本文選題：骨形成蛋白　切入點：轉(zhuǎn)化成因子β　出處：《第三軍醫(yī)大學學報》2014年02期

【摘要】：目的分析聯(lián)合應(yīng)用不同劑量TGF-β1對BMP9誘導(dǎo)間充質(zhì)干細胞成骨分化過程的影響,尋找最佳的促進BMP9成骨的TGF-β1劑量,為聯(lián)合不同細胞因子促進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化學發(fā)光法和組織化學染色法檢測堿性磷酸酶(alkaline phosphatase,ALP)表達及活性;茜素紅S染色檢測鈣鹽沉積;實時熒光定量PCR和Western blot法分別檢測成骨相關(guān)基因Ⅰ型膠原(collagenⅠ,COLⅠ)、骨橋素(osteopontin,OPN)、骨鈣素(osteocalcin,OCN)的mRNA轉(zhuǎn)錄表達水平和蛋白表達水平改變;流式細胞儀檢測細胞周期;臺盼藍拒染法計數(shù)活細胞監(jiān)測細胞增殖。結(jié)果 5 ng/mL TGF-β1與BMP9聯(lián)合處理較BMP9單獨處理能更早、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單獨處理與對照組間無明顯差異。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單獨處理使COLⅠ、OPN、OCN的mRNA轉(zhuǎn)錄水平和蛋白表達水平明顯增高,COLⅠ增高變化發(fā)生較早和顯著;TGF-β1應(yīng)用劑量達到20 ng/mL出現(xiàn)基因轉(zhuǎn)錄和蛋白表達受到抑制,但COLⅠ升高趨勢仍能維持。BMP9促進細胞增殖,TGF-β1對該效應(yīng)起抑制作用,細胞周期轉(zhuǎn)化表現(xiàn)G0/G1期阻滯。結(jié)論在BMP9誘導(dǎo)間充質(zhì)干細胞成骨分化中,適量的TGF-β1聯(lián)合應(yīng)用具有促進作用;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，

本文編號：1726958