关于三维共培养体系中血管化的研究  

Experimental research of vascularization in three - dimensional co - cultured system

作　　者：杨逸禧 杨波[1] 娄爱菊[3] 张铮[5] 林启旺 何可人[4] 蔡梓红 尹飚[1] 黎双庆 杨富国[1] 郭元[2] 王簕[1] 

机构地区：[1]广州医科大学附属第三医院骨科,510150 [2]广州医科大学附属第三医院口腔科,510150 [3]广州医学院荔湾医院风湿科,510170 [4]广州医科大学临床医学系,510089 [5]第二炮兵总医院心内科,北京100088

出　　处：《中华实验外科杂志》2017年第11期1880-1884,共5页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金（31200726、31570980）;广州市市属高校科研项目（1201430092）;广东省公益研究与能力建设专项资金项目（2014A020212347）;广东省医学科研基金（B2014195）;广州医科大学学生校外科研项目（2013A022、2013A031、2013A032）;广东省科技厅社会发展领域项目（2014A020212355）;广东省研究生教育创新计划项目（2015SFKC30）

摘　　要：目的探讨人脐静脉内皮细胞（hUVECs）与人骨髓间充质干细胞（BMSCs）在三维共培养体系中成类血管网状结构的可行性。方法分别用绿色荧光蛋白和红色荧光蛋白转染第4代hUVECs和第4代人骨髓间充质干细胞，将标记后的细胞植入Ⅰ型鼠尾胶原于培养皿内共培养。添加含有体积分数为5%胎牛血清的内皮细胞培养基（ECM）1 ml。共培养2、4、7 d及单纯细胞培养7 d后用激光扫描共聚焦显微镜观察两种细胞相互成网的状态及其两者生长情况。通过MetaXpress软件系统中血管生成分析模块（Angiogenesis）计算出生成的类血管网状结构的长度值，并利用DNA萃取法分析细胞的活力与增殖水平，反转录-聚合酶链反应（RT-PCR）检测与Western blot分析血管内皮生长因子（VEGF） mRNA水平及VEGF蛋白水平。结果共培养2 d后BMSCs明显开始拉伸，呈长梭状，细胞之间无聚集趋势；hUVECs拉伸情况不明显，仅有少数细胞有轻微拉伸。4 d后BMSCs持续拉伸状态并有明显聚集趋势，hUVECs拉伸情况明显并有开始有聚集趋势，但两种细胞之间联系不明显，可见类血管状结构形成。7 d后两种细胞均拉伸、聚集，两者相互交错并有管状结构形成。DNA含量测定显示细胞数量在7 d内有明显增殖，增殖幅度与单纯培养的凝胶支架差异无统计学意义。共培养后2、4、7 d对共培养的材料支架进行RT-PCR检测与Western blot分析，结果显示，相对于2 d，4 d与7 d VEGF mRNA相对表达量分别增长（2.1±0.1）倍及（5.2±0.2）倍；7 d后单纯hUVECs及共培养的VEGF mRNA相对表达量分别为单纯BMSCs培养的（2.4±0.1）倍及（8.8±0.2）倍。结论被荧光蛋白标记后的hUVECs和BMSCs在Ⅰ型鼠尾胶原内可保持其荧光并存活，两者在此共培养体系中可进行三维生长与自由拉伸。7 d后可见其明显的相互作用并形成类血管网状结构。可见使用此共培养体系是�Objective To investigate the feasibility of human umbilical vein endothelial cells （hUVECs） and human bone marrow mesenchymal stem cells （BMSCs） forming vascular network like structure in three - dimensional co - cultured system. Methods Green fluorescent protein and red fluores- cent protein to were used to transt＇ect the 4th generation human umbilical vein endothelial cells and the 4th generation bone marrow mesenchymal stem cells respectively, which were implanted into type I rat tail col- lagen to co - culture in the dish including a volume fraction of 5% extracellular matrix （ECM） fatal bovine serun（FBS） medium 1 ml. Through confocal laser scanning microscope, the state and development of two cells form network structures and both growth after co -cultured 2 d, 4 d, 7 d were observed. MetaXpress software system was used to calculate the length of the vascular network like structure and analyze cell via- bility using DNA extraction method by DNA extraction. Results 2 days later, BMSCs started stretching, growing fusiform and showing no aggregation between cells, hUVECs did not dramatically stretch, only a few cells slightly stretched. 4 days later BMSCs continued stretching and aggregating, while hUVECs stretched considerably and had started to gather, but there was no obvious link between the two cells. 7 days later two cells stretched and aggregated. Both interlaced and formed tubular structure and vessel - like structures extendingobviously. Determination of DNA content displayed the number of cell proliferation in 7 days significantly, as well as the mRNA and protein level of vascular endothelial growth factor （VEGF）. Conclusion The labeled hUVECs and BMSCs maintain their fluorescence and survive in type I rat tail collagen. They both can grow and extend free in this three - dimensional co - culture system. They show significant interaction to form vascular network like structure 7 days after co - culture. The results of the present study suggest that this co -cultured system is effe

关 键 词：人脐静脉内皮细胞 人骨髓间充质干细胞 Ⅰ型鼠尾胶原 类血管网状结构 

分 类 号：R318[医药卫生—生物医学工程]