and out of the nucleus was labeled with immunofluorescence staining and the percentage of positive cells (i.e. nuclear mobility) of p-Smad1 nuclear migration was calculated. Nuclear protein and Cytoplasmic protein, western blotting were isolated from smooth muscle cells (SMC). The total amount of p-Smad1 in the cell and the relative content of p-Smad1 in and out of the cell nucleus were analyzed by. Cell Counting Kit-WST-8 kit to detect cell proliferation. Results the total amount of p-Smad1 in the cells, the relative content and nuclear mobility in the nucleus of BMP-2 group were significantly higher than those in the control group (p0.05), the nuclear mobility and relative content of p-Smad1 in the BMP-2 PDGF-BB group were significantly lower than those in the BMP-2 group (p0.05), but the relative content of p-Smad1 in the nucleus in the BMP-2 PDGF-BB group was significantly lower than that in the BMP-2 group (p0.05). The total amount of intracellular p-Smad1 in BMP-2 PDGF-BB Y-27632 group and BMP-2 PDGF-BB U0126 group was similar to that in BMP-2 group (p0.05). The OD value of BMP-2 group was significantly lower than that of control group (p0.05), and that of PDGF-BB BMP-2 group was significantly lower than that of PDGF-BB BMP-2 group, while that of BMP-2 PDGF-BB U0126 group was significantly lower than that of BMP-2 group (p0.05), while that of BMP-2 PDGF-BB Y-27632 group and BMP-2 PDGF-BB U0126 group was similar to BMP-2 group (p0.05). The OD value of BMP-2 PDGF-BB Y-27632 group and BMP-2 PDGF-BB U0126 group was lower than that of PDGF-BB BMP-2 group (P0.05). Conclusion in rat pulmonary artery smooth muscle cells, Rho kinase activated by PDGF-BB inhibits the nuclear migration of p-Smad1 induced by BMP-2 through MEK/ERK1/2, promotes the proliferation of smooth muscle cells and leads to pulmonary vascular remodeling.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
本文编号：2292052
[Abstract]:Aim to investigate the effect of Rho kinase on nuclear migration of p-Smad1 and its signal transduction pathway, and to explore the mechanism of its role in pulmonary vascular remodeling. Materials and methods Rat pulmonary artery smooth muscle cells were isolated and cultured. PDGF-BB was used to activate the Rho kinase signaling pathway in pulmonary smooth muscle cells, and BMP-2 was used to initiate the BMP signal pathway. Rho kinase inhibitor Y-27632 was used to intervene with U0126. The cultured cells were divided into five groups: (1) control group; (2) BMP-2 group; (3) BMP-2 PDGF-BB group; (4) BMP-2 PDGF-BB Y-27632 group; (5) BMP-2 PDGF-BB U0126 group. The distribution of p-Smad1 in and out of the nucleus was labeled with immunofluorescence staining and the percentage of positive cells (i.e. nuclear mobility) of p-Smad1 nuclear migration was calculated. Nuclear protein and Cytoplasmic protein, western blotting were isolated from smooth muscle cells (SMC). The total amount of p-Smad1 in the cell and the relative content of p-Smad1 in and out of the cell nucleus were analyzed by. Cell Counting Kit-WST-8 kit to detect cell proliferation. Results the total amount of p-Smad1 in the cells, the relative content and nuclear mobility in the nucleus of BMP-2 group were significantly higher than those in the control group (p0.05), the nuclear mobility and relative content of p-Smad1 in the BMP-2 PDGF-BB group were significantly lower than those in the BMP-2 group (p0.05), but the relative content of p-Smad1 in the nucleus in the BMP-2 PDGF-BB group was significantly lower than that in the BMP-2 group (p0.05). The total amount of intracellular p-Smad1 in BMP-2 PDGF-BB Y-27632 group and BMP-2 PDGF-BB U0126 group was similar to that in BMP-2 group (p0.05). The OD value of BMP-2 group was significantly lower than that of control group (p0.05), and that of PDGF-BB BMP-2 group was significantly lower than that of PDGF-BB BMP-2 group, while that of BMP-2 PDGF-BB U0126 group was significantly lower than that of BMP-2 group (p0.05), while that of BMP-2 PDGF-BB Y-27632 group and BMP-2 PDGF-BB U0126 group was similar to BMP-2 group (p0.05). The OD value of BMP-2 PDGF-BB Y-27632 group and BMP-2 PDGF-BB U0126 group was lower than that of PDGF-BB BMP-2 group (P0.05). Conclusion in rat pulmonary artery smooth muscle cells, Rho kinase activated by PDGF-BB inhibits the nuclear migration of p-Smad1 induced by BMP-2 through MEK/ERK1/2, promotes the proliferation of smooth muscle cells and leads to pulmonary vascular remodeling.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
【共引文献】
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