神经源性尿路功能障碍大鼠输尿管平滑肌细胞超微结构和SKca表达研究

Ureteral ultrastructure and expression of SKca in ureterai smooth muscle cell of rats with neuropathic urinary tract dysfunction

目的探讨大鼠神经源性尿路功能障碍（neurogenic urinary tract dysfunction，NUTD）输尿管平滑肌细胞（ureteral smooth muscle cells，USMC）超微结构改变及小电导钙激活钾通道（small-conductance Ca^2＋-activated K^＋channels，SKca）表达变化。方法选取45只SD大鼠随机平均分为NUTD组、实验对照组（EC组）和空白对照组（BC组）。其中NUTD组给予破坏S，～。脊髓节段，术后1周行影像尿动力学检查，证实存在逼尿肌无收缩但无膀胱输尿管发生反流；EC组仅咬除棘突，未破坏脊髓；术后1周行影像尿动力学检查，证实无明显膀胱尿道功能障碍。模型建立后6周三组大鼠均行影像尿动力学检查，输尿管功能和形态学及USMC超微结构观察，并应用荧光定量PCR和Western-blot检测USMC SKca mRNA和蛋白表达情况。结果模型建立后6周NUTD组大鼠膀胱均表现为无逼尿肌过度活动、膀胱逼尿肌无收缩。30min内NUTD组产生尿量为（0．90±0．17）ml，EC组为（0．86±0．18）ml，BC组为（0．94±0．15）ml三组间差异无统计学意义；但NUTD组输尿管蠕动频率（16．23±2．35）次／5min显著低于EC组（21．80±1．97）次／5min和BC组（20．47±2．48）次／5min，差异有统计学意义（P〈O．05）。NUTD组USMC中SKca2和SKca3蛋白表达显著上调，mRNA表达相对于BC组平均分别上调4．15和4．56倍，差异有统计学意义（P〈0．01）。电镜下可见NUTD组USMC排列散乱，线粒体体积增大水肿、数量增多，线粒体嵴增粗、髓样变，部分细胞可见线粒体内膜和嵴膜的大量破坏。结论USMC超微结构改变和SKca表达水平上调可能是输尿管原发性功能障碍的重要机制之一。

Objective To explore the changes of ureteral ultrastructure in rats with neuropathic urinary tract dysfunction （NUTD） and the expression of small-conductance Ca^2＋-activated K^＋ channels （SKca） in ureteral smooth muscle cell. Methods A total of 45 rats weighted 200 g were randomly divided into 3 groups of NUTD, experimental control （EC） and blank control （BC）. The NUTD group underwent spinal cord transection at the first lumbar level and a destruction of sacral cord. EC group had a mere removal of spinous process at the same position, an exposure of spinal cord and there was no transection; BC group had no operation. One week later, video-urodynamics showed that there was indeed acontractile detrusor （ACD）, no expanded bladder capacity and vesicoureteral reflux （VUR） in rats from NUTD group and no significant urinary tract dysfunction in rats from experimental control and blank control groups. Video-urodynamics, observation of ureteral ultrastructure and morphology were performed at Week 6 post-operation. The expressions of mRNA and protein of SKca in ureteral smooth muscle cell （USMC） were measured by real-time fluorescent quantitative polymerase chain reaction （PCR） and Western blot after 6 weeks. Meanwhile fine structures of ureteral smooth muscle cell were observed by electron microscopy. Results Acontractile detrusor （ACD） and expanded bladder capacity were found in all rats from NUTD group without vesicoureteral reflux. No significant difference existed in urine volume per 30 min among three groups （NUTD group： 0. 90 ±0. 17 ml, EC group： 0. 86 ±0. 18 ml, BC group： 0. 94 ± 0. 15 ml）. The 5-rain peristaltic frequency of left ureter was significantly lower in NUTD group （16. 65 ±2. 35） than those of EC group （21.80 ±1.97） and BC group（20. 47 ±2. 48, P〈0. 05）. No abnormal gross changes occurred in ureteral lamina epithelium, lamina propria and muscular layer among three groups. Compared with BC group, the mRNA expressions of SKca2 and SKca3 were up-regulated for 4. 15 and 4. 56 folds in NUTD group respectively （P〈0. 01）. And the protein expressions of SKca2 and SKca3 also significantly increased （P〈0. 01）. Under electron microscopy, the permutations of smooth muscle were scattered and there were plenty of expanded and edematous mitochondria with crest medullary enlargement; extensive damage of intima and ridge membrane. Conclusions A down- regulation of SKca and altered ultrastructure in USMC may play an important role in primary upper urinary tract dysfunction.