本文關(guān)鍵詞：細胞因子組合對大鼠間充質(zhì)干細胞基質(zhì)金屬蛋白酶表達及細胞遷移影響的實驗研究　出處：《華中科技大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 間充質(zhì)干細胞 促紅細胞生成素 粒細胞集落刺激因子 基質(zhì)金屬蛋白酶 遷移 細胞外調(diào)節(jié)蛋白激酶

【摘要】：目的:研究促紅細胞生成素(EPO)聯(lián)合粒細胞集落刺激因子(G-CSF)對大鼠間充質(zhì)干細胞基質(zhì)金屬蛋白酶表達及細胞遷移的影響。 方法:采用經(jīng)典的全骨髓貼壁法培養(yǎng)MSC,通過成骨、成脂肪等多向誘導(dǎo)分化以及流式細胞儀分析其表面標記(CD90,CD29,CD34,CD45,CD44)等鑒定MSC特征;P3代MSC分別給予不同濃度的EPO、G-CSF及EPO和G-CSF組合,RT-PCR方法檢測MSC MMP和TIMP mRNA的表達。Transwell和Wound healing實驗評價細胞的遷移能力。免疫印跡法檢測ERK1/2蛋白的改變。 結(jié)果:①培養(yǎng)出的P3代MSC,CD90、CD29呈陽性,CD34、CD45、CD44呈陰性,具有成脂肪、成骨等多向分化能力。 ②不同濃度EPO作用于MSC12h后,MMP-2的表達較空白對照組明顯增加,以1IU/ml時表達最強(P0. 05),MMP-9、TIMP-1、TIMP-2的表達無明顯差異。 ③不同濃度G-CSF作用于MSC12h后,MMP-2的表達較空白對照組明顯增加,在1ng/ml時接近峰值(P0. 05),MMP-9、TIMP-1、TIMP-2的表達無明顯差異。 ④給予EPO(1IU/ml)、G-CSF(0.1ng/ml、1ng/ml)及EPO(1IU/ml)和G-CSF(0.1ng/ml、1ng/ml)組合作用于MSC12h后,MMP-2的表達均有增加,與空白對照組及單因子組相比較,EPO 1IU/ml+G-CSF 0.1ng/ml時表達增強(P0. 05),MMP-9、TIMP-1、TIMP-2的表達無明顯差異。 ⑤給予EPO(1IU/ml)、G-CSF(0.1ng/ml、1ng/ml)及EPO(1IU/ml)和G-CSF(0.1ng/ml、1ng/ml)組合作用于MSC12h后,間充質(zhì)干細胞侵襲的數(shù)目均有增加,與空白對照組及單因子組相比較,在EPO 1IU/ml+G-CSF 0.1ng/ml時侵襲細胞的數(shù)目最多(P0. 05)。 ⑥給予EPO(1IU/ml)、G-CSF(0.1ng/ml、1ng/ml)及EPO(1IU/ml)和G-CSF(0.1ng/ml、1ng/ml)組合作用于MSC18h后,間充質(zhì)干細胞遷移的面積均有增加,與空白對照組及單因子組相比較,在EPO 1IU/ml+G-CSF 0.1ng/ml時遷移的面積最明顯(P0. 05)。 ⑦給予EPO(1IU/ml)、G-CSF(0.1ng/ml、1ng/ml)及EPO(1IU/ml)和G-CSF(0.1ng/ml、1ng/ml)組合作用于MSC3h后,ERK1/2的表達均有增加,與空白對照組及單因子組相比較,在EPO 1IU/ml+G-CSF 0.1ng/ml時表達最強(P0. 05)。 結(jié)論:EPO和G-CSF聯(lián)合作用可通過刺激間充質(zhì)干細胞MMP-2的表達促進MSC的遷移,并且與ERK1/2信號通路有關(guān)。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

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本文編號：1393215