rhBMP-2和rhBMP-7诱导兔骨髓间充质干细胞成骨的实验研究

发布时间：2018-04-22 17:11

本文选题：重组人骨形态发生蛋白2 + 重组人骨形态发生蛋白7　；参考：《广州医学院》2010年硕士论文

【摘要】：目的:利用具有诱导成骨活性的重组人骨形态发生蛋白2(rhBMP-2)和重组人骨形态发生蛋白7(rhBMP-7)在体外诱导兔骨髓间充质干细胞(BM-MSC)成骨,观察BM-MSC的生长形态特征和增殖情况,并检测相关的成骨指标,从而了解在外源生长因子的刺激下BM-MSC的生长形态特征及不同种类和浓度的rhBMP的体外诱导成骨能力,为后续利用骨髓间充质干细胞进行骨质修复奠定实验基础。方法:抽取兔股骨骨髓,利用改进的全骨髓贴壁细胞分离法从骨髓中分离出兔BM-MSC,体外培养扩增,观察其生长形态特征,并用含10ng/ml、50ng/ml、100ng/ml的rhBMP-2和rhBMP-7的培养液分别对BM-MSC进行体外成骨诱导,每天观察其形态变化,并于第2、4、6、8天进行细胞计数,比较其增殖情况。同时在加入rhBMP后的第4、7、10、14、18天收集细胞培养上清液,定量检测成骨指标碱性磷酸酶和骨钙素,同时设仅加基础培养液培养的BM-MSC作为对照组。结果: 1.采用改进的全骨髓贴壁细胞分离法成功分离培养了兔BM-MSC。对传2代BM-MSC进行荧光免疫染色,CD44表达阳性,CD45表达阴性,对原代及传4代的细胞用流式细胞仪进行分析,CD29和CD44表达阳性,CD45表达阴性,与间充质干细胞表型相符合。 2. BM-MSC在rhBMP-2和rhBMP-7的诱导下定向分化为成骨样细胞。培养过程中,可见贴壁细胞逐渐由长梭形转变为多角形,出现细胞聚集,胞质变深,含粗大颗粒。 3.与对照组比较,10ng/ml rhBMP-2、10ng/ml rhBMP-7和100ng/ml rhBMP-7组细胞数无明显差别,对BM-MSC的增殖无促进作用。而50ng/ml rhBMP-2、100ng/ml rhBMP-2和50ng/ml rhBMP-7组与对照组比较,细胞数明显增多,对BM-MSC的增殖有着促进作用,100ng/ml rhBMP-2对BM-MSC的增殖最强。 4.对收集的第4、7、10、14、18天的细胞培养上清液进行成骨指标ALP活性和OC含量的检测,显示100ng/ml的rhBMP-2的诱导成骨活性最高。结论:通过改进的全骨髓贴壁细胞分离培养法能获得较高纯度的具有间充质细胞表型的兔BM-MSC,以此获得的BM-MSC在体外培养条件下可大量增殖。rhBMP-2和rhBMP-7具有促进增殖和诱导成骨作用,可使BM-MSC定向向成骨细胞分化。这为后续应用BM-MSC修复动物的牙骨缺损奠定了良好的实验基础。
[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.
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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