摘要
目的:建立从膀胱癌患者的分离并培养膀胱平滑肌细胞的实验技术。方法:取一小块无明显肿瘤生长的膀胱组织,分离并培养膀胱平滑肌细胞;动态观察细胞形态变化、生长增殖情况以及平滑肌肌动蛋白(SMA)、结蛋白(Desmin)和广谱细胞角蛋白(AE1/AE3)的表达。结果:接种24 h后即有长梭形细胞贴壁生长,10天后长至80%融合,呈典型的"峰谷"样形态;传代后1天为潜伏期,2～6天为指数生长期,然后进入融合平台期,需再次传代。第2代细胞的SMA和Desmin表达阳性率分别高达(99.0±0.8)%和(97.0±2.1)%,不表达AE1/AE3。随着传代次数的增加,细胞去分化,细胞形态变成短梭状或椭圆形;SMA和Desmin的表达开始下降,传至第5代时,SMA和Desmin阳性率分别降至(78.0±3.3)%和(74.0±2.6)%;至第7代时,SMA和Desmin阳性率降至(51.0±3.0)%和(49.0±2.6)%。第7代细胞经血清饥饿培养48 h后,细胞又能再分化,形态转变成长梭状,SMA和Desmin阳性率可分别升至(90.0±3.5)%和(88.0±2.5)%,具有显著性差异。结论:本研究所培养的人膀胱平滑肌细胞具有较高的纯度,血清饥饿能促进去分化的细胞再分化,能为构建组织工程膀胱提供种子细胞。
Objective：To develop a new method of isolation and culture of human bladder smooth muscle cell （HBSMC） from bladder cancer patients for engineering bladder tissues in vitro. Methods：A small piece of no significant tumor growth in bladder was taken , Isolated and cultured bladder smooth muscle cells. The cell growth rates were investigated. The morphologic characteristics were observed over several passages, and the immunophenotypie levels of smooth muscle actin （SMA）, desmin and pancytokeratin AE1/AE3 were also examined. Resuits：The HBSMC attached to the plates 24 hour post-seeding and assumed elongate spindle morphology. After 10 days in culture, the cells grew into 80 % confluences and displayed the characteristic hills and valleys pattern. Subcultured HBSMC showed an initial lag phase of one day followed by an exponential growth phase of 5 days, leading to a confluence plateau phase at day 7. The HBSMC showed negative staining for AE1/AE3. The high purity of the cultured HBSMC was indicated by the results that （99.0±0. 8）% and （97.0±2. 1）% of the cells stained positive for SMA and desmin respectively at 2nd passage. The cultured cells gradually dedifferentiated over several passages and changed to short spindle or oval shape. The SMA and desmin also reduced to （78.0± 3.3） % and （74.0±2.6） %respectively at the 5th passage, and （51.0±3.0）% and （49.0±2.6） % at the 7th passage. After serum starvation for 48 hours, the dedifferentiated HBSMC had become to elongate spindle shape and the expres sion of SMA and desmin dramatically increased to （90± 3.5）% and （88.0±2. 5）%. Conclusions： We successfully established the techniques for isolation and culture of high purity HBSMC without the contamination of human urothelial cells. Serum starvation resulted in redifferentiation of differentiated HBSMC.
出处
《临床泌尿外科杂志》
北大核心
2010年第4期299-302,共4页
Journal of Clinical Urology
基金
南京市卫生局医学科技发展重点资助项目(批准号:ZKX07008)
关键词
组织工程
血清饥饿
膀胱平滑肌细胞
种子细胞
tissue engineering
serum starvation
bladder smooth muscle cell
cell source
