细胞因子组合对大鼠间充质干细胞基质金属蛋白酶表达及细胞迁移影响的实验研究

发布时间：2018-01-07 15:36

本文关键词：细胞因子组合对大鼠间充质干细胞基质金属蛋白酶表达及细胞迁移影响的实验研究　出处：《华中科技大学》2010年硕士论文　论文类型：学位论文

【摘要】：目的:研究促红细胞生成素(EPO)联合粒细胞集落刺激因子(G-CSF)对大鼠间充质干细胞基质金属蛋白酶表达及细胞迁移的影响。方法:采用经典的全骨髓贴壁法培养MSC,通过成骨、成脂肪等多向诱导分化以及流式细胞仪分析其表面标记(CD90,CD29,CD34,CD45,CD44)等鉴定MSC特征;P3代MSC分别给予不同浓度的EPO、G-CSF及EPO和G-CSF组合,RT-PCR方法检测MSC MMP和TIMP mRNA的表达。Transwell和Wound healing实验评价细胞的迁移能力。免疫印迹法检测ERK1/2蛋白的改变。结果:①培养出的P3代MSC,CD90、CD29呈阳性,CD34、CD45、CD44呈阴性,具有成脂肪、成骨等多向分化能力。 ②不同浓度EPO作用于MSC12h后,MMP-2的表达较空白对照组明显增加,以1IU/ml时表达最强(P0. 05),MMP-9、TIMP-1、TIMP-2的表达无明显差异。 ③不同浓度G-CSF作用于MSC12h后,MMP-2的表达较空白对照组明显增加,在1ng/ml时接近峰值(P0. 05),MMP-9、TIMP-1、TIMP-2的表达无明显差异。 ④给予EPO(1IU/ml)、G-CSF(0.1ng/ml、1ng/ml)及EPO(1IU/ml)和G-CSF(0.1ng/ml、1ng/ml)组合作用于MSC12h后,MMP-2的表达均有增加,与空白对照组及单因子组相比较,EPO 1IU/ml+G-CSF 0.1ng/ml时表达增强(P0. 05),MMP-9、TIMP-1、TIMP-2的表达无明显差异。 ⑤给予EPO(1IU/ml)、G-CSF(0.1ng/ml、1ng/ml)及EPO(1IU/ml)和G-CSF(0.1ng/ml、1ng/ml)组合作用于MSC12h后,间充质干细胞侵袭的数目均有增加,与空白对照组及单因子组相比较,在EPO 1IU/ml+G-CSF 0.1ng/ml时侵袭细胞的数目最多(P0. 05)。 ⑥给予EPO(1IU/ml)、G-CSF(0.1ng/ml、1ng/ml)及EPO(1IU/ml)和G-CSF(0.1ng/ml、1ng/ml)组合作用于MSC18h后,间充质干细胞迁移的面积均有增加,与空白对照组及单因子组相比较,在EPO 1IU/ml+G-CSF 0.1ng/ml时迁移的面积最明显(P0. 05)。 ⑦给予EPO(1IU/ml)、G-CSF(0.1ng/ml、1ng/ml)及EPO(1IU/ml)和G-CSF(0.1ng/ml、1ng/ml)组合作用于MSC3h后,ERK1/2的表达均有增加,与空白对照组及单因子组相比较,在EPO 1IU/ml+G-CSF 0.1ng/ml时表达最强(P0. 05)。结论:EPO和G-CSF联合作用可通过刺激间充质干细胞MMP-2的表达促进MSC的迁移,并且与ERK1/2信号通路有关。
[Abstract]:Aim: to study the effects of erythropoietin (EPO) combined with granulocyte colony stimulating factor (G-CSF) on the expression of matrix metalloproteinases and cell migration in rat mesenchymal stem cells. Methods: MSCs were cultured by classical whole bone marrow adherent method. Osteogenesis, adipose-induced differentiation and flow cytometry were used to analyze the surface marker CD90, CD29 and CD34. CD45, CD44, etc., to identify the characteristics of MSC; P3 generation MSC was given different concentrations of G-CSF, EPO and G-CSF. Detection of the expression of MSC MMP and TIMP mRNA by RT-PCR. Transwell and Wound. The migration ability of cells was evaluated by healing assay. The changes of ERK1/2 protein were detected by Western blot. Results CD90 CD29 was positive in P3 generation cultured at 1: 1. CD34, CD45, CD44 and CD44 were negative and had the ability to differentiate into fat and osteogenesis. 2the expression of MMP-2 in MSC12h treated with different concentrations of EPO was significantly higher than that in control group, and the strongest expression of MMP-9 was found in 1IU- / ml group. There was no significant difference in the expression of TIMP-1 and TIMP-2. (3) the expression of MMP-2 in MSC12h treated with G-CSF at different concentrations was significantly higher than that in control group, and the expression of MMP-2 was close to the peak value at 1 ng / ml. There was no significant difference in the expression of TIMP-1 and TIMP-2. (4) give EPO1 / ml / ml G-CSFN 0.1 ng / ml and EPO1 / ml / ml) and G-CSF / 0.1 ng / ml. The expression of MMP-2 was increased after the combination of 1ng / ml with MSC12h, which was compared with the blank control group and single factor group. There was no significant difference in the expression of MMP-9, TIMP-1and TIMP-2 at 0.1 ng / ml G-CSF of EPO 1 IUU. (5) give EPO1 / ml / ml G-CSFN 0.1 ng / ml and EPO1 / ml / ml) and G-CSF / 0.1 ng / ml. The number of invasion of mesenchymal stem cells increased after 1 ng / ml combination of MSC12h, compared with blank control group and single factor group. The maximum number of invasive cells at 0.1 ng / ml G-CSF at 1 IUU / ml of EPO was P0. 05. (6) give EPO1 / ml / ml G-CSFN 0.1 ng / ml and EPO1 / ml / ml) and G-CSF / 0.1 ng / ml. The migration area of mesenchymal stem cells increased after 1 ng / ml combination of MSC18h, compared with blank control group and single factor group. The migration area was the most obvious at 0.1 ng / ml G-CSF 0.1 ng / ml in EPO 1 IUU / ml. (7) give EPO1 IUP / ml / ml G-CSFN 0.1ng / ml and EPO-1IUP / ml) and G-CSF / 0.1ngr / ml. The expression of ERK 1 / 2 was increased after the combination of 1ng / ml with MSC3h, which was compared with the blank control group and single factor group. The strongest expression was at 0.1 ng / ml G-CSF of EPO 1 IUU / ml. Conclusion the combined action of MMP-2 and G-CSF can promote the migration of MSC by stimulating the expression of MMP-2 in mesenchymal stem cells, and may be related to the ERK1/2 signaling pathway.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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