Department of Urology, Oita University Faculty of Medicine, Oita, Japan.
J Urol. 2010 Jun;183(6):2445-50. doi: 10.1016/j.juro.2010.01.063. Epub 2010 Apr 18.
A decrease in the human urethral rhabdosphincter is reported with aging due to apoptosis, which may be a cause of urinary incontinence in the elderly population. To explore this mechanism we investigated the effects of tumor necrosis factor-alpha (Upstate, Temecula, California) on human urethral rhabdosphincter satellite cells.
Human urethral rhabdosphincter satellite cells were cultured and selected by magnetic affinity cell sorting, extended their life span. Apoptosis induction was examined by flow cytometry and immunocytochemistry. Caspase cascade activation was determined by Western blot analysis. After tumor necrosis factor receptor expression was confirmed we determined the tumor necrosis factor signaling pathway.
Tumor necrosis factor-alpha inhibited human urethral rhabdosphincter satellite cell proliferation. It caused some cells to stain positive for annexin V-fluorescein isothiocyanate but not for propidium iodide, suggesting the induction of early phase apoptosis. Flow cytometry revealed an increased sub-G1 fraction. Western blot analysis showed activation of caspase-8 and 3, and cleavage of poly (adenosine diphosphate-ribose) polymerase. Tumor necrosis factor receptor expression at the mRNA and protein levels was confirmed by reverse transcriptase-polymerase chain reaction and Western blot analysis, respectively. IkappaBalpha phosphorylation was noted within 2 to 5 minutes after tumor necrosis factor-alpha treatment. The tumor necrosis factor-alpha antagonist etanercept (Wyeth, Collegeville, Pennsylvania) inhibited IkappaBalpha activation and reversed tumor necrosis factor-alpha effects on human urethral rhabdosphincter satellite cells.
Since tumor necrosis factor-alpha induces growth inhibition and apoptosis of human urethral rhabdosphincter satellite cells via tumor necrosis factor receptor activation, it may be involved in age related decreases in the number of human urethral rhabdosphincter cells and be a causative factor for urinary incontinence in the elderly population.
据报道，由于细胞凋亡，人类尿道横纹括约肌随年龄增长而减少，这可能是老年人口尿失禁的一个原因。为了探讨这一机制，我们研究了肿瘤坏死因子-α（Upstate，加利福尼亚州Temecula）对人尿道横纹括约肌卫星细胞的影响。
通过磁性亲和细胞分选培养和选择人尿道横纹括约肌卫星细胞，延长其寿命。通过流式细胞术和免疫细胞化学检测细胞凋亡诱导。通过 Western blot 分析确定半胱天冬酶级联激活。在确认肿瘤坏死因子受体表达后，我们确定了肿瘤坏死因子信号通路。
肿瘤坏死因子-α抑制人尿道横纹括约肌卫星细胞增殖。它使一些细胞对 Annexin V-异硫氰酸荧光素呈阳性染色，但对碘化丙啶呈阴性染色，提示诱导早期凋亡。流式细胞术显示亚 G1 期细胞比例增加。Western blot 分析显示半胱天冬酶-8 和 3 的激活，以及多聚（腺苷二磷酸核糖）聚合酶的裂解。通过逆转录-聚合酶链反应和 Western blot 分析分别证实了肿瘤坏死因子受体在 mRNA 和蛋白质水平的表达。用肿瘤坏死因子-α处理后 2 至 5 分钟，即观察到 IkappaBalpha 磷酸化。肿瘤坏死因子-α拮抗剂依那西普（Wyeth，宾夕法尼亚州 Collegeville）抑制 IkappaBalpha 激活并逆转肿瘤坏死因子-α对人尿道横纹括约肌卫星细胞的作用。
由于肿瘤坏死因子-α通过肿瘤坏死因子受体激活诱导人尿道横纹括约肌卫星细胞生长抑制和凋亡，它可能参与与年龄相关的人尿道横纹括约肌细胞数量减少，并可能是老年人口尿失禁的一个致病因素。
