本文選題：Notch信號(hào) + 角質(zhì)形成細(xì)胞　； 參考：《第四軍醫(yī)大學(xué)》2012年碩士論文

【摘要】：目的： 探討調(diào)節(jié)Notch信號(hào)對(duì)角質(zhì)形成細(xì)胞增殖分化及分泌纖維化相關(guān)因子的影響，及對(duì)成纖維細(xì)胞增殖及分泌膠原的影響。 方法： 應(yīng)用DAPT及Jagged1-Fc融合蛋白刺激角質(zhì)形成細(xì)胞，其中DAPT阻斷Notch信號(hào)，Jagged1-Fc融合蛋白活化Notch信號(hào)，MTT法檢測(cè)角質(zhì)形成細(xì)胞的增殖情況，繪制生長(zhǎng)曲線，免疫熒光檢測(cè)角質(zhì)形成細(xì)胞分化情況（K19、Involucrine）；建立角質(zhì)形成細(xì)胞血清刺激模型，以模擬體內(nèi)創(chuàng)傷愈合過(guò)程，Real time PCR檢測(cè)Notch受體配體及下游分子（Notch1、Jagged1、P21及P63）的表達(dá)，Real time PCR及ELISA檢測(cè)纖維化相關(guān)因子表達(dá)（TGF-β1、TGF-β2、IGF-1、CTGF、EGF及VEGF）；應(yīng)用不同濃度的DAPT及Jagged1-Fc融合蛋白對(duì)角質(zhì)形成細(xì)胞血清刺激模型進(jìn)行預(yù)處理，Realtime PCR檢測(cè)Notch下游分子及纖維化相關(guān)因子的表達(dá)，包括P21、P63、TGF-β1、TGF-β2、IGF-1、CTGF、EGF及VEGF；應(yīng)用合適濃度的DAPT（10uM）及Jagged1-Fc(1ug/ml)融合蛋白對(duì)角質(zhì)形成細(xì)胞血清刺激模型進(jìn)行預(yù)處理，血清刺激后不同時(shí)間收集標(biāo)本（0h、1h、6h、12h及24h），Realtime PCR及ELISA檢測(cè)Notch下游分子及纖維化相關(guān)因子的表達(dá)(P21、P63、TGF-β1、TGF-β2、IGF-1、CTGF、EGF、VEGF)；應(yīng)用Notch信號(hào)處理過(guò)的角質(zhì)形成細(xì)胞條件培養(yǎng)基刺激成纖維細(xì)胞，MTT法檢測(cè)成纖維細(xì)胞的增殖情況，繪制生長(zhǎng)曲線，Real time PCR檢測(cè)膠原（COLⅠ及COLⅢ）表達(dá)。 結(jié)果： DAPT能夠明顯促進(jìn)角質(zhì)形成細(xì)胞增殖并抑制細(xì)胞分化，Jagged1-Fc融合蛋白則促進(jìn)了角質(zhì)形成細(xì)胞的分化而抑制細(xì)胞增殖；血清明顯促進(jìn)角質(zhì)形成細(xì)胞Notch受體配體表達(dá)及Notch信號(hào)的活化，其中Notch1及Jagged1的表達(dá)明顯升高，并呈現(xiàn)時(shí)間依賴(lài)性，P21表達(dá)升高，6h到達(dá)表達(dá)高峰，P63表達(dá)降低，6h下降幅度最大。此外，血清也刺激了纖維化相關(guān)因子的表達(dá)（TGF-β1、TGF-β2、IGF-1、CTGF、EGF、VEGF）；Jagged1-Fc融合蛋白促進(jìn)了P21表達(dá)，抑制了P63表達(dá)，同時(shí)進(jìn)一步促進(jìn)了纖維化相關(guān)因子（TGF-β1、TGF-β2、IGF-1、CTGF、EGF、VEGF）的表達(dá)，并呈現(xiàn)劑量依賴(lài)性，DAPT則使纖維化相關(guān)因子表達(dá)維持在基礎(chǔ)水平；DAPT處理的角質(zhì)形成細(xì)胞條件培養(yǎng)基能明顯抑制成纖維細(xì)胞的增殖及膠原的表達(dá)，而Jagged1-Fc融合蛋白處理的角質(zhì)形成細(xì)胞條件培養(yǎng)基則促進(jìn)了成纖維細(xì)胞的增殖。 結(jié)論： Notch信號(hào)活化可能參與增生性瘢痕的形成過(guò)程，，其具體機(jī)制可能是Notch參與了增生性瘢痕的角質(zhì)形成細(xì)胞的異常增殖與分化的異常調(diào)節(jié)，進(jìn)而導(dǎo)致了角質(zhì)形成細(xì)胞的分泌功能異常。阻斷Notch信號(hào)能通過(guò)改變角質(zhì)形成細(xì)胞的生物學(xué)活性，進(jìn)而抑制成纖維細(xì)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

本文編號(hào)：1760216