中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2007年
50期
10181-10184
,共4页
刘斌钰%李宁毅%樊功为%袁荣涛%金晓明%陈立强
劉斌鈺%李寧毅%樊功為%袁榮濤%金曉明%陳立彊
류빈옥%리저의%번공위%원영도%금효명%진립강
骨髓间充质干细胞%诱导分化%细胞培养%鉴定
骨髓間充質榦細胞%誘導分化%細胞培養%鑒定
골수간충질간세포%유도분화%세포배양%감정
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120～150 g,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5×107 L-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,9 d后开始密集重叠生长,21～28 d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21～28 d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.
揹景:許多分離純化骨髓間充質榦細胞操作繁瑣、費用昂貴,且對細胞的活性影響較大,許多研究都緻力于尋找有效且價格低廉的培養鑒定方法.目的:採用全骨髓貼壁培養法對大鼠骨髓間充質榦細胞經體外進行成骨誘導和分化,併進行細胞鑒定.設計:觀察對比實驗.單位:青島大學醫學院.材料:實驗于2005-11/2007-03在青島大學醫學院口腔研究室及分子生物學實驗室完成,選用20隻生後三四週Wistar大鼠,SPF級,雌雄不拘,體質量120～150 g,由青島市實驗動物中心提供.實驗過程中對動物的處置符閤動物倫理學標準.胎牛血清購自杭州四季清生物技術有限公司,堿性燐痠酶檢測試劑盒由南京建成生物工程研究所提供,逆轉錄試劑盒為PROMEGA產品,引物由上海生工公司閤成.方法:採用全骨髓培養法分離培養成年大鼠骨髓間充質榦細胞,用2.5 g/L的胰蛋白酶消化後分瓶,以5×107 L-1的密度接種于6孔培養闆,誘導分化組加入誘導分化培養液,對照組加入等量基礎培養液培養.①倒置相差顯微鏡觀察細胞誘導分化結果及鈣結節形成情況.②採用鈣結節Von Kossa染色、鈣結節茜素紅染色進行誘導後細胞的生物學特性檢測.③採用重氮鹽法染色觀察堿性燐痠酶活性.④RT-PCR檢測細胞內成骨細胞轉錄因子、骨鈣素、成骨細胞特異性基因mRNA的錶達.主要觀察指標:①細胞誘導分化結果.②大鼠骨髓間充質榦細胞誘導後細胞的生物學特性.③堿性燐痠酶活性.④成骨細胞轉錄因子、骨鈣素、成骨細胞特異性基因mRNA的錶達.結果:①誘導分化組加入誘導分化培養液後,9 d後開始密集重疊生長,21～28 d齣現較多散在的緻密圓形礦化結節.對照組細胞雖密集重疊生長,但不形成礦化結節.②誘導分化組成骨誘導21～28 d形成明顯的圓形或卵圓形肉眼可見的鈣化結節.Von Kossa染色為黑色沉澱,茜素紅染色為橙紅色結節狀,對照組未見鈣結節形成.③誘導分化組誘導2週細胞堿性燐痠酶活性明顯增高,對照組活性較弱.④誘導分化組經誘導後成骨細胞轉錄因子、骨鈣素、成骨細胞特異性基因mRNA的錶達均較彊.結論:大鼠的骨髓間充質榦細胞經全骨髓培養法體外誘導和分化,錶現齣與典型的成骨細胞相似的形態特徵和生物學特性.
배경:허다분리순화골수간충질간세포조작번쇄、비용앙귀,차대세포적활성영향교대,허다연구도치력우심조유효차개격저렴적배양감정방법.목적:채용전골수첩벽배양법대대서골수간충질간세포경체외진행성골유도화분화,병진행세포감정.설계:관찰대비실험.단위:청도대학의학원.재료:실험우2005-11/2007-03재청도대학의학원구강연구실급분자생물학실험실완성,선용20지생후삼사주Wistar대서,SPF급,자웅불구,체질량120～150 g,유청도시실험동물중심제공.실험과정중대동물적처치부합동물윤리학표준.태우혈청구자항주사계청생물기술유한공사,감성린산매검측시제합유남경건성생물공정연구소제공,역전록시제합위PROMEGA산품,인물유상해생공공사합성.방법:채용전골수배양법분리배양성년대서골수간충질간세포,용2.5 g/L적이단백매소화후분병,이5×107 L-1적밀도접충우6공배양판,유도분화조가입유도분화배양액,대조조가입등량기출배양액배양.①도치상차현미경관찰세포유도분화결과급개결절형성정황.②채용개결절Von Kossa염색、개결절천소홍염색진행유도후세포적생물학특성검측.③채용중담염법염색관찰감성린산매활성.④RT-PCR검측세포내성골세포전록인자、골개소、성골세포특이성기인mRNA적표체.주요관찰지표:①세포유도분화결과.②대서골수간충질간세포유도후세포적생물학특성.③감성린산매활성.④성골세포전록인자、골개소、성골세포특이성기인mRNA적표체.결과:①유도분화조가입유도분화배양액후,9 d후개시밀집중첩생장,21～28 d출현교다산재적치밀원형광화결절.대조조세포수밀집중첩생장,단불형성광화결절.②유도분화조성골유도21～28 d형성명현적원형혹란원형육안가견적개화결절.Von Kossa염색위흑색침정,천소홍염색위등홍색결절상,대조조미견개결절형성.③유도분화조유도2주세포감성린산매활성명현증고,대조조활성교약.④유도분화조경유도후성골세포전록인자、골개소、성골세포특이성기인mRNA적표체균교강.결론:대서적골수간충질간세포경전골수배양법체외유도화분화,표현출여전형적성골세포상사적형태특정화생물학특성.
BACKGROUND: Many operations for isolating, purifying and identifying bone marrow mesenchymal stem calls (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.SETTING: 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 ×107 L-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: ① Induction and differentiation results of cells. ② Biological characteristics of cells induced by rat BMSCs. ③ ALP 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.
