本文關(guān)鍵詞： 單核巨噬細(xì)胞集落刺激因子 創(chuàng)面愈合 成纖維細(xì)胞 MMP-2 MMP-9　出處：《上海交通大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：研究目的探討單核巨噬細(xì)胞集落刺激因子(GM-CSF)對(duì)人皮膚成纖維細(xì)胞MMP-2、MMP-9表達(dá)的影響以及其分子生物學(xué)機(jī)制。研究方法1)ELISA、明膠酶譜及Western Blot檢測(cè)不同GM-CSF濃度(0、10、50、100、500ng/ml)刺激下MMP-2和MMP-9在成纖維細(xì)胞中的表達(dá)情況;2)免疫熒光及Western Blot檢測(cè)GM-CSF及SP600125(JNK抑制劑)對(duì)成纖維細(xì)胞內(nèi)JNK/c-Jun磷酸化水平的影響,RT-PCR分別檢測(cè)GM-CSF或SP600125+GM-CSF對(duì)成纖維細(xì)胞內(nèi)MMP-2/-9 m RNA合成的影響,ELISA分別檢測(cè)GM-CSF或SP600125+GM-CSF處理成纖維細(xì)胞后(6、12、24、48h)MMP-2/-9的濃度;3)cck-8試劑盒檢測(cè)GM-CSF對(duì)成纖維細(xì)胞增殖的影響,細(xì)胞劃痕實(shí)驗(yàn)檢測(cè)GM-CSF對(duì)成纖維細(xì)胞遷移能力的影響。研究結(jié)果1)GM-CSF處理后,人皮膚成纖維細(xì)胞MMP-2/-9的表達(dá)均明顯高于對(duì)照組(P0.05),MMP-2/-9的表達(dá)量及活性與GM-CSF濃度呈正相關(guān);2)GM-CSF處理后,成纖維細(xì)胞內(nèi)JNK/c-Jun的磷酸化水平明顯高于對(duì)照組(P0.05);MMP-2/-9的m RNA轉(zhuǎn)錄水平顯著高于對(duì)照組(P0.05);SP600125預(yù)處理后,與GM-CSF組相比,c-Jun的磷酸化明顯被抑制(P0.05),MMP-2/-9的m RNA水平則顯著降低(P0.05);GM-CSF處理細(xì)胞后,上清中MMP-2/-9的濃度在24小時(shí)達(dá)到最高值,24-48小時(shí)逐漸降低,但均顯著高于對(duì)照組(P0.05);在SP600125+GM-CSF組,MMP-2的表達(dá)低于對(duì)照組,而MMP-9的表達(dá)明顯高于對(duì)照組(P0.05);3)GM-CSF對(duì)成纖維細(xì)胞的增殖無(wú)明顯影響(P0.05);GM-CSF能夠促進(jìn)成纖維細(xì)胞的遷移(P0.05),而SP600125則能抑制成纖維細(xì)胞的遷移(P0.05);結(jié)論:GM-CSF通過(guò)活化JNK/c-Jun信號(hào)通路上調(diào)成纖維細(xì)胞MMP-2和MMP-9的合成、表達(dá)與分泌,并促進(jìn)成纖維細(xì)胞的遷移。
[Abstract]:Objective to investigate the effect of mononuclear macrophage colony stimulating factor (GM-CSF) on human skin fibroblasts (MMP-2). The effect of MMP-9 expression and its molecular biological mechanism. Methods 1. The different concentrations of GM-CSF were detected by gelatinase spectrum and Western Blot. The expression of MMP-2 and MMP-9 in fibroblasts stimulated by 50ng / ml; 2) the effects of immunofluorescence and Western Blot on the phosphorylation of JNK/c-Jun in fibroblasts were detected by GM-CSF and SP600125(JNK inhibitors. The effects of GM-CSF or SP600125 GM-CSF on the synthesis of MMP-2/-9 m RNA in fibroblasts were detected by RT-PCR. ELISA was used to detect the concentration of MMP-2 / -9 in fibroblasts treated with GM-CSF or SP600125 GM-CSF for 48 h. The effect of GM-CSF on the proliferation of fibroblasts was detected by 3Ccck-8 kit. The effect of GM-CSF on the migration of fibroblasts was detected by cell scratch assay. The expression of MMP-2/-9 in human skin fibroblasts was significantly higher than that in the control group. The expression and activity of MMP-2 / -9 in human skin fibroblasts were positively correlated with the concentration of GM-CSF. 2the phosphorylation level of JNK/c-Jun in fibroblasts treated with GM-CSF was significantly higher than that in control group (P 0.05). The m RNA transcription level of MMP-2/-9 was significantly higher than that of control group (P 0.05). Compared with GM-CSF group, the phosphorylation of c-Jun in SP600125 group was significantly inhibited (P 0.05). The m RNA level of MMP-2/-9 decreased significantly (P 0.05). After treated with GM-CSF, the concentration of MMP-2/-9 in the supernatant gradually decreased at 24 h to 48 h, but was significantly higher than that of the control group (P 0.05). The expression of MMP-2 in SP600125 GM-CSF group was lower than that in control group, while the expression of MMP-9 was significantly higher than that in control group (P 0.05). 3GM-CSF had no significant effect on the proliferation of fibroblasts. GM-CSF could promote the migration of fibroblasts (P 0.05), while SP600125 could inhibit the migration of fibroblasts (P 0.05). Conclusion the synthesis, expression and secretion of MMP-2 and MMP-9 in fibroblasts can be upregulated by the activation of JNK/c-Jun signaling pathway, and the migration of fibroblasts can be promoted by WGM CSF.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R641

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