犬内皮与平滑肌细胞的短期静态联合培养

Short-time static co-culture of canine smooth muscle cells and endothelial cells

目的:为获得组织工程化自体血管,观察体外静态培养条件下犬内皮细胞与平滑肌细胞联合培养组织学及形态学的特征。方法:实验于2004-07/2005-06在首都医科大学宣武医院外科院级实验室完成。①实验材料:雄性杂种犬,3个月龄,体质量8～12kg。②实验方法:贴块法及酶解法对犬内皮细胞及平滑肌细胞进行原代分离培养及扩增,将第Ⅱ代平滑肌细胞以1×109L-1的密度种植于胶原膜上培养13d,再将第Ⅱ代内皮细胞接种于生长平滑肌细胞的胶原膜上2d。③实验评估:行苏木精-伊红染色同时扫描电镜和透射电镜观察平滑肌细胞和内皮细胞在胶原载体上联合培养后的形态。结果:苏木精-伊红染色见平滑肌细胞较均匀的分布于支架材料表面及内部;扫描电镜下,平滑肌细胞可以在胶原载体材料上生长,增殖明显并在短期内形成多层细胞。内皮细胞与平滑肌细胞联合培养2d就可在平滑肌细胞层表面获得连续的单层内皮细胞层。结论:在短期静态培养条件下犬的血管平滑肌细胞和内皮细胞可以在胶原载体材料上形成具有两层细胞结构的组织工程化动脉血管组织片。

AIM：To observe the histological and morphological features of static co-culture of canine endothelial cell （EC） and smooth muscle cell （SMC） in vitro. METHODS： The experiment was performed in the Surgical Laboratory of Xuanwu Hospital, Capital Medical University from July 2004 to June 2005. ①Canine SMCs and ECs were isolated from the blood vessel of male hybrid canine of 3 months old and 8-12 kg and cultured by tissue explants-adherence methods and enzyme hydrolysis. The second passage of SMCs （1×10^9 L^-1） were seeded on collagen scaffold and co-cultured for 13 days. Afterwards, the second passage of ECs were seeded on the surface of collagen scaffold with SMCs and cultured for 2 days. ②The co-culture of canine SMCs and ECs on collagen scaffold was observed by H-E staining under scanning and transmission electron microscope. RESULTS： H-E staining showed that SMCs grew well on the surface and interior of collagen scaffold. By the scanning electron microscope study, SMCs grew on the collagen scaffold and proliferated into multilayer cells during short time. The continuous monolayer of ECs was found on the surface of SMCs after 2 days co-culture. CONCLUSION： Tissue engineered arterial blood patch containing canine SMCs and ECs could be obtained via static short-time co-culture of two cells on collagen scaffold.