摘要
目的:观察由骨髓间充质干细胞诱导的内皮细胞与自体骨髓间充质干细胞共培养时对骨髓间充质干细胞成骨作用的影响。方法:实验于2005-03/11在复旦大学附属中山医院外科中心实验室完成。①使用密度梯度离心法分离兔骨髓间充质干细胞。②取传两三代细胞分组分别进行如下操作:骨髓间充质干细胞组仅应用基础培养液(DMEM-LG培养基、体积分数为0.1的胎牛血清)常规培养;骨髓间充质干细胞成骨诱导组在基础培养液中加入成骨诱导剂;内皮细胞诱导组在内皮细胞生长培养基作用下将骨髓间充质干细胞诱导为内皮细胞;诱导的骨髓间充质干细胞与内皮细胞联合培养组将骨髓间充质干细胞和诱导而来的内皮细胞按1∶1的比例接种于培养板中,加入成骨诱导培养液。③通过干细胞形态、免疫荧光、细胞增殖、碱性磷酸酶活性、骨钙素含量等从酶学、组织学、生化等不同方面观察诱导的内皮细胞对骨髓间充质干细胞成骨活性及细胞生长情况的影响。结果:①免疫细胞荧光染色结果:内皮细胞诱导组培养诱导的细胞成为内皮细胞。②倒置相差显微镜、苏木精-伊红染色结果:均显示联合培养组骨髓间充质干细胞和诱导的内皮细胞混合生长良好。③四甲基偶氮唑盐法检测结果:各组细胞增殖差异无显著性(P>0.05)。④碱性磷酸酶活性:除内皮细胞诱导组外,其他3组细胞的活性均随时间的延长而逐渐增高,联合培养组增长最明显,第12天时联合培养组细胞碱性磷酸酶活性已达到(618.46±28.34)nkat/L,高于其他各组(P<0.05)。⑤骨钙素检测结果:在培养的第7天、第14天,联合培养组的骨钙素分泌量分别为2.03,3.17μg/L,明显高于其他各组(P<0.01)。结论:由骨髓间充质干细胞诱导的内皮细胞能够增强骨髓间充质干细胞的成骨活性,提高骨髓间充质干细胞的增殖能力。
AIM： To study the influence of co-culture of endothelial cell and autologous marrow mesenchymal stem cells （MSCs） induced by MSCs on osteogenic ability of MSCs.
METHODS： The experiment was conducted in the Central Laboratory of Department of Surgery, Zhongshan Hospital, Fudan University from March to November 2005. ①Marrow MSCs were obtained by gradient centrifuge method. ②The adhesive cells were preserved to passage culture and divided into MSCs group cultured in basic culture fluid （DMEM-LG medium containing 0.1 volume fraction FBS）; induced MSCs group cultured in medium containing osteogenic inductor; induced endothelial cells group, in which the MSCs were induced into endothelial cells based on endothelial cell medium; and co-cultured induced IMSCs and induced endothelial cells group, in which the MSCs and induced endothelial cells were s.eed in culture plate at a ratio of 1：1 and the osteogenic inductor was added. ③The effect of induced endothelial cells on MSCs, osteogenic potential and cellular compatibility were determined by enzymology, histology, biochemistry such as cell morphology, cytoimmunochemical staining, cell proliferation, alkaline phosphatase （ALPase） activity, osteocalcin synthesis and so on. RESULTS： ① Result of cytoimmunochemical staining： The MSCs of induced endothelial cells group were induced to endothelial cells. ② Results of inverted microscope and HE staining： The cellular compatibility of MSCs and endothelial cells was good. ③Results of MTT： There was no significant difference in proliferation among each group （P 〉 0.05）. ④ALP activity： Except the induced endothelial cells group, the activity in other groups were all gradually increased during culture, especially the co-cultured group, in which the ALP activity was increased to （618.46±28.34） nkat/L at the 12^th day, significantly higher than other group （P 〈 0. 05）. ⑤Results of osteocalcin synthesis： The osteocalcin content of the co-cultured group at the 7^th and 14^th day was 2.03 and 3.17 μg/L, respectively, which was obviously higher than any other group （P 〈 0. 01）.
CONCLUSION： The endothelial cells induced by MSCs can increase the osteogenic activity and proliferated ability of MSCs.
出处
《中国临床康复》
CSCD
北大核心
2006年第33期68-71,i0004,共5页
Chinese Journal of Clinical Rehabilitation
基金
国家自然科学基金资助项目(30271262)
教育部留学回国基金资助项目2003(406)~~
