法舒地尔对尿道瘢痕成纤维细胞细胞外基质合成的作用及机制

发布时间：2018-04-11 07:11

本文选题：法舒地尔 + Rho激酶抑制剂　；参考：《福建医科大学》2014年硕士论文

【摘要】：目的本研究通过Rho激酶抑制剂—法舒地尔对TGF-β1体外刺激下尿道成纤维细胞的增殖、细胞外基质合成以及参与尿道疤痕收缩的细胞骨架运动相关蛋白，如α-肌动蛋白(α-SMA)、磷酸化肌球蛋白轻链（myosin light chain phosphorylation，p-MLC）、 LIM激酶1(LIM kinase， LIMK-1)、磷酸化丝切蛋白(Cofilinphosphorylation，p-Cofilin)，以及I和III胶原蛋白的影响,进一步揭示尿道瘢痕发病机理，为各种纤维化疾病的发病机制提供新的理论和方法。方法取原代人尿道瘢痕成纤维细胞进行培养。实验分成8个组，培养液中分别加入TGF-β1(10ng/ml)，法舒地尔（12.5μmol/L，25μmol/L，50μmol/L），TGF-β1（10ng/ml）+法舒地尔（12.5μmol/L，25μmol/L，50μmol/L）以及正常对照组。用MTT比色法检测细胞活力；用流式细胞仪检测细胞的凋亡率；用Western-blot检测各组α-SMA、p-MLC、LIMK-1、p-Cofilin，以及I和III胶原蛋白的表达。结果 TGF-β1能显著增加尿道成纤维细胞活力，能诱导α-SMA、p-MLC、LIMK-1、p-Cofilin，以及I和III胶原蛋白的表达，，且随TGF-β1浓度的升高其诱导作用呈逐渐增强趋势。随着法舒地尔干预浓度增加，尿道成纤维细胞活力减弱；α-SMA、p-MLC、LIMK-1、p-Cofilin，以及I和III胶原蛋白的表达下降；流式细胞仪检测显示在无TGF-β1作用下，较高浓度法舒地尔组早期凋亡率高于对照组(P0.05)；而加入TGF-β1作用下，法舒地尔对尿道成纤维细胞早期凋亡有明显的浓度依赖性。结论法舒地尔可能通过Rho/Rock信号通路抑制TGF-β1诱导的人尿道成纤维细胞的增殖、转化和分泌细胞外基质（ECM）等生物学行为，并诱导其早期凋亡。使细胞骨架运动相关蛋白α-SMA、p-MLC、LIMK-1、p-Cofilin表达下降，且成剂量依赖性。
[Abstract]:objective
Through the study of Rho kinase inhibitor fasudil on TGF- - beta 1 in vitro stimulation urethral fibroblasts proliferation, cytoskeleton associated protein synthesis of extracellular matrix and participate in urethral scar contraction, such as alpha actin (alpha -SMA), phosphorylation of myosin light chain (myosin light chain phosphorylation, p-MLC), LIM kinase 1 (LIM, kinase, LIMK-1), phosphorylated cofilin (Cofilinphosphorylation, p-Cofilin), and the effects of I and III collagen, further reveal the pathogenesis of urethral scar, provide a new theory and method for the pathogenesis of various fibrotic diseases.
Method
The primary human keloid fibroblasts were cultured. The experiment was divided into 8 groups, were cultured with TGF- beta 1 (10ng/ml), fasudil (12.5 mol/L, 25 mol/L, 50 mol/L), TGF- beta 1 (10ng/ml) + fasudil (12.5 mol/L, 25 mol/L. 50 mol/L) and normal control group. MTT assay was used to detect cell viability; apoptosis by flow cytometry; were measured with Western-blot p-MLC, alpha -SMA, LIMK-1, p-Cofilin, and the expression of I and III collagen.
Result
TGF- beta 1 can significantly increase urethral fibroblasts induced by alpha activity, -SMA, p-MLC, LIMK-1, p-Cofilin, and the expression of I and collagen III, and with the increase in the concentration of TGF- beta 1 induced effect has been gradually increased. With the increase of fasudil concentration, urethral fibroblast activity; alpha -SMA. P-MLC, LIMK-1, p-Cofilin, I and III decreased and the expression of collagen; flow cytometry showed that in the absence of TGF- beta 1 under high concentration of fasudil group early apoptosis rate was higher than the control group (P0.05); and TGF- beta 1 under the effect of fasudil fiber early apoptosis was concentration dependent the urethra.
conclusion
Fasudil may through Rho/Rock signaling pathway inhibition of TGF- beta 1 induced human urethral fibroblasts proliferation, transformation and secretion of extracellular matrix (ECM) and the biological behavior, and induce early apoptosis. The cytoskeleton associated protein alpha -SMA, p-MLC, LIMK-1, p-Cofilin expression decreased in dose-dependent manner.

【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R699.6

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