摘要
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 operations for isolating, purifying and identifying bone marrow mesenchymal stem cells （BMSCs） are complicated and cost much. Also they have great effect on cell activity. Whether whole bone marrow adherent culture can avoid above-mentioned disadvantages remains unclear. At present, many studies huve been done to confirm an effective and low cost method for isolating, purifying and identifying such cells.
OBJECTIVE： This study is to in vitro induce and differentiate rat BMSCs by whole bone marrow adherent culture and to identify the cells.
DESIGN： A controlled observational experiment
SEI-FING： Qingdao University Medical College
MATERIALS： This study was carried out in the Laboratory of Oral Cavity and Laboratory of Molecular Biology （provincial level） Qingdao University Medical College between November 2005 and March 2007. Twenty Wistar rats of either gender, aged 3 to 4 weeks, of SPF grade, weighing 120-150 g, were provided by the Qingdao Laboratory Center. The protocol was carried out in accordance with animal ethics guidelines for the use and care of animals. Fetal bovine serum （FBS, Hangzhou Sijiqing Bioengineering Material Research Institute）, alkaline phosphatase （ALP） kit （Nanjing Jiancheng Bioengineering Research Institute）, reverse transcription kit （American Promega Corporation） and primer （Shanghai Bioengineering Co.,Ltd.） were used in this study.
METHODS： Adult rat BMSCs were isolated and cultured by whole bone marrow adherent culture, They were digested with 2.5 g/L trypse and inoculated at a density of 5 ×10^7L^-1 in 6-well culture plate. Then, the cells were divided into experimental group and control group. Inducing culture medium was added to experimental group, and the same amount of basic culture medium was added to control group, ① Cell differentiation and calcium tuberculation were observed under the inverted microscope. ② Biological characteristics of induced cells were detected by calcium tubercle Von Kossa and alizarin Bordeaux. ③ALP activity was detected by diazo salt staining. ④Human core binding factor alpha subunit-1 （Cbf α-1）, osteocalcin （OCN） and osteoblast-specific Osterix （OSX） mRNA expressions were detected by reverse transcription-polymerase chain reaction （RT-PCR）.
MAIN OUTCOME MEASURES： ①Induction and differentiation results of cells. ②Biological characteristics of cells induced by rat BMSCs, ③ALP activity. ④ Cbf α-1, OCN and OSX expressions.
RESULTS： ①Inducing culture medium was added in the serial subcultivation, About 9 days later, cell clones were connected to each other. On about 21 to 28 days, some pykno-round mineralized tubercles appeared. Meanwhile, control cells were connected to each other, but they did not form the tubercle. ② In the experimental group, when MSCs were induced for 21 to 28 days, obvious round or oval calcified tubercles were seen by naked eyes. The results of Von Kossa staining exhibited black sediments, and those of alizarin Bordeaux staining exhibited salmon tubercles. Calcium tubercles were not found in the control group. ③The ALP activity after 2 weeks of induction was obviously increased in the experimental group, but was relatively weak in the control group, ④In the experimental group, Cbf α-1, OCN and OSX expressions were significantly increased after induction.
CONCLUSION： After being in vitro induced and differentiated by whole bone marrow adherent culture, rat BMSCs exhibited morphological and biological characteristics similar to typical osteoblasts.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第50期10181-10184,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金(30471899)~~
