心瓣膜组织工程中种子细胞原代联合培养的实验研究  

Primary co-culture of mixed vascular cells for tissue engineered heart valves

作　　者：成少飞[1] 杨岷[1] 陈长志[1] 王学宁[1] 叶清[1] 王维俊[1] 童菊芳[1] 

机构地区：[1]上海交通大学医学院附属仁济医院胸心外科,200127

出　　处：《国际心血管病杂志》2006年第5期342-345,共4页International Journal of Cardiovascular Disease

基　　金：教育部留学回国人员科研基金(编号:03-406-01)

摘　　要：目的：探讨体外原代联合培养、扩增，鉴定同一来源的人血管内皮细胞、平滑肌细胞、成纤维细胞的方法，为组织工程心脏瓣膜的构建提供理想的种子细胞。方法：无菌条件下取冠状动脉旁路移植术中剩余大隐静脉3cm～8cm，组织块培养法原代培养混合血管细胞，DMEM培养液培养、扩增混合血管细胞。倒置显微镜、免疫组化方法对内皮细胞、平滑肌细胞、成纤维细胞进行鉴定。结果：倒置显微镜显示原代细胞及经传代培养后的细胞，呈现内皮样细胞、成纤维样细胞混合生长的特征。免疫细胞化学检测示内皮细胞Ⅷ因子、平滑肌细胞α-肌动蛋白、成纤维细胞纤维连接蛋白相关抗原呈阳性反应。细胞经3～4次传代，细胞数量可增殖达到8×10^6～10×10^6。结论：使用组织块法原代培养可获得内皮细胞、平滑肌细胞、成纤维细胞，三种细胞经体外联合培养、传代可以达到组织工程心脏瓣膜种子细胞的足够数量。Objective： To evaluate the methods of primary co-culture and identification of the human endothelial cells （ECs）, myofibroblasts and smooth muscle cells for tissue engineered heart valves（TEHVs）, and to prepare seed cells for the construction of TEHVs. Methods： Mixed human vascular cells were obtained by primary culture method. A segment of 3-8cm great saphenous vein of patients undergoing coronary artery bypass graft in our institution was used for culture. The vein was freed of adventitia and fat. The vessel media and intima were minced into pieces of about 3 mm× 3mm, then transferred into culture flask. DMEM culture medium and 20% fetal bovine serum were added into flask. The tissue pieces were cultured in a humidified incubator at stable, controlled gas and temprature （5 % CO2, 37 ℃, humidified air）. After 5-10 days the cells germinated from the tissues, about l week later the primary cell populations reached confluency and were passaged several times with 0.25 % Trypsin- 0.02 % EDTA solution. Morphology and proliferation of the cells were observed with inverted phase contrast microscope. The type of cells was evaluated by immunocytochemistry with yon Willebrand factor, Fibronectin and α-smooth muscle actin antibodies. Results： After 4-5 weeks,the primary cells were passaged 3-5 times and the number of cells achieved 8×10^6～10×10^6. Under inverted phase contrast microscope, mixed cells like endothelial cells and myofibroblasts were observed. By immunocytochernistry method, a mixture of endothelial cells, myofibroblasts and smooth muscle cells were demonstrated. Conclusions： Mixed human vascular cells were obtained by primary culture method with human great saphenous vein. They can be co-cultured successfully and be amplified to a sufficient number for the seeding procedure of TEHVs.

关 键 词：组织工程 心脏瓣膜 细胞培养 混合血管细胞 

分 类 号：R318[医药卫生—生物医学工程]