rhBMP-2调节人脂肪间充质干细胞VEGF表达的生物学规律及其机制的研究

发布时间：2018-03-10 18:49

本文选题：组织工程　切入点：重组人骨形态发生蛋白2　出处：《新疆医科大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的：rhBMP-2是构建血管化组织工程骨理想的生长因子，本课题拟研究不同诱导浓度以及不同诱导时间点rhBMP-2调节人脂肪间充质干细胞表达VEGF的生物学规律并探讨p38信号通路在此过程中的调节作用。方法：从成人脂肪组织中分离培养脂肪间充质干细胞，培养至三代。采用RT-PCR及ELISA从基因水平和蛋白水平检测不同诱导浓度（0ng/ml，50ng/ml，100ng/ml）以及不同诱导时间点（3h，6h，12h，18h，24h，36h，48h）rhBMP-2诱导下人脂肪间充质干细胞表达VEGF的规律。采用Western-blot检测比较不同诱导浓度（0ng/ml，50ng/ml，100ng/ml，200ng/ml）及不同诱导时间点（3h，6h，，24h，36h）p38信号通路磷酸化的水平并研究其与VEGF表达量的的相关性。实验中加入不同浓度（5μM,10μM）p38特异性阻断剂sb203580后采用RT-PCR以及ELISA检测人脂肪间充质干细胞VEGF表达的变化。结果：相同时间点下当rhBMP-2诱导浓度为100ng/ml时VEGF的表达量最高。3h-6h和18h-24h为诱导组和对照组表达VEGF的两个高峰时间段，其中3h-6h时诱导组VEGF的表达量要低于对照组（P0.05）而在18h-24h诱导组VEGF的表达量高于对照组（P0.05）。p38信号通路磷酸化的水平与VEGF的表达量密切相关，两者表现出相似的变化趋势。p38阻断剂sb203580可以抑制VEGF的表达，且浓度越高抑制效果越明显，15μmol/ml组VEGF的表达几乎被完全抑制。结论：rhBMP-2调节人脂肪间充质干细胞表达VEGF具有浓度依赖性和时间依赖性。rhBMP-2最适诱导浓度为100ng/ml，3h-6h和18h-24h是利用rhBMP-2促进组织工程骨血管化的关键时间点。p38信号通路参与到rhBMP-2促进人脂肪间充质干细胞表达VEGF的过程当中并起着重要的调控作用。
[Abstract]:Objective: rhBMP-2 is an ideal growth factor for vascularized tissue engineering bone. The aim of this study was to investigate the biological regulation of rhBMP-2 on the expression of VEGF in human adipose mesenchymal stem cells at different concentrations and at different time points and to explore the regulatory role of p38 signaling pathway in this process. Adipose mesenchymal stem cells, RT-PCR and ELISA were used to detect the expression of VEGF in human adipose mesenchymal stem cells induced by RT-PCR and ELISA at different levels of gene and protein. The expression of VEGF in human adipose mesenchymal stem cells was detected by Western-blot. 0 ng / ml / ml 50 ng / ml / ml 100 ng / ml / ml 100 ng / ml / ml) and different induction time points at 3 h ~ 6 h ~ 6 h ~ (24) h ~ (24) h ~ (36) h ~ (38) signal pathway phosphorylation and its correlation with the expression of VEGF. In the experiment, 5 渭 M 10 渭 M ~ (38) specific blocker sb2035 80 was added to detect the dry fat mesenchyme of human adipose tissue by RT-PCR and ELISA. Results: at the same time, when the concentration of rhBMP-2 was 100ng / ml, the expression of VEGF was the highest. 3h-6h and 18h-24 h were the two peak periods of the expression of VEGF in the induction group and control group. At 3h-6 h, the expression of VEGF in the induced group was lower than that in the control group (P0.05), but the expression level of VEGF in the 18h-24 h group was higher than that in the control group. The phosphorylation level of the signal pathway of P0.05N. P38 was closely related to the expression of VEGF. The expression of VEGF was inhibited by p38 antagonist sb203580. The higher the concentration of rhBMP-2 was, the more obvious was the inhibition of VEGF expression in 15 渭 mol/ml group. Conclusion the effect of rhBMP-2 on the expression of VEGF in human adipose mesenchymal stem cells is in a concentration-and time-dependent manner. The optimal concentration of rhBMP-2 is 100ng / ml 3h-6h and 18h-24 h, respectively. The key point of rhBMP-2 in promoting vascularization of tissue engineering bone. P38 signaling pathway is involved in the process of rhBMP-2 promoting the expression of VEGF in human adipose mesenchymal stem cells and plays an important regulatory role.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R329.2

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