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目的:本课题组前期工作已经证明阻断Notch信号对角质形成细胞的分泌功能有所影响,使其分泌的多种促纤维化因子发生改变。本实验将探讨在复合培养的条件下,通过调节角质形成细胞中的Notch信号,了解其对成纤维细胞collagen-1合成的影响。方法:运用角质形成细胞-成纤维细胞复合培养的模型,于复合培养前3天进行血清刺激或者γ-分泌酶抑制剂DAPT阻断角质形成细胞中的Notch信号,即在角质形成细胞分化前进行干预。然后提升至气液交界面使其达到复层生长和终末分化,后与成纤维细胞共培养,观察阻断Notch信号后的角质形成细胞对成纤维细胞合成collagen-1的影响。结果:在分化前,给予角质形成细胞血清刺激,在复合培养0小时,Notch-1、Jagged-1表达明显升高(P<0.05),在复合培养第一天,p21表达上调、p63表达下降(P<0.05),表明在复合培养时,角质形成细胞中的Notch信号明显活化。通过在血清刺激前给予DAPT预处理,在复合培养0小时,角质形成细胞中Notch信号下游分子p21和p63的表达恢复至无血清刺激水平(P<0.05),表明DAPT组确实阻断了角质形成细胞中的Notch信号。而DAPT组的成纤维细胞collagen-1的表达相对于血清刺激组明显下降,而与无血清组无明显差异,表明阻断Notch信号后的角质形成细胞在复合培养条件下,确实能够抑制成纤维细胞collagen-1的表达,使其合成collagen-1的量恢复至无血清刺激水平。结论:DAPT能够阻断Notch信号的活化,用阻断Notch信号后的角质形成细胞与成纤维细胞共培养,能够明显抑制成纤维细胞合成Ⅰ型胶原的能力,从而抑制瘢痕的增生。
OBJECTIVE: The previous work of our group has demonstrated that the blockade of Notch signaling has an impact on the secretory function of keratinocytes, resulting in the alteration of a variety of pro-fibrotic factors secreted by them. In this study, we investigated the effects of Notch signaling in keratinocytes on collagen-1 synthesis in fibroblasts under complex culture conditions. METHODS: Using a model of keratinocyte-fibroblast complex culture, serum stimulation or DAPT, a gamma-secretase inhibitor, was used to block Notch signaling in keratinocytes 3 days prior to compound culture, ie, prior to keratinocyte differentiation Intervention. Then it was promoted to the gas-liquid interface to reach the level of stratification and terminal differentiation. Then it was co-cultured with fibroblasts to observe the effects of Notch signaling-induced keratinocytes on collagen-1 synthesis by fibroblasts. Results: Before differentiation, keratinocytes were stimulated with serum and the expression of Notch-1 and Jagged-1 was significantly increased at 0 hour (P <0.05). On the first day of compound culture, p21 expression was up-regulated and p63 expression was decreased (P <0.05), indicating that Notch signaling in keratinocytes is significantly activated at the time of co-culture. Expression of p21 and p63 downstream of Notch signaling in keratinocytes returned to serum-free stimulus levels (P <0.05) by DAPT pretreatment before serum stimulation, indicating that DAPT did block keratinocyte formation at 0 h Notch signal in cells. However, the expression of collagen-1 in fibroblasts of DAPT group was significantly lower than that in serum-stimulated group, but no significant difference with serum-free group, indicating that Keratinocytes blocking Notch signaling could indeed inhibit fibroblasts under compound culture conditions The expression of cellular collagen-1 restored the amount of collagen-1 synthesized to serum-free stimulus. CONCLUSION: DAPT can block the activation of Notch signaling. Co-culture of Notch signaling with keratinocytes and fibroblasts can significantly inhibit the ability of fibroblasts to synthesize type I collagen and inhibit the proliferation of scar.