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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 establish an experimental technique for the separation and culture of bladder smooth muscle cells from patients with bladder cancer. Methods: A small piece of bladder tissue with no obvious tumor growth was isolated and cultured. The changes of cell morphology, growth and proliferation, smooth muscle actin (SMA), Desmin and broad-spectrum cytokeratin (AE1 / AE3) expression. Results: Fusiform cells grew adherently 24 hours after inoculation, and reached the peak of 80% confluence after 10 days, with a typical “peak-valley” like morphology; one day after passage and one exponential growth 2-6 days Period, and then enter the integration platform, need to pass on again. The expression of SMA and Desmin in the second generation cells were up to (99.0 ± 0.8)% and (97.0 ± 2.1)% respectively, but not AE1 / AE3. The expression of SMA and Desmin began to decline with the passage of times, and the positive rates of SMA and Desmin decreased to (78.0 ± 3.3) % And (74.0 ± 2.6)%, respectively. By the seventh passage, the positive rates of SMA and Desmin decreased to (51.0 ± 3.0)% and (49.0 ± 2.6)%, respectively. After cultured for 7 h, the cells were redifferentiated and transformed into long spindle. The positive rates of SMA and Desmin increased to (90.0 ± 3.5)% and (88.0 ± 2.5)%, respectively, with significant difference difference. CONCLUSION: Human bladder smooth muscle cells cultured in this study have high purity. Serum starvation can promote the dedifferentiation of dedifferentiated cells and provide seed cells for tissue engineering bladder.