本文選題：膀胱粘膜上皮細(xì)胞株 + 膀胱平滑肌細(xì)胞株��； 參考：《昆明醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的： 了解ASICla和ASIC3在膀胱尿路上皮細(xì)胞株(SV-HUC-1)及膀胱平滑肌胞株(HBdSMC)表達(dá)的情況,并觀察阿米洛利及細(xì)胞培養(yǎng)液pH值改變對(duì)兩種細(xì)胞株ASICla和ASIC3表達(dá)的影響及對(duì)細(xì)胞電流變化的影響。 方法： 1、對(duì)人膀胱尿路上皮細(xì)胞株(SV-HUC-1)和膀胱平滑肌細(xì)胞株(HBdSMC)進(jìn)行傳代培養(yǎng),并利用熒光免疫檢測(cè)其中ASICla和ASIC3蛋白的表達(dá)； 2、培養(yǎng)液中加入酸敏感離子通道抑制劑阿米洛利后,檢測(cè)SV-HUC-1細(xì)胞株、HBdSMC細(xì)胞株ASICla和ASIC3蛋白表達(dá) 3、改變胞外H+濃度并加入ASIC抑制劑阿米洛利,使用膜片鉗技術(shù)檢測(cè)人膀胱尿路上皮細(xì)胞電流變化情況。 結(jié)果： 免疫熒光染色顯示：(1)人膀胱尿路上皮細(xì)胞株及膀胱平滑肌細(xì)胞株上含有ASICla和ASIC3蛋白表達(dá)；且ASICla和ASIC3蛋白主要表達(dá)于膀胱尿路上皮細(xì)胞株上,膀胱平滑肌細(xì)胞株中ASICla和ASIC3蛋白的表達(dá)遠(yuǎn)不及膀胱尿路上皮細(xì)胞株(P0.01)。人膀胱尿路上皮細(xì)胞株中,ASIC3蛋白表達(dá)要明顯高于ASICla(P0.05);人膀胱平滑肌細(xì)胞株中,ASICla和ASIC3蛋白表達(dá)無(wú)明顯差異(P0.05)。 (2)加入ASIC抑制劑阿米洛利后,人膀胱尿路上皮細(xì)胞株上ASICla的表達(dá)降低(P0.05,),ASIC3表達(dá)明顯降低(P0.01)；膀胱平滑肌細(xì)胞株中ASICla和ASIC3蛋白表達(dá)無(wú)明顯變化(P0.05)。 (3)膜片鉗檢測(cè)提示：人膀胱尿路上皮細(xì)胞株上ASICs隨胞外H+濃度上升而產(chǎn)生相應(yīng)的電流,H+濃度越大,電流強(qiáng)度越高(R=0.8320.06)。加入ASICs的特異性抑制劑阿米洛利后,相同pH值下人膀胱尿路上皮細(xì)胞株上ASICs所引起的電流強(qiáng)度明顯減弱(P0.05)。 結(jié)論： 1.人膀胱尿路上皮細(xì)胞株及膀胱平滑肌細(xì)胞株上均含有ASICs, ASICla與ASIC3在人膀胱平滑肌細(xì)胞株內(nèi)的蛋白表達(dá)較少且相差無(wú)幾,而在膀胱尿路上皮細(xì)胞株中ASIC3蛋白表達(dá)明顯比ASICla高。 2.酸敏感離子通道抑制劑阿米洛利可以抑制膀胱尿路上皮細(xì)胞株中ASICla和ASIC3的蛋白表達(dá)；對(duì)膀胱平滑肌細(xì)胞株中ASICs的影響不明顯； 3.人膀胱膀胱尿路上皮細(xì)胞株上表達(dá)ASICs受H+的影響較大,其過度的蛋白表達(dá)及所引起的電流變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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R694.5

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