本文選題：法舒地爾 + Rho激酶抑制劑��； 參考：《福建醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的 本研究通過Rho激酶抑制劑—法舒地爾對(duì)TGF-β1體外刺激下尿道成纖維細(xì)胞的增殖、細(xì)胞外基質(zhì)合成以及參與尿道疤痕收縮的細(xì)胞骨架運(yùn)動(dòng)相關(guān)蛋白，如α-肌動(dòng)蛋白(α-SMA)、磷酸化肌球蛋白輕鏈（myosin light chain phosphorylation，p-MLC）、 LIM激酶1(LIM kinase， LIMK-1)、磷酸化絲切蛋白(Cofilinphosphorylation，p-Cofilin)，以及I和III膠原蛋白的影響,進(jìn)一步揭示尿道瘢痕發(fā)病機(jī)理，為各種纖維化疾病的發(fā)病機(jī)制提供新的理論和方法。 方法 取原代人尿道瘢痕成纖維細(xì)胞進(jìn)行培養(yǎng)。實(shí)驗(yàn)分成8個(gè)組，培養(yǎng)液中分別加入TGF-β1(10ng/ml)，法舒地爾（12.5μmol/L，25μmol/L，50μmol/L），TGF-β1（10ng/ml）+法舒地爾（12.5μmol/L，25μmol/L，50μmol/L）以及正常對(duì)照組。用MTT比色法檢測(cè)細(xì)胞活力；用流式細(xì)胞儀檢測(cè)細(xì)胞的凋亡率；用Western-blot檢測(cè)各組α-SMA、p-MLC、LIMK-1、p-Cofilin，以及I和III膠原蛋白的表達(dá)。 結(jié)果 TGF-β1能顯著增加尿道成纖維細(xì)胞活力，能誘導(dǎo)α-SMA、p-MLC、LIMK-1、p-Cofilin，以及I和III膠原蛋白的表達(dá)，，且隨TGF-β1濃度的升高其誘導(dǎo)作用呈逐漸增強(qiáng)趨勢(shì)。隨著法舒地爾干預(yù)濃度增加，尿道成纖維細(xì)胞活力減弱；α-SMA、p-MLC、LIMK-1、p-Cofilin，以及I和III膠原蛋白的表達(dá)下降；流式細(xì)胞儀檢測(cè)顯示在無TGF-β1作用下，較高濃度法舒地爾組早期凋亡率高于對(duì)照組(P0.05)；而加入TGF-β1作用下，法舒地爾對(duì)尿道成纖維細(xì)胞早期凋亡有明顯的濃度依賴性。 結(jié)論 法舒地爾可能通過Rho/Rock信號(hào)通路抑制TGF-β1誘導(dǎo)的人尿道成纖維細(xì)胞的增殖、轉(zhuǎn)化和分泌細(xì)胞外基質(zhì)（ECM）等生物學(xué)行為，并誘導(dǎo)其早期凋亡。使細(xì)胞骨架運(yùn)動(dòng)相關(guān)蛋白α-SMA、p-MLC、LIMK-1、p-Cofilin表達(dá)下降，且成劑量依賴性。
[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.

【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R699.6

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