不同劑量TGF-β1在BMP9誘導(dǎo)間充質(zhì)干細(xì)胞成骨分化中的作用
發(fā)布時(shí)間:2018-04-09 15:08
本文選題:骨形成蛋白 切入點(diǎn):轉(zhuǎn)化成因子β 出處:《第三軍醫(yī)大學(xué)學(xué)報(bào)》2014年02期
【摘要】:目的分析聯(lián)合應(yīng)用不同劑量TGF-β1對(duì)BMP9誘導(dǎo)間充質(zhì)干細(xì)胞成骨分化過(guò)程的影響,尋找最佳的促進(jìn)BMP9成骨的TGF-β1劑量,為聯(lián)合不同細(xì)胞因子促進(jìn)組織工程骨再生尋找有效途徑。方法應(yīng)用鼠間充質(zhì)干細(xì)胞株C3H10T1/2,以重組腺病毒法將其導(dǎo)入BMP9,再分別施加0、5、10、20 ng/mL TGF-β1蛋白處理,建立BMP9聯(lián)合TGF-β1誘導(dǎo)間充質(zhì)干細(xì)胞成骨分化模型。條件培養(yǎng)基法獲取含活性良好的TGF-β1蛋白上清液;改良SEAP化學(xué)發(fā)光法和組織化學(xué)染色法檢測(cè)堿性磷酸酶(alkaline phosphatase,ALP)表達(dá)及活性;茜素紅S染色檢測(cè)鈣鹽沉積;實(shí)時(shí)熒光定量PCR和Western blot法分別檢測(cè)成骨相關(guān)基因Ⅰ型膠原(collagenⅠ,COLⅠ)、骨橋素(osteopontin,OPN)、骨鈣素(osteocalcin,OCN)的mRNA轉(zhuǎn)錄表達(dá)水平和蛋白表達(dá)水平改變;流式細(xì)胞儀檢測(cè)細(xì)胞周期;臺(tái)盼藍(lán)拒染法計(jì)數(shù)活細(xì)胞監(jiān)測(cè)細(xì)胞增殖。結(jié)果 5 ng/mL TGF-β1與BMP9聯(lián)合處理較BMP9單獨(dú)處理能更早、更多地提高ALP活性和引起鈣鹽沉積,持續(xù)處理17 d后鈣鹽沉積結(jié)果不再顯示明顯差異,TGF-β1單獨(dú)處理與對(duì)照組間無(wú)明顯差異。隨TGF-β1劑量增加,ALP活性和鈣鹽沉積效應(yīng)受到抑制。5 ng/mL TGF-β1與BMP9聯(lián)合處理較BMP9單獨(dú)處理使COLⅠ、OPN、OCN的mRNA轉(zhuǎn)錄水平和蛋白表達(dá)水平明顯增高,COLⅠ增高變化發(fā)生較早和顯著;TGF-β1應(yīng)用劑量達(dá)到20 ng/mL出現(xiàn)基因轉(zhuǎn)錄和蛋白表達(dá)受到抑制,但COLⅠ升高趨勢(shì)仍能維持。BMP9促進(jìn)細(xì)胞增殖,TGF-β1對(duì)該效應(yīng)起抑制作用,細(xì)胞周期轉(zhuǎn)化表現(xiàn)G0/G1期阻滯。結(jié)論在BMP9誘導(dǎo)間充質(zhì)干細(xì)胞成骨分化中,適量的TGF-β1聯(lián)合應(yīng)用具有促進(jìn)作用;BMP9與TGF-β1在誘導(dǎo)成骨分化中可能存在協(xié)同作用關(guān)系。
[Abstract]:Objective to investigate the effects of different doses of TGF- 尾 1 on the osteogenic differentiation of mesenchymal stem cells (MSCs) induced by BMP9, to find the best dose of TGF- 尾 1 for promoting bone regeneration in tissue engineering with different cytokines.Methods the mouse mesenchymal stem cell line C3H10T1 / 2 was introduced into BMP9 by recombinant adenovirus method, and then treated with 0H101020 ng/mL TGF- 尾 1 protein respectively. The osteogenic differentiation model of mesenchymal stem cells induced by BMP9 combined with TGF- 尾 1 was established.The supernatant of TGF- 尾 1 protein with good activity was obtained by conditioned medium method, the expression and activity of alkaline phosphatase (ALP) were detected by modified SEAP chemiluminescence assay and histochemical staining, and calcium salt deposition was detected by alizarin red S staining.Real-time fluorescence quantitative PCR and Western blot were used to detect the changes of mRNA transcription and protein expression of osteoblast-associated gene type 鈪,
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