调节Notch信号对角质形成细胞及成纤维细胞活性的影响

发布时间：2018-04-16 19:07

  本文选题：Notch信号 + 角质形成细胞　； 参考：《第四军医大学》2012年硕士论文

【摘要】：目的： 探讨调节Notch信号对角质形成细胞增殖分化及分泌纤维化相关因子的影响，及对成纤维细胞增殖及分泌胶原的影响。 方法： 应用DAPT及Jagged1-Fc融合蛋白刺激角质形成细胞，其中DAPT阻断Notch信号，Jagged1-Fc融合蛋白活化Notch信号，MTT法检测角质形成细胞的增殖情况，绘制生长曲线，免疫荧光检测角质形成细胞分化情况（K19、Involucrine）；建立角质形成细胞血清刺激模型，以模拟体内创伤愈合过程，Real time PCR检测Notch受体配体及下游分子（Notch1、Jagged1、P21及P63）的表达，Real time PCR及ELISA检测纤维化相关因子表达（TGF-β1、TGF-β2、IGF-1、CTGF、EGF及VEGF）；应用不同浓度的DAPT及Jagged1-Fc融合蛋白对角质形成细胞血清刺激模型进行预处理，Realtime PCR检测Notch下游分子及纤维化相关因子的表达，包括P21、P63、TGF-β1、TGF-β2、IGF-1、CTGF、EGF及VEGF；应用合适浓度的DAPT（10uM）及Jagged1-Fc(1ug/ml)融合蛋白对角质形成细胞血清刺激模型进行预处理，血清刺激后不同时间收集标本（0h、1h、6h、12h及24h），Realtime PCR及ELISA检测Notch下游分子及纤维化相关因子的表达(P21、P63、TGF-β1、TGF-β2、IGF-1、CTGF、EGF、VEGF)；应用Notch信号处理过的角质形成细胞条件培养基刺激成纤维细胞，MTT法检测成纤维细胞的增殖情况，绘制生长曲线，Real time PCR检测胶原（COLⅠ及COLⅢ）表达。 结果： DAPT能够明显促进角质形成细胞增殖并抑制细胞分化，Jagged1-Fc融合蛋白则促进了角质形成细胞的分化而抑制细胞增殖；血清明显促进角质形成细胞Notch受体配体表达及Notch信号的活化，其中Notch1及Jagged1的表达明显升高，并呈现时间依赖性，P21表达升高，6h到达表达高峰，P63表达降低，6h下降幅度最大。此外，血清也刺激了纤维化相关因子的表达（TGF-β1、TGF-β2、IGF-1、CTGF、EGF、VEGF）；Jagged1-Fc融合蛋白促进了P21表达，抑制了P63表达，同时进一步促进了纤维化相关因子（TGF-β1、TGF-β2、IGF-1、CTGF、EGF、VEGF）的表达，并呈现剂量依赖性，DAPT则使纤维化相关因子表达维持在基础水平；DAPT处理的角质形成细胞条件培养基能明显抑制成纤维细胞的增殖及胶原的表达，而Jagged1-Fc融合蛋白处理的角质形成细胞条件培养基则促进了成纤维细胞的增殖。 结论： Notch信号活化可能参与增生性瘢痕的形成过程，，其具体机制可能是Notch参与了增生性瘢痕的角质形成细胞的异常增殖与分化的异常调节，进而导致了角质形成细胞的分泌功能异常。阻断Notch信号能通过改变角质形成细胞的生物学活性，进而抑制成纤维细胞的活性。
[Abstract]:Objective:To investigate the effects of regulating Notch signal on the proliferation, differentiation and secretion of fibrosis related factors of keratinocytes, and on the proliferation and secretion of collagen in fibroblasts.Methods:DAPT and Jagged1-Fc fusion proteins were used to stimulate keratinocytes. The proliferation of keratinocytes was detected by Notch signal activated by DAPT, and the growth curve was plotted.The differentiation of keratinocytes was detected by immunofluorescence, and the serum stimulation model of keratinocytes was established.Real time PCR was used to detect the expression of Notch receptor ligand and its downstream molecule Notch1Jagged1P21 and P63 in vivo. Real time PCR and ELISA were used to detect the expression of fibrosis related factors TGF- 尾 2IGF-1 CTGF-1 and VEGF; and different concentrations of DAPT and Jagged1-Fc fusion proteins were used for diagonal formation.The expression of Notch downstream molecules and fibrosis related factors were detected by pretreatment with Realtime PCR in the model of cell serum stimulation.The model of serum stimulation of keratinocytes was pretreated with the fusion protein P21P63TGF- 尾 1TGF- 尾 2IGF-1CTGFEGF and VEGF; with suitable concentration of DAPT 10uM) and Jagged1-FcCU 1ugr.ml.Serum samples were collected at different times after stimulation for 12 and 24 hours, respectively, to detect the expression of Notch downstream molecules and fibrosis related factors (TGF- 尾 _ 2IGF-1CTGF- 尾 _ 2IGF-1CTGF- 尾 _ 2IGF-1 CTGFF-1 CTGFF-1), and to detect the expression of Notch downstream molecules and fibroblast-associated factors by ELISA. The keratinocyte conditioned medium treated with Notch signal was used to stimulate fibroblast culture medium to detect the expression of VEGF-尾 _ 2TGF- 尾 _ 2IGF-1 CTGF- EGF-1; to detect the proliferation of fibroblasts stimulated by Notch signal-treated keratinocyte conditioned medium.The proliferation of fibroblasts,The expression of Col 鈪

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