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背景:许多分离纯化骨髓间充质干细胞操作繁琐、费用昂贵,且对细胞的活性影响较大,许多研究都致力于寻找有效且价格低廉的培养鉴定方法。目的:采用全骨髓贴壁培养法对大鼠骨髓间充质干细胞经体外进行成骨诱导和分化,并进行细胞鉴定。设计:观察对比实验。单位:青岛大学医学院。材料:实验于2005-11/2007-03在青岛大学医学院口腔研究室及分子生物学实验室完成,选用20只生后三四周Wistar大鼠,SPF级,雌雄不拘,体质量120～150g,由青岛市实验动物中心提供。实验过程中对动物的处置符合动物伦理学标准。胎牛血清购自杭州四季清生物技术有限公司,碱性磷酸酶检测试剂盒由南京建成生物工程研究所提供,逆转录试剂盒为PROMEGA产品,引物由上海生工公司合成。方法:采用全骨髓培养法分离培养成年大鼠骨髓间充质干细胞,用2.5 g/L的胰蛋白酶消化后分瓶,以5×10~7L~(-1)的密度接种于6孔培养板,诱导分化组加入诱导分化培养液,对照组加入等量基础培养液培养。①倒置相差显微镜观察细胞诱导分化结果及钙结节形成情况。②采用钙结节Von Kossa染色、钙结节茜素红染色进行诱导后细胞的生物学特性检测。③采用重氮盐法染色观察碱性磷酸酶活性。④RT-PCR检测细胞内成骨细胞转录因子、骨钙素、成骨细胞特异性基因mRNA的表达。主要观察指标:①细胞诱导分化结果。②大鼠骨髓间充质干细胞诱导后细胞的生物学特性。③碱性磷酸酶活性。④成骨细胞转录因子、骨钙素、成骨细胞特异性基因mRNA的表达。结果:①诱导分化组加入诱导分化培养液后,9d后开始密集重叠生长,21～28d出现较多散在的致密圆形矿化结节。对照组细胞虽密集重叠生长,但不形成矿化结节。②诱导分化组成骨诱导21～28d形成明显的圆形或卵圆形肉眼可见的钙化结节。Von Kossa染色为黑色沉淀,茜素红染色为橙红色结节状,对照组未见钙结节形成。③诱导分化组诱导2周细胞碱性磷酸酶活性明显增高,对照组活性较弱。④诱导分化组经诱导后成骨细胞转录因子、骨钙素、成骨细胞特异性基因mRNA的表达均较强。结论:大鼠的骨髓间充质干细胞经全骨髓培养法体外诱导和分化,表现出与典型的成骨细胞相似的形态特征和生物学特性。
BACKGROUND: Many isolated and purified bone marrow mesenchymal stem cells are cumbersome, expensive and have a large impact on cell activity. Many studies have been devoted to finding effective and inexpensive culture identification methods. OBJECTIVE: To induce osteogenic differentiation and differentiation of rat bone marrow mesenchymal stem cells in vitro using whole bone marrow adherence culture method and identify cells. Design: observe the contrast experiment. Unit: Qingdao University School of Medicine. MATERIALS: The experiment was performed at the Laboratory of Oral Laboratory and Molecular Biology, Qingdao University Medical College from November 2005 to March 2007 with 20 Wistar rats of three weeks and three weeks after birth. SPF was used in both male and female. The body weight was 120 ~ 150g , Provided by Qingdao Experimental Animal Center. The handling of animals during the experiment is in line with animal ethics standards. Fetal bovine serum was purchased from Hangzhou Sijiqing Biotechnology Co., Ltd. Alkaline phosphatase detection kit was provided by Nanjing Jiancheng Institute of Bioengineering. The reverse transcription kit was PROMEGA, and the primers were synthesized by Shanghai Biotechnology Co., Ltd. Methods: Adult rat bone marrow mesenchymal stem cells were isolated and cultured by whole bone marrow culture. After digestion with 2.5 g / L trypsin, the cells were seeded into 6-well plates at a density of 5 × 10 ~ 7L ~ (-1) The induced differentiation group was added into the induced differentiation medium and the control group was added with the same amount of basal medium. ① inverted phase contrast microscope to observe the results of cell differentiation and calcium formation. ② Using calcium nodules Von Kossa staining, calcium nodular alizarin red staining of cells after the biological characteristics of the test. ③ using the diazo method staining alkaline phosphatase activity. ④ RT-PCR detection of intracellular osteoblast transcription factor, osteocalcin, osteoblast-specific gene mRNA expression. MAIN OUTCOME MEASURES: ① Cells induced differentiation. ② The biological characteristics of rat bone marrow mesenchymal stem cells after induction. ③ alkaline phosphatase activity. ④ osteoblasts transcription factor, osteocalcin, osteoblast-specific gene mRNA expression. Results: ① After induction and differentiation, the supernatant grew intensively and overlaid after 9 days, and more dense and round mineralized nodules appeared in 21 ~ 28 days. Although the control group of cells overlap intensive growth, but does not form mineralized nodules. ② induced differentiation of bone formation induced 21 ~ 28d to form a clear round or oval visible calcified nodules. Von Kossa stained black, alizarin red stained orange-red nodules, and no calcium nodules formed in the control group. (3) Alkaline phosphatase activity induced by differentiation group was significantly higher than that of control group at 2 weeks. ④ After inducing differentiation, the expression of osteoblast transcription factor, osteocalcin and osteoblast-specific gene mRNA were all strong. CONCLUSION: Rat bone marrow-derived mesenchymal stem cells are induced and differentiated in vitro by whole-bone culture and exhibit similar morphological and biological characteristics as typical osteoblasts.