犬膀胱平滑肌细胞的体外连续培养  

SERIAL CULTIVATION OF CANINE SMOOTH MUSCLE CELLS FROM URINARY BLADDERS

作　　者：智伟[1,2] 杨志明[1] 陈晓禾[1] 李秀群[1] 韩平[3] 谭美云[1] 唐耘熳[4] 邓力[1,2] 

机构地区：[1]四川大学华西医院生物治疗国家重点实验室.干细胞与组织工程研究室,成都610041 [2]华西药学院 [3]华西医院泌尿外科 [4]华西医院小儿外科

出　　处：《中国修复重建外科杂志》2008年第12期1476-1480,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：教育部新世纪优秀人才支持计划(NCET-07-0575);中国博士后科学基金资助项目(20070410389)~~

摘　　要：目的通过体外培养不同代次的犬膀胱平滑肌细胞,比较其生物学特征,探讨多次传代后的平滑肌细胞作为种子细胞的可行性,为构建组织工程膀胱和尿道筛选种子细胞提供方法和依据。方法取体重10～12kg的12月龄雄性犬膀胱肌层,混合酶消化法获取平滑肌细胞,并于含10%小牛血清的培养基中培养,观察比较不同代次细胞的形态变化及生长、增殖过程,电镜下观察细胞超微结构,并通过免疫组织化学方法检测特征性蛋白的表达。结果体外培养的平滑肌细胞单层生长至亚融合状态后,倒置相差显微镜下细胞呈长梭形,并呈峰-谷样结构,可连续传至12代。生长曲线显示第7代以前的细胞具有相似的增殖特点,约每40小时为1次生长周期,透射电镜下可见平滑肌细胞特有的密体结构,平滑肌肌动蛋白免疫组织化学染色为阳性;第7代后细胞增殖逐渐迟滞,至第10代细胞内肌丝及密体结构消失。结论犬膀胱平滑肌细胞可在体外连续培养,通过适当的纯化方法可获得大量高纯度的平滑肌细胞。第7代以前的平滑肌细胞均可作为种子细胞,用于构建组织工程膀胱和尿道。Objective To provide an ideal seed cell for tissue engineered urinary bladder and urethra by serially culturing canine smooth muscle cells from urinary bladder in vitro and compare biological characteristics of different passages of cells. Methods Bladder smooth muscle cells of 12-month-old male dogs weighing 10-12 kg were isolated from adult dogs＇ urinary bladders by collagenase and trypsin digestion and serially cultured in DMEM medium supplemented with 10% serum of newborn bovines. Morphology and proliferation of the cells were observed and the serially-cultured cells were identified with the transmission electron microscope and immunohistochemistry. Results The cells appeared spindle in parallel rows when they grew to the degree of subconfluence, and showed the ＂peak-valley＂ structure under the inverted phase contrast microscope. The cells could be proliferated serially to the 12th passage in vitro. The growth curve showed the cells before the 7th passage had the similar proliferation characteristics and the growth cycle was about 40 hours. The TEM showed myofilament and the dense body in cytoplasm of smooth muscle cells. Smooth muscle actin was positive by immunohistochemical staining. After the 7th passage, the cells＇ growth became slow, and myofilament and the dense body in cytoplasm vanished. Conclusion The canine smooth muscle cells from urinary bladder can be serially cultured in vitro and highly purified and largely proliferated by the appropriate method. The cells before the 7th passage can be used as optimal seed cells for tissue engineered urinary bladder and urethra.

关 键 词：组织工程膀胱 平滑肌细胞 细胞培养 犬 

分 类 号：R318[医药卫生—生物医学工程]