激发型共刺激分子CD40信号通路对人脐静脉内皮细胞的影响及其机制

发布时间：2018-01-19 08:51

  本文关键词： 脐静脉内皮细胞 平滑肌样细胞 激发型抗CD40单抗5C11 转分化 TGF-β受体　出处：《苏州大学》2007年硕士论文　论文类型：学位论文

【摘要】： 内皮细胞向平滑肌样细胞的转分化近年来受到越来越多的重视。这种转分化可能在肺纤维化、动脉粥样硬化及肾脏纤维化等疾病中起重要作用。内皮细胞上有CD40分子的高表达,激发型CD40信号可以导致内皮细胞功能失调。CD40信号通路对内皮细胞转分化的影响未见相关报道。转化生长因子β(TGF-β)是目前认为作用最强的促纤维化生长因子,其在肾小管上皮细胞转分化中被认为是最重要的,但在内皮细胞转分化中的作用还不清楚。 目的:应用抗CD40激发型单克隆抗体5C11作用于体外培养的脐静脉内皮细胞,通过流式细胞仪、免疫细胞化学等方法观察内皮细胞能否向平滑肌样细胞转化,同时探讨转化生长因子-β(TGF-β)在转化中的作用。 方法: 1)MTT比色法:把处于对数生长期生长状态良好(细胞呈铺路石样)的HUVEC接种到96孔板,加入不同浓度的5C11,同时设阴性对照组。检测5C11对HUVEC促增殖或抑制的影响。2)细胞爬片免疫化学法:把生长良好的细胞接种于6孔培养板,每孔放一灭菌盖玻片,培养后,分组加入5C11,浓度为5 ug/ml,同时设阴性对照组,继续培养。分别于24h、48h、72h取出玻片,观察内皮细胞的转化情况。3)流式细胞仪检测经5C11刺激后的内皮细胞中TGF-β的Ⅱ型受体的表达情况。4)流式细胞仪检测经5C11刺激后的内皮细胞是否发生凋亡。 结果: 1)MTT法检测结果:应用MTT法检测5C11对HUVEC抑制或增殖的影响,实验数据应用统计分析软件分析无统计学意义(P0.05)。流式细胞仪检测细胞凋亡:内皮细胞经过5C11刺激72H后应用流式细胞仪未检测到细胞凋亡的发生。2)抗CD40单克隆抗体5C11不同浓度刺激对HUVEC的影响:细胞免疫组织化学显示,经过5C11刺激后HUVEC正常血管内皮细胞的标志分子Flk1随着刺激时间的延长表达下调,肌成纤维细胞的标志分子α-SMA随着刺激时间的延长表达上调(P0.05)。3)流式细胞仪检测经5C11刺激后HUVEC上转化生长因子-β(TGF-β)Ⅱ型受体的表达是随着刺激时间的延长表达上调。 结论:综上所述,我们的实验证实经5C11刺激后,正常内皮细胞的标志物Flk1呈下调表达,肌成纤维细胞的标志蛋白平滑肌肌动蛋白(α-SMA)的表达上调,提示我们激发型CD40信号通路可促使内皮细胞向平滑肌样细胞转分化,并且这一过程伴有内皮细胞上TGF-βⅡ型受体的上调表达,提示TGF-β在内皮细胞转分化中起重要作用。这为临床治疗及预防肾脏纤维化、动脉粥样硬化及肺纤维化等各种相关的疾病提供了新的靶点。
[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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