本文選題：組織工程　切入點(diǎn)：重組人骨形態(tài)發(fā)生蛋白2　出處：《新疆醫(yī)科大學(xué)》2013年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的：rhBMP-2是構(gòu)建血管化組織工程骨理想的生長(zhǎng)因子，本課題擬研究不同誘導(dǎo)濃度以及不同誘導(dǎo)時(shí)間點(diǎn)rhBMP-2調(diào)節(jié)人脂肪間充質(zhì)干細(xì)胞表達(dá)VEGF的生物學(xué)規(guī)律并探討p38信號(hào)通路在此過(guò)程中的調(diào)節(jié)作用。方法：從成人脂肪組織中分離培養(yǎng)脂肪間充質(zhì)干細(xì)胞，培養(yǎng)至三代。采用RT-PCR及ELISA從基因水平和蛋白水平檢測(cè)不同誘導(dǎo)濃度（0ng/ml，50ng/ml，100ng/ml）以及不同誘導(dǎo)時(shí)間點(diǎn)（3h，6h，12h，18h，24h，36h，48h）rhBMP-2誘導(dǎo)下人脂肪間充質(zhì)干細(xì)胞表達(dá)VEGF的規(guī)律。采用Western-blot檢測(cè)比較不同誘導(dǎo)濃度（0ng/ml，50ng/ml，100ng/ml，200ng/ml）及不同誘導(dǎo)時(shí)間點(diǎn)（3h，6h，，24h，36h）p38信號(hào)通路磷酸化的水平并研究其與VEGF表達(dá)量的的相關(guān)性。實(shí)驗(yàn)中加入不同濃度（5μM,10μM）p38特異性阻斷劑sb203580后采用RT-PCR以及ELISA檢測(cè)人脂肪間充質(zhì)干細(xì)胞VEGF表達(dá)的變化。結(jié)果：相同時(shí)間點(diǎn)下當(dāng)rhBMP-2誘導(dǎo)濃度為100ng/ml時(shí)VEGF的表達(dá)量最高。3h-6h和18h-24h為誘導(dǎo)組和對(duì)照組表達(dá)VEGF的兩個(gè)高峰時(shí)間段，其中3h-6h時(shí)誘導(dǎo)組VEGF的表達(dá)量要低于對(duì)照組（P0.05）而在18h-24h誘導(dǎo)組VEGF的表達(dá)量高于對(duì)照組（P0.05）。p38信號(hào)通路磷酸化的水平與VEGF的表達(dá)量密切相關(guān)，兩者表現(xiàn)出相似的變化趨勢(shì)。p38阻斷劑sb203580可以抑制VEGF的表達(dá)，且濃度越高抑制效果越明顯，15μmol/ml組VEGF的表達(dá)幾乎被完全抑制。結(jié)論：rhBMP-2調(diào)節(jié)人脂肪間充質(zhì)干細(xì)胞表達(dá)VEGF具有濃度依賴(lài)性和時(shí)間依賴(lài)性。rhBMP-2最適誘導(dǎo)濃度為100ng/ml，3h-6h和18h-24h是利用rhBMP-2促進(jìn)組織工程骨血管化的關(guān)鍵時(shí)間點(diǎn)。p38信號(hào)通路參與到rhBMP-2促進(jìn)人脂肪間充質(zhì)干細(xì)胞表達(dá)VEGF的過(guò)程當(dāng)中并起著重要的調(diào)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.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R329.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 陳濱;裴國(guó)獻(xiàn);王珂;唐光輝;;組織工程骨修復(fù)山羊負(fù)重骨大段骨缺損的長(zhǎng)期觀察[J];南方醫(yī)科大學(xué)學(xué)報(bào);2006年06期

2 田少奇;王麗;孫康;夏長(zhǎng)所;唐東起;;人臍血間充質(zhì)干細(xì)胞的分離培養(yǎng)及其多向分化潛能的實(shí)驗(yàn)研究[J];中國(guó)骨與關(guān)節(jié)損傷雜志;2009年02期

3 裴國(guó)獻(xiàn),金丹;骨組織工程研究進(jìn)展[J];中華創(chuàng)傷骨科雜志;2004年01期

4 鞠秀麗,黃志偉,時(shí)慶,侯懷水,段春紅;體外擴(kuò)增臍血間充質(zhì)干細(xì)胞的生物學(xué)特性和誘導(dǎo)分化能力的研究[J];中華兒科雜志;2005年07期

5 干耀愷;戴\戎;張蒲;湯亭亭;郝永強(qiáng);唐堅(jiān);史定偉;王友;孫月華;朱振安;;應(yīng)用富集骨髓干細(xì)胞技術(shù)治療骨缺損[J];中華骨科雜志;2006年11期

6 金丹,陳濱,裴國(guó)獻(xiàn),王珂,唐光輝,魏寬海;筋膜瓣促組織工程骨再血管化及山羊長(zhǎng)段骨缺損的修復(fù)[J];中華實(shí)驗(yàn)外科雜志;2005年03期

7 馬云勝;羅美;劉樹(shù)輝;秦書(shū)儉;;人臍血基質(zhì)細(xì)胞生物學(xué)特性的實(shí)驗(yàn)研究[J];中國(guó)臨床解剖學(xué)雜志;2009年02期

8 蘇云;孟祥俊;孫強(qiáng);于小光;徐爽;;BMP/VEGF/PLGA復(fù)合物促進(jìn)兔骨折愈合的實(shí)驗(yàn)研究[J];中國(guó)老年學(xué)雜志;2009年23期

9 李建軍;韓冬;孫鴻斌;王歡;徐莘香;;基因修飾的生物可降解人工骨修復(fù)骨缺損的血管化研究[J];中國(guó)修復(fù)重建外科雜志;2006年09期

10 侯天勇;羅飛;劉杰;邊巴羅布;許建中;;凍干組織工程骨與組織工程骨成骨活性比較研究[J];中國(guó)修復(fù)重建外科雜志;2010年07期

本文編號(hào)：1594607