不同比例血管内皮细胞和骨髓间充质干细胞共培养系统成骨性能研究  

Effect of different proportions of bone marrow mesenchymal stem cells and endothelial cells on osteogenesis

作　　者：刘蓉[1] 刘江峰[1] 郭吕华[1] 田爱峰[1] 

机构地区：[1]广州医科大学附属口腔医院·广州口腔病研究所·口腔医学重点实验室,510140

出　　处：《中华口腔医学杂志》2015年第11期675-680,共6页Chinese Journal of Stomatology

基　　金：广东省技术研发与升级改造专项资金项目计划（20138021800278）

摘　　要：目的 观察血管内皮细胞与骨髓间充质干细胞共培养时,不同比例共培养系统的成骨性能,为体外构建血管化骨组织工程奠定基础.方法 全骨髓离心贴壁法分离大鼠骨髓间充质干细胞.骨髓间充质干细胞的内皮向诱导（M199＋体积分数为0.1的胎牛血清＋体积分数为0.01的青霉素/链霉素＋2 μg/L的碱性成纤维细胞生长因子bFGF＋10 μg/L的血管内皮生长因子VEGF）.建立不同比例骨髓间充质干细胞和血管内皮细胞的共培养系统（10∶0、10∶1、8∶2、7∶3、5∶5、3∶7、2∶8、1∶10、0∶10）.通过干细胞和内皮细胞的形态、免疫荧光、碱性磷酸酶活性、成骨基因表达等从酶学、组织学、基因等不同方面观察各个比例血管内皮细胞对骨髓间充质干细胞成骨活性的影响.结果 第3代骨髓间充质干细胞的生长曲线显示：骨髓间充质干细胞的细胞倍增时间为39.9 h.免疫荧光染色结果显示,骨髓间充质干细胞表面特异性标志、内皮细胞表面特异性标志免疫荧光染色阳性.倒置相差显微镜观察结果显示,联合培养组不同比例的骨髓间充质干细胞和血管内皮细胞混合生长良好.共培养组茜素红染色结果显示,共培养比例为7∶3时钙结节计数为19.0±3.0,共培养比例为5∶5时钙结节计数为20.8±2.9,两者之间差异无统计学意义（P＞0.05）;但均显著高于其他共培养组（P＜0.01）.成骨诱导7d,共培养比例为10∶0、10∶1、8∶2、7∶3和5∶5共培养组酶活性分别为：16.84±0.82、15.86±3.10、16.37±1.33、17.99±1.98和17.49±0.87,差异无统计学意义（P＞0.05）,但均显著高于其他共培养组（P＜0.05）;随着诱导时间延长,碱性磷酸酶活性整体升高,7：3共培养组（33.74±0.99）略高于5∶5共培养组（31.09±0.87）,二者均显著高于其他共培养组（P＜0.01）.定量PCR检测结果显示,7∶3共培养组成骨相关基因OCN和RUNX2的表达均显著高于其他Objective To evaluate the effect of co-culture system of bone marrow mesenchymal stem cells（BMSC） and vascular endothelial cells（EC） on osteogenesis.Methods BMSC were isolated by whole bone marrow centrifugal adherent method.Then BMSC were induced into EC with induced medium.Co-culture system in different proportions of BMSC and EC（10∶0, 10∶1, 8∶2, 7∶3, 5∶5, 3∶7, 2∶8, 1∶10, 0∶10） were further evaluated.The cell growth level of BMSC was examined.The CD44 expression of BMSC and von willebrand factor（vWF） expression of vascular EC were examined by immunofluorescence.Furthermore,calcium nodules exhibited by alizarin red staining, alkaline phosphatase activity, and the expression of osteogenic genes by reverse transcription-quantitative PCR（RT-qPCR） were observed to validate the osteogenesis of co-culture system.Results The growth curve of P3 passage of BMSC demonstrated the doubling time of BMSC was 39.9 h.The positive specific markers of BMSC and EC showed efficient induction.Although the calcium nodules ratio of the co-culture[group 7∶3（19.0±3.0） and group 5∶5（20.8±2.9）] was not significantly different（P〉0.05）, but higher than that of other co-culture groups with a significant difference（P〈0.01）.Alkaline phosphatase activity was increased with prolonged induction of osteogenic medium.While alkaline phosphatase activity of group 10： 0（16.84±0.82）, group 10∶1（15.86±3.10）, group 8∶2（16.37± 1.33）, group 7∶3（17.99± 1.98）, and group 5∶5（17.49±0.87） did not show significant difference after osteogenic induction for 7 days（P〉0.05）, but significantly higher than that of other co-culture groups（P〈0.05）.The co-culture ratio of 7∶3（33.74±0.99） was slightly higher than that of 5∶5 （31.09±0.87）, but significantly higher than that of other groups（P〈0.01）.Moreover, the osteocalcin（OCN） and runt-related transcription factor 2（RUNX2） expression of group 7∶3 was significantly higher than that of

关 键 词：骨髓间充质干细胞 内皮细胞 血管内皮生长因子 碱性成纤维细胞因子 共培养 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]