【作者】 才晓君；

【导师】 季晓平；

【作者基本信息】 山东大学 ， 内科学， 2005， 硕士

【摘要】 目的:探讨大鼠自体骨髓间充质干细胞在体外分离、培养方法,观察骨髓间充质干细胞的生物学特性;探讨对培养的骨髓间充质干细胞进行体外药物定向诱导,使其分化为心肌样细胞的方法及心肌样细胞的鉴定方法。 方法:共选取wistar雄性大鼠15只,体重180±20g,分为两组,一组7只,用Percoll分离培养法分离、培养;另一组8只,用全骨髓培养法分离、培养。15只大鼠均腹腔内注射戊巴比妥钠（30 mg/kg）麻醉,无菌条件下断离股骨,一组7只用PBS缓沖洗股骨干及干骺端,然后用Percoll液（密度为1.073g/ml）进行密度梯度离心后,取分界面的浑浊层,.用DMEM洗细胞2遍,接种到一次性塑料培养瓶中培养;另一组8只用DMEM培养液冲洗股骨干及干骺端,接种于培养瓶中,贴壁分离出骨髓间充质干细胞。将骨髓间充质干细胞混入培养体系（DMEM+15%特等优质胎牛血清+青霉素、链霉素各100 μg/ml;5%CO₂,37℃）,扩增。待原代培养的细胞增殖到80%时,用0.25%牛胰蛋白酶消化细胞使之成为单细胞悬液,进行传代培养。 取生长良好的第三代骨髓间充质干细胞,胰酶消化后加入5-氮胞苷5μmol/L作用24小时,按正常培养条件培养,细胞长满培养瓶时（约5-6d）1:2.5传代,传代后进行同样的诱导、培养,倒置显微镜观察各期细胞形态的变化和生长发育情况。利用免疫荧光术、透射电镜等技术对诱导后的细胞进行鉴定。 结果:全骨髓培养法较Percoll密度梯度离心法培养的骨髓间充质干细胞纯山东大学硕士学位论文度高,细胞增殖能力强,在体外培养时细胞均一性较好,方法简单易行;spmol/L的5一氮胞昔为诱导骨髓间充质干细胞向心肌样细胞分化较为适合的浓度;免疫荧光可见a一Aetin,eardiae troponinl（eTnl）阳性细胞,透射电镜观察到部分细胞形成肌丝样结构,细胞中可见到丰富的线粒体和糖原颗粒。 结论:1全骨髓培养法是一种简便易行、有效分离骨髓间充质干细胞的方法;2 spmol/L的5一氮胞营为诱导骨髓间充质干细胞向心肌样细胞分化较为适合的浓度;3骨髓间充质干细胞具有多能分化特性,体外培养的骨髓间充质干细胞可在5一氮胞昔诱导下具有心肌细胞结构特点的心肌样细胞分化,为心肌细胞移植打下坚实的细胞基础。关键词:骨髓间充质干细胞、心肌细胞、全骨髓培养法、细胞分化、5一氮胞普、免疫荧光术更多还原

【Abstract】 Objective: To investigate the method of rat mesenchymal stem cells separated and cultured in vitro and to observe the biologic characteristics;To investigate the method by which rat mesenchymal stem cells can be induced into cardiomyocytes and the method which can identified them.Method: 15 Wistar rats,male,weighting 180±20g were separated into two groups randomly.One group is 7 rats,another is 8 rats.All of the rats were anesthetized with pentobarbital(30mg/kg body weight)by anintraperitoneal injection.Then the rats were come off the thighbones . The stem and epiphysis of thigbones of 7 rats were rinsed with PBS ,then rMSCs were isolated by Percoll separating medium(1.073g/ml) and cultured in culture bottles.Another 8 rats were rinsed with DMEM of 15% FBS,100ug/ml penicillin and 100ug/ml streptomycin.Finally,the rinsed water were inoculated in culture bottles and were cultured in vitro,observing rMSCs biologic characteristics.Then rMSCs were induced by 5-Aza 5umol/L for 24 hours.We identified that some of the induced rMSCs had differentiated into cardiomyocytes by means of transmission electron microscopy technique,immunofluorescent technique.Results: rMSCS isolated by the whole bone marrow culture had higher purity .better uniform and ability of proliferation in the process of culture in vitro than those of Percoll isolated method.5umol/L 5-Aza is an optimal concentration for rMSCs to differentite into cardiomyocytes.Some of the induced rMSCs were positive expression of a-Actin,cardiac troponinl (cTnl).Electron microscopy revealedmyofilament-like structure,rich glycogen granules and anumber of mitochondria.Conclusion: 1 The method of whole bone marrow culture is a well-referenced medium for density gradient centrifugation of rMSCs;2 The optimal conditions for rMSCs to differentiated into cardiomyocytes is 5umol/L 5-Aza;3 Mscs are mutipotential stem cells and they can be induced into cardiomyocytes by 5-Aza in vitro which have the characterestics of cardie myocytes in structure.更多还原