ASICs在膀胱粘膜上皮细胞及膀胱平滑肌细胞上表达的意义

发布时间：2018-05-16 01:18

  本文选题：膀胱粘膜上皮细胞株 + 膀胱平滑肌细胞株　； 参考：《昆明医科大学》2014年硕士论文

【摘要】：目的： 了解ASICla和ASIC3在膀胱尿路上皮细胞株(SV-HUC-1)及膀胱平滑肌胞株(HBdSMC)表达的情况,并观察阿米洛利及细胞培养液pH值改变对两种细胞株ASICla和ASIC3表达的影响及对细胞电流变化的影响。 方法： 1、对人膀胱尿路上皮细胞株(SV-HUC-1)和膀胱平滑肌细胞株(HBdSMC)进行传代培养,并利用荧光免疫检测其中ASICla和ASIC3蛋白的表达； 2、培养液中加入酸敏感离子通道抑制剂阿米洛利后,检测SV-HUC-1细胞株、HBdSMC细胞株ASICla和ASIC3蛋白表达 3、改变胞外H+浓度并加入ASIC抑制剂阿米洛利,使用膜片钳技术检测人膀胱尿路上皮细胞电流变化情况。 结果： 免疫荧光染色显示：(1)人膀胱尿路上皮细胞株及膀胱平滑肌细胞株上含有ASICla和ASIC3蛋白表达；且ASICla和ASIC3蛋白主要表达于膀胱尿路上皮细胞株上,膀胱平滑肌细胞株中ASICla和ASIC3蛋白的表达远不及膀胱尿路上皮细胞株(P0.01)。人膀胱尿路上皮细胞株中,ASIC3蛋白表达要明显高于ASICla(P0.05);人膀胱平滑肌细胞株中,ASICla和ASIC3蛋白表达无明显差异(P0.05)。 (2)加入ASIC抑制剂阿米洛利后,人膀胱尿路上皮细胞株上ASICla的表达降低(P0.05,),ASIC3表达明显降低(P0.01)；膀胱平滑肌细胞株中ASICla和ASIC3蛋白表达无明显变化(P0.05)。 (3)膜片钳检测提示：人膀胱尿路上皮细胞株上ASICs随胞外H+浓度上升而产生相应的电流,H+浓度越大,电流强度越高(R=0.8320.06)。加入ASICs的特异性抑制剂阿米洛利后,相同pH值下人膀胱尿路上皮细胞株上ASICs所引起的电流强度明显减弱(P0.05)。 结论： 1.人膀胱尿路上皮细胞株及膀胱平滑肌细胞株上均含有ASICs, ASICla与ASIC3在人膀胱平滑肌细胞株内的蛋白表达较少且相差无几,而在膀胱尿路上皮细胞株中ASIC3蛋白表达明显比ASICla高。 2.酸敏感离子通道抑制剂阿米洛利可以抑制膀胱尿路上皮细胞株中ASICla和ASIC3的蛋白表达；对膀胱平滑肌细胞株中ASICs的影响不明显； 3.人膀胱膀胱尿路上皮细胞株上表达ASICs受H+的影响较大,其过度的蛋白表达及所引起的电流变化均可被阿米洛利抑制。
[Abstract]:Objective: To investigate the expression of ASICla and ASIC3 in bladder urinary tract epithelial cell line (SV-HUC-1) and bladder smooth muscle cell line (HBdSMC), and to observe the effect of amiloride and pH on the expression of ASICla and ASIC3 and the change of cell current. Methods: 1. Human bladder urothelial cell line SV-HUC-1 and bladder smooth muscle cell line HBdSMC) were subcultured, and the expression of ASICla and ASIC3 were detected by fluorescence immunoassay. 2. The expression of ASICla and ASIC3 protein in SV-HUC-1 cell line was detected after the addition of Aminolol, an acid sensitive ion channel inhibitor, to the culture medium. 3. Changes of extracellular H concentration and amiloride, an inhibitor of ASIC, were used to detect the current changes of human urinary tract epithelial cells by patch clamp technique. Results: Immunofluorescence staining showed that ASICla and ASIC3 protein were expressed in human urinary tract epithelial cell line and bladder smooth muscle cell line, and ASICla and ASIC3 protein were mainly expressed in bladder urinary tract epithelial cell line. The expression of ASICla and ASIC3 protein in bladder smooth muscle cell line was much lower than that in bladder urothelial cell line P0.01. The expression of ASIC3 protein in human urinary tract epithelial cell line was significantly higher than that in human bladder smooth muscle cell line P0.05.The expression of ASICla protein and ASIC3 protein in human bladder smooth muscle cell line had no significant difference (P 0.05). (2) after the addition of ASIC inhibitor amilolol, the expression of ASICla in human urinary tract epithelial cell line decreased significantly, while the expression of ASICla and ASIC3 protein in bladder smooth muscle cell line did not change significantly. The results of patch clamp test showed that the higher the concentration of ASICs and the higher the current intensity, the higher the current intensity of ASICs on human bladder urothelial cell line was 0.8320.06 with the increase of extracellular H concentration. After adding amilolol, a specific inhibitor of ASICs, the current intensity induced by ASICs on human bladder urothelial cell line at the same pH value was significantly decreased (P 0.05). Conclusion: 1. ASICs were found in human urinary tract epithelial cell line and bladder smooth muscle cell line. The expression of ASIC3 protein in human bladder smooth muscle cell line was similar to that in human bladder smooth muscle cell line, but the expression of ASIC3 protein in bladder urinary tract epithelial cell line was significantly higher than that in ASICla cell line. 2. Aminolol, an acid sensitive ion channel inhibitor, inhibited the expression of ASICla and ASIC3 protein in bladder urothelial cell line, but had no significant effect on ASICs in bladder smooth muscle cell line. 3. The expression of ASICs on human bladder urothelial cell line was greatly affected by H, and its excessive protein expression and current changes could be inhibited by amiloride.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R694.5

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