本文關(guān)鍵詞： 臍靜脈內(nèi)皮細(xì)胞 平滑肌樣細(xì)胞 激發(fā)型抗CD40單抗5C11 轉(zhuǎn)分化 TGF-β受體　出處：《蘇州大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 內(nèi)皮細(xì)胞向平滑肌樣細(xì)胞的轉(zhuǎn)分化近年來(lái)受到越來(lái)越多的重視。這種轉(zhuǎn)分化可能在肺纖維化、動(dòng)脈粥樣硬化及腎臟纖維化等疾病中起重要作用。內(nèi)皮細(xì)胞上有CD40分子的高表達(dá),激發(fā)型CD40信號(hào)可以導(dǎo)致內(nèi)皮細(xì)胞功能失調(diào)。CD40信號(hào)通路對(duì)內(nèi)皮細(xì)胞轉(zhuǎn)分化的影響未見(jiàn)相關(guān)報(bào)道。轉(zhuǎn)化生長(zhǎng)因子β(TGF-β)是目前認(rèn)為作用最強(qiáng)的促纖維化生長(zhǎng)因子,其在腎小管上皮細(xì)胞轉(zhuǎn)分化中被認(rèn)為是最重要的,但在內(nèi)皮細(xì)胞轉(zhuǎn)分化中的作用還不清楚。 目的:應(yīng)用抗CD40激發(fā)型單克隆抗體5C11作用于體外培養(yǎng)的臍靜脈內(nèi)皮細(xì)胞,通過(guò)流式細(xì)胞儀、免疫細(xì)胞化學(xué)等方法觀察內(nèi)皮細(xì)胞能否向平滑肌樣細(xì)胞轉(zhuǎn)化,同時(shí)探討轉(zhuǎn)化生長(zhǎng)因子-β(TGF-β)在轉(zhuǎn)化中的作用。 方法: 1)MTT比色法:把處于對(duì)數(shù)生長(zhǎng)期生長(zhǎng)狀態(tài)良好(細(xì)胞呈鋪路石樣)的HUVEC接種到96孔板,加入不同濃度的5C11,同時(shí)設(shè)陰性對(duì)照組。檢測(cè)5C11對(duì)HUVEC促增殖或抑制的影響。2)細(xì)胞爬片免疫化學(xué)法:把生長(zhǎng)良好的細(xì)胞接種于6孔培養(yǎng)板,每孔放一滅菌蓋玻片,培養(yǎng)后,分組加入5C11,濃度為5 ug/ml,同時(shí)設(shè)陰性對(duì)照組,繼續(xù)培養(yǎng)。分別于24h、48h、72h取出玻片,觀察內(nèi)皮細(xì)胞的轉(zhuǎn)化情況。3)流式細(xì)胞儀檢測(cè)經(jīng)5C11刺激后的內(nèi)皮細(xì)胞中TGF-β的Ⅱ型受體的表達(dá)情況。4)流式細(xì)胞儀檢測(cè)經(jīng)5C11刺激后的內(nèi)皮細(xì)胞是否發(fā)生凋亡。 結(jié)果: 1)MTT法檢測(cè)結(jié)果:應(yīng)用MTT法檢測(cè)5C11對(duì)HUVEC抑制或增殖的影響,實(shí)驗(yàn)數(shù)據(jù)應(yīng)用統(tǒng)計(jì)分析軟件分析無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。流式細(xì)胞儀檢測(cè)細(xì)胞凋亡:內(nèi)皮細(xì)胞經(jīng)過(guò)5C11刺激72H后應(yīng)用流式細(xì)胞儀未檢測(cè)到細(xì)胞凋亡的發(fā)生。2)抗CD40單克隆抗體5C11不同濃度刺激對(duì)HUVEC的影響:細(xì)胞免疫組織化學(xué)顯示,經(jīng)過(guò)5C11刺激后HUVEC正常血管內(nèi)皮細(xì)胞的標(biāo)志分子Flk1隨著刺激時(shí)間的延長(zhǎng)表達(dá)下調(diào),肌成纖維細(xì)胞的標(biāo)志分子α-SMA隨著刺激時(shí)間的延長(zhǎng)表達(dá)上調(diào)(P0.05)。3)流式細(xì)胞儀檢測(cè)經(jīng)5C11刺激后HUVEC上轉(zhuǎn)化生長(zhǎng)因子-β(TGF-β)Ⅱ型受體的表達(dá)是隨著刺激時(shí)間的延長(zhǎng)表達(dá)上調(diào)。 結(jié)論:綜上所述,我們的實(shí)驗(yàn)證實(shí)經(jīng)5C11刺激后,正常內(nèi)皮細(xì)胞的標(biāo)志物Flk1呈下調(diào)表達(dá),肌成纖維細(xì)胞的標(biāo)志蛋白平滑肌肌動(dòng)蛋白(α-SMA)的表達(dá)上調(diào),提示我們激發(fā)型CD40信號(hào)通路可促使內(nèi)皮細(xì)胞向平滑肌樣細(xì)胞轉(zhuǎn)分化,并且這一過(guò)程伴有內(nèi)皮細(xì)胞上TGF-βⅡ型受體的上調(diào)表達(dá),提示TGF-β在內(nèi)皮細(xì)胞轉(zhuǎn)分化中起重要作用。這為臨床治療及預(yù)防腎臟纖維化、動(dòng)脈粥樣硬化及肺纖維化等各種相關(guān)的疾病提供了新的靶點(diǎn)。
[Abstract]:In recent years, more and more attention has been paid to the transdifferentiation of endothelial cells to smooth muscle cells, which may be due to pulmonary fibrosis. Atherosclerosis and renal fibrosis and other diseases play an important role. Endothelial cells have high expression of CD40 molecules. The effect of CD40 signaling pathway on endothelial cell transdifferentiation has not been reported. Transforming growth factor 尾 (TGF- 尾). It is considered to be the most effective fibrogenic growth factor. It is considered to be the most important in renal tubular epithelial cell transdifferentiation, but its role in endothelial cell transdifferentiation is unclear. Objective: to apply monoclonal antibody 5C11, a monoclonal antibody against CD40, to culture umbilical vein endothelial cells (HUVEC) in vitro, and to use flow cytometry (FCM). Immunocytochemistry was used to observe whether endothelial cells could be transformed into smooth muscle like cells, and to explore the role of transforming growth factor- 尾 -TGF- 尾 in the transformation. Methods: 1MTT colorimetric method: HUVEC in logarithmic growth phase was inoculated into 96 well plate with different concentrations of 5C11. At the same time, the negative control group was set up. The effect of 5C11 on the proliferation or inhibition of HUVEC. 2) Immunochemistry method: the well-growing cells were inoculated into the 6-well culture plate and one sterilizing slide was placed in each hole. After culture, 5C11 was added at the concentration of 5ugml, and the negative control group was set up. The slides were taken out at 24 h, 48 h and 72 h, respectively. Observation of endothelial cell transformation. 3) flow cytometry was used to detect the expression of TGF- 尾 type 鈪，

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