GM-CSF诱导成纤维细胞MMP-2和MMP-9的表达及机制研究

发布时间：2018-01-27 14:17

本文关键词： 单核巨噬细胞集落刺激因子创面愈合成纤维细胞 MMP-2 MMP-9　出处：《上海交通大学》2015年硕士论文　论文类型：学位论文

【摘要】：研究目的探讨单核巨噬细胞集落刺激因子(GM-CSF)对人皮肤成纤维细胞MMP-2、MMP-9表达的影响以及其分子生物学机制。研究方法1)ELISA、明胶酶谱及Western Blot检测不同GM-CSF浓度(0、10、50、100、500ng/ml)刺激下MMP-2和MMP-9在成纤维细胞中的表达情况;2)免疫荧光及Western Blot检测GM-CSF及SP600125(JNK抑制剂)对成纤维细胞内JNK/c-Jun磷酸化水平的影响,RT-PCR分别检测GM-CSF或SP600125+GM-CSF对成纤维细胞内MMP-2/-9 m RNA合成的影响,ELISA分别检测GM-CSF或SP600125+GM-CSF处理成纤维细胞后(6、12、24、48h)MMP-2/-9的浓度;3)cck-8试剂盒检测GM-CSF对成纤维细胞增殖的影响,细胞划痕实验检测GM-CSF对成纤维细胞迁移能力的影响。研究结果1)GM-CSF处理后,人皮肤成纤维细胞MMP-2/-9的表达均明显高于对照组(P0.05),MMP-2/-9的表达量及活性与GM-CSF浓度呈正相关;2)GM-CSF处理后,成纤维细胞内JNK/c-Jun的磷酸化水平明显高于对照组(P0.05);MMP-2/-9的m RNA转录水平显著高于对照组(P0.05);SP600125预处理后,与GM-CSF组相比,c-Jun的磷酸化明显被抑制(P0.05),MMP-2/-9的m RNA水平则显著降低(P0.05);GM-CSF处理细胞后,上清中MMP-2/-9的浓度在24小时达到最高值,24-48小时逐渐降低,但均显著高于对照组(P0.05);在SP600125+GM-CSF组,MMP-2的表达低于对照组,而MMP-9的表达明显高于对照组(P0.05);3)GM-CSF对成纤维细胞的增殖无明显影响(P0.05);GM-CSF能够促进成纤维细胞的迁移(P0.05),而SP600125则能抑制成纤维细胞的迁移(P0.05);结论:GM-CSF通过活化JNK/c-Jun信号通路上调成纤维细胞MMP-2和MMP-9的合成、表达与分泌,并促进成纤维细胞的迁移。
[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.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R641

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