P物质对骨髓基质干细胞来源的成骨细胞与内皮细胞体外联合培养体系的影响  

Effect of substance P on co-cultured osteoblasts and vascular endothelial cells induced from bone marrow stromal cells in rabbits

作　　者：李富航[1] 靳宇飞[1] 毕龙[1] 樊俊俊[1] 户刚[1] 裴国献[1] 

机构地区：[1]第四军医大学西京医院骨科,西安710032

出　　处：《中华创伤骨科杂志》2014年第5期427-432,共6页Chinese Journal of Orthopaedic Trauma

基　　金：国家自然科学基金（81272011）;国家自然科学基金青年项目（81301569）

摘　　要：目的 探讨P物质对骨髓基质干细胞（BMSCs）来源的成骨细胞（OB）与内皮细胞（EC）体外联合培养的影响. 方法 分别建立BMSCs来源的OB与EC单独培养体系,以及按2∶1的比例体外直接或间接共培养体系.实验组：各培养体系细胞内同时加入1＆#215;10-8 mol/L P物质（n=6）,对照组：各培养体系不添加P物质（n=6）.培养48 h后采用流式细胞仪分析细胞的增殖与活性,并采用酶联免疫吸附法测定碱性磷酸酶（ALP）的表达,实时定量聚合酶链式反应检测骨钙素（OC）mRNA基因的表达.比较两组不同培养方式细胞功能的变化. 结果 与对照组比较,实验组OB单独培养、间接共培养时细胞G1期减少,G2/M期明显增高,差异均有统计学意义（P＜0.05）.实验组OB单独培养、间接共培养、直接共培养的细胞ALP增殖活性[（2.877 ±0.188）、（3.053±0.179）、（4.345±0.305）]及OCmRNA的表达量[（1.682±0.155）、（3.188±0.213）、（3.720 ±0.194）]均明显高于对照组[（2.600±0.233）、（2.842±0.120）、（3.220±0.191）;（1.360±0.141）、（2.272±0.143）、（2.507 ±0.176）],差异均有统计学意义（P＜0.05）;而EC单独培养的ALP增殖活性和OC mRNA的表达量两组间比较差异均无统计学意义（P＞0.05）.实验组间接共培养、直接共培养的ALP增殖活性及OC mRNA的表达量较OB单独培养明显增高,且直接共培养较间接共培养也有所提高,差异均有统计学意义（P＜0.05）. 结论 BMSCs来源的OB与EC共培养细胞相容性良好,P物质可显著促进共培养体系中OB的增殖与活性.Objective To study the effect of substance P （SP） on the co-cultured osteoblasts （OB） and endothelial cells （EC） induced from bone marrow stromal cells （BMSCs） in vitro in rabbits.Methods Four culture systems （OB,EC,indirect and direct co-culture of OB and EC at the ratio of 2∶ 1） were respectively established.Of each system,the experimental group was simultaneously added with 1 ＆#215; 10-8mol/L SP （n =6） while the control was not.After culture for 48 hours,the cell cycles were analyzed by flow cytometry to detect the cell proliferation and activity; the expression of ALP （Alkaline phosphatase） was measured by ELISA; the mRNA expression of OC （osteocalcin） gene in osteoblasts was detected by real-time quantitative PCR.The changing of cell function was compared between the 2 groups in each cell culture system.Results In systems of mere OB culture and indirect co-culture,the cells were significantly decreased at phase G1 and significantly increased at phase G2/M in the experimental groups compared with the controls （P 〈 0.05）.In systems of mere OB culture and indirect and direct co-cuhures,the experimental groups had significantly higher ALP expression （2.877 ±0.188,3.053 ±0.179 and 4.345 ±0.305） and mRNA expression of OC （1.682 ±0.155,3.188 ± 0.213 and 3.720 ± 0.194） than the controls respectively （2.600±0.233,2.842 ±0.120 and 3.220 ±0.191; 1.360 ±0.141,2.272 ±0.143 and 2.507 ±0.176） （P 〈 0.05）.In the system of mere EC culture,there were no significant differences in ALP expression or mRNA expression of OC between the 2 groups （P 〉 0.05）.In the experimental groups,indirect and direct co-cultures produced significantly higher ALP expression and mRNA expression of OC than mere OB culture,and so did the direct co-culture than the indirect co-culture （P 〈 0.05）.Conclusions There is good cellular compatibility between the endothelial cells and osteoblasts induced from BMSCs when the 2 kinds of cells are co-cultured.SP can significantly

关 键 词：骨髓细胞 P物质 成骨细胞 内皮细胞 共同培养技术 

分 类 号：R683[医药卫生—骨科学]