本文選題：重組人骨形態(tài)發(fā)生蛋白2 + 重組人骨形態(tài)發(fā)生蛋白7��； 參考：《廣州醫(yī)學(xué)院》2010年碩士論文

【摘要】：目的:利用具有誘導(dǎo)成骨活性的重組人骨形態(tài)發(fā)生蛋白2(rhBMP-2)和重組人骨形態(tài)發(fā)生蛋白7(rhBMP-7)在體外誘導(dǎo)兔骨髓間充質(zhì)干細(xì)胞(BM-MSC)成骨,觀察BM-MSC的生長形態(tài)特征和增殖情況,并檢測相關(guān)的成骨指標(biāo),從而了解在外源生長因子的刺激下BM-MSC的生長形態(tài)特征及不同種類和濃度的rhBMP的體外誘導(dǎo)成骨能力,為后續(xù)利用骨髓間充質(zhì)干細(xì)胞進(jìn)行骨質(zhì)修復(fù)奠定實(shí)驗(yàn)基礎(chǔ)。 方法:抽取兔股骨骨髓,利用改進(jìn)的全骨髓貼壁細(xì)胞分離法從骨髓中分離出兔BM-MSC,體外培養(yǎng)擴(kuò)增,觀察其生長形態(tài)特征,并用含10ng/ml、50ng/ml、100ng/ml的rhBMP-2和rhBMP-7的培養(yǎng)液分別對BM-MSC進(jìn)行體外成骨誘導(dǎo),每天觀察其形態(tài)變化,并于第2、4、6、8天進(jìn)行細(xì)胞計(jì)數(shù),比較其增殖情況。同時(shí)在加入rhBMP后的第4、7、10、14、18天收集細(xì)胞培養(yǎng)上清液,定量檢測成骨指標(biāo)堿性磷酸酶和骨鈣素,同時(shí)設(shè)僅加基礎(chǔ)培養(yǎng)液培養(yǎng)的BM-MSC作為對照組。 結(jié)果: 1.采用改進(jìn)的全骨髓貼壁細(xì)胞分離法成功分離培養(yǎng)了兔BM-MSC。對傳2代BM-MSC進(jìn)行熒光免疫染色,CD44表達(dá)陽性,CD45表達(dá)陰性,對原代及傳4代的細(xì)胞用流式細(xì)胞儀進(jìn)行分析,CD29和CD44表達(dá)陽性,CD45表達(dá)陰性,與間充質(zhì)干細(xì)胞表型相符合。 2. BM-MSC在rhBMP-2和rhBMP-7的誘導(dǎo)下定向分化為成骨樣細(xì)胞。培養(yǎng)過程中,可見貼壁細(xì)胞逐漸由長梭形轉(zhuǎn)變?yōu)槎嘟切?出現(xiàn)細(xì)胞聚集,胞質(zhì)變深,含粗大顆粒。 3.與對照組比較,10ng/ml rhBMP-2、10ng/ml rhBMP-7和100ng/ml rhBMP-7組細(xì)胞數(shù)無明顯差別,對BM-MSC的增殖無促進(jìn)作用。而50ng/ml rhBMP-2、100ng/ml rhBMP-2和50ng/ml rhBMP-7組與對照組比較,細(xì)胞數(shù)明顯增多,對BM-MSC的增殖有著促進(jìn)作用,100ng/ml rhBMP-2對BM-MSC的增殖最強(qiáng)。 4.對收集的第4、7、10、14、18天的細(xì)胞培養(yǎng)上清液進(jìn)行成骨指標(biāo)ALP活性和OC含量的檢測,顯示100ng/ml的rhBMP-2的誘導(dǎo)成骨活性最高。 結(jié)論:通過改進(jìn)的全骨髓貼壁細(xì)胞分離培養(yǎng)法能獲得較高純度的具有間充質(zhì)細(xì)胞表型的兔BM-MSC,以此獲得的BM-MSC在體外培養(yǎng)條件下可大量增殖。rhBMP-2和rhBMP-7具有促進(jìn)增殖和誘導(dǎo)成骨作用,可使BM-MSC定向向成骨細(xì)胞分化。這為后續(xù)應(yīng)用BM-MSC修復(fù)動物的牙骨缺損奠定了良好的實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Objective: to investigate the growth and proliferation of BM-MSC in rabbit bone marrow mesenchymal stem cells (BM-MSCs) induced by recombinant human bone morphogenetic protein (rhBMP-2) and recombinant human bone morphogenetic protein (rhBMP-7). In order to understand the morphological characteristics of BM-MSC under the stimulation of exogenous growth factors and the osteogenic ability of different kinds and concentrations of rhBMP in vitro, the related osteogenic indexes were detected. To lay an experimental foundation for bone repair with bone marrow mesenchymal stem cells. Methods: rabbit bone marrow was extracted from rabbit femur bone marrow. Rabbit BM-MSCs were isolated from bone marrow by modified whole bone marrow adherent cell separation method. The growth morphology of BM-MSCS was observed in vitro. Osteogenesis of BM-MSC was induced by rhBMP-2 and rhBMP-7 containing 10ng / ml 50ng / ml BM-MSC and 100ng / ml rhBMP-7, respectively. The morphological changes were observed every day, and the cell count was carried out on day 2, 4, 6 and 8 to compare the proliferation of the cells. At the same time, the supernatants of cell culture were collected on day 18 after adding rhBMP. The osteogenic indexes of alkaline phosphatase and osteocalcin were measured quantitatively, and the BM-MSC cultured in basic medium was used as control group. Results: 1. Rabbit BM-MSCs were successfully isolated and cultured by an improved whole bone marrow adherent cell separation method. The positive expression of CD44 and CD45 was detected by fluorescence immunostaining in the second generation of BM-MSC, and the positive expression of CD29 and CD44 in primary and fourth passage cells was detected by flow cytometry, which was consistent with the phenotype of mesenchymal stem cells (mesenchymal stem cells). 2. BM-MSC was induced by rhBMP-2 and rhBMP-7 to differentiate into osteoblast-like cells. During the culture process, adherent cells gradually changed from long fusiform to polygonal, cell aggregation, cell depth, including coarse particles. 3. Compared with the control group, there was no significant difference in the number of cells between 10ng / ml rhBMP-2ng / ml rhBMP-7 and 100ng/ml rhBMP-7 group, and there was no effect on the proliferation of BM-MSC. In the 50ng/ml rhBMP-2100ng/ml rhBMP-2 and 50ng/ml rhBMP-7 groups, the number of cells was significantly increased, and the proliferation of BM-MSC was promoted by 100ng / ml rhBMP-2. The proliferation of BM-MSC was strongest in the 50ng/ml rhBMP-2100ng/ml rhBMP-2 and 50ng/ml rhBMP-7 groups. 4. The activity of ALP and the content of OC in the supernatant of the culture supernatant of the cells collected on day 4, 7, 10, 14 and 18 were determined. The results showed that rhBMP-2 of 100ng/ml had the highest osteogenic activity. Conclusion: a high purity rabbit BM-MSC with mesenchymal cell phenotype can be obtained by the improved method of whole bone marrow adherent cell isolation and culture. The BM-MSC obtained by this method can promote proliferation and induce osteogenesis in a large amount under the condition of culture in vitro, rhBMP-2 and rhBMP-7 can promote proliferation and osteogenesis. BM-MSC can differentiate into osteoblasts. This provides a good experimental basis for the subsequent application of BM-MSC in the restoration of dental bone defects in animals.
【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

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