【作者】 鄢俊安；

【导师】 宋波；

【作者基本信息】 第三军医大学 ， 外科学， 2009， 博士

【摘要】 众所周知,膀胱肌组织能够产生不受神经系统控制的自发性动作电位,但目前其机制尚未明了。前期的研究中已经证实了膀胱存在ICC,简单记录了Ih电流。这些结果都不能证实ICC在膀胱中到底起到何种作用。Ih电流（在心脏称为If电流）在胃肠道和心脏窦房结的研究中被认为是“起搏电流”,而HCN通道是Ih电流的分子基础。我们前期的系列研究中已经提示ICC上可能存在HCN通道,但是其电生理特征还尚未明确。同时,既往的实验方法中采用培养的ICC进行电生理实验,效果并不理想,其主要原因是培养后的ICC进行膜片钳实验时难度很大,而且其所测参数并不一定代表了真实生理状态下的情况。因此本实验的目的:建立一套稳定、可靠的急性分离方法以获得活性更好的ICC;观察ICC上HCN通道的表达,完善HCN通道基本的电生理参数,为进一步的研究提供实验基础和理论依据。主要实验结果与结论如下:1.建立了稳定可靠的急性分离方法,所得到的细胞经免疫组织化学实验方法确认是ICC;其酶消化方案为:Ⅱ型胶原蛋白酶2.0mg/ml,胰酶抑制剂2.0mg/ml,木瓜蛋白酶1.0mg/ml,BSA1.0mg/ml,其消化时间15min。2.采用c-Kit和HCN抗原的免疫荧光双标法,证明了c-Kit阳性的ICC细胞上具有HCN通道抗原的阳性表达。3.ICC上记录到了Ih电流,其激活电压位于-140mV～－90mV之间,电流幅度为300-800pA,具有电压依赖性,并能被ZD7288（20μM）和Cs～＋（500μM）阻断。4.Ih电流反转电位42±2mV,PNa/P_K为0.42,膜电容为10-40pF;胞外ZD7288阻断Ih电流具有浓度依赖性,其IC50为24.7±6.5μmol/L,且其阻断效应不可逆;胞外Cs⁺阻断Ih电流具有浓度依赖性,其IC50为232.5±24.8μmol/L,且其阻断效应可逆。5.胞外钠钾对HCN通道皆有影响,具有浓度依赖性,其浓度的改变会影响HCN通道的激活和电导;胞外给予TTX、TEA及Ba²⁺对Ih电流无明显影响。6.胞外钙可明显影响HCN通道的激活时间,具有浓度依赖性;胞内cAMP可影响HCN通道的激活时间,具有浓度依赖性。更多还原

【Abstract】 As we all know that spontaneous action potential（AP） can be recorded from muscular tissue of bladder, even if its nerval control is removed. But till now its mechanism is unknown to us.It has been confirmed the existence of the ICC in bladder on early study. The Ih current was recorded so simply. All of these results could not confirm which kind of role the ICC may play on functions.The Ih current is regarded as "pacemaker current" on the study of gastrointestinal tract and sinus node. HCN channels are the molecular basis of the Ih current. Our series of preliminary studies have prompted the existence of HCN channels on ICC in bladder. However, its electrophysiological characteristics have not yet been clear. Additionally, the method of using cultured ICCs was unsatisfactory in electrophysiological experiments. For the most main reason, It’s very difficult to clamp patch on cultured ICCs. The test parameters could not meet the actual that is under physiological conditions.So the aim of our study: To find the method to get better ICCs by acute separation.To observe the expression of HCN by by c-Kit and HCN antigen using double-labeled immunofluorescence （IF）. To get the basic electrophysiologic parameters of HCN. To provide the theoretical basis and experimental basis in further research.Main results and conclusions:1. We have got the stable and reliable method for better ICCs by acute separation. The Program for its enzymatic digestion:ⅡCollagenase 2.0mg/ml; Trypsin inhibitor 2.0mg/ml ;Papain 1.0mg/ml; BSA 1.0mg/ml. The time of digestion is 15 min.2. We have confirmed the existence of HCN by c-Kit and HCN antigen using double-labeled immunofluorescence （IF）.3. Whole-cell patch clamp recorded a inward current which began to slowly active between -90mV～-140mV in voltage and time dependent manner without deactivation. The current range is 300-800pA. Ih current were blocked by 20μM ZD7288 and 500uM Cs⁺.4. The reversal potential of Ih was 42±2mV, Na+ versus K+（PNa/PK） was 0.42, and membrane capacitance was 10-40pF. The extracellular ZD7288 blocked Ih current with concentration-dependent, and its IC50 was 24.7±6.5uM. the blocking effect of ZD7288 is not reversible. The extracellular Cs⁺ blocked Ih current with concentration-dependent, and its IC50 was 232.5±24.8μM. the blocking effect of Cs⁺ is reversible.5. Both the extracellular Na⁺ and K⁺ could impact activation and conductance of HCN with concentration-dependent. There were no significant effect on Ih current by adding extracellular drug such as TTX, TEA, and Ba²⁺.6. The extracellular Ca2+ could significantly affect the activation time of HCN with concentration-dependent. The intracellular cAMP could significantly affect the activation time of HCN with concentration-dependent.更多还原