【作者】 朱凡；

【导师】 张玉芹；

【作者基本信息】 武汉科技大学 ， 生理学， 2008， 硕士

【摘要】 第一部分目的:探讨建立P2X4受体基因异体表达的可行性,为研究P2X4受体的功能及调控机制奠定基础。方法:利用体外转录cRNA技术,将克隆至真核表达载体pcDNA3的P2X4的cDNA转录成P2X4的cRNA,然后通过显微注射技术将该cRNA注入去滤泡膜的成熟非洲爪蟾卵母细胞的胞质中进行表达,并用双微电极电压钳技术检测所表达受体的功能。结果:P2X4受体能成功地在大多数爪蟾卵母细胞上表达,外加ATP可使这些细胞产生ATP诱导电流,该电流具有浓度依赖性和饱和现象。结论:爪蟾卵母细胞可成功表达外源性的离子型P2X4受体。第二部分目的:研究P2X4受体介导的ATP-激活电流(ATP-activated current ,IATP)的特征。方法:应用双电极电压钳技术记录表达在非洲爪蟾卵母细胞上的P2X4受体介导的IATP。结果:(1)大鼠P2X4受体可高效表达于非洲爪蟾卵母细胞。(2)外加1μmol/L,10μmol/L和100μmol/L ATP时,IATP激活相的时间常数τon分别是2.58±0.60 s, 1.80±0.47 s and 1.05±0.24 s。IATP失敏相的时间常数τdes分别是9.54±1.56 s,6.52±1.24 s and 4.11±0.78 s。(3)ATP作用的量-效关系:EC50为15.9±1.7μmol/L,Hill系数为0.95±0.07。(4)浓度为100μmol/L的PPADS和Suramin不改变IATP的幅值。(5)IATP呈电压依赖性和内向整流特性。(6)加药时间分别为20 s,30 s,45 s和60 s的情况下,IATP幅值并无显著性差异。结论:以上结果增进了我们对P2X4受体介导的ATP-激活电流特征的认识,为进一步研究P2X4受体的功能和调控机制奠定基础。第三部分目的:研究铝对大鼠P2X4受体功能的调制作用及其机制。方法:应用双电极电压钳记录表达在非洲爪蟾卵母细胞上的P2X4受体介导电流(IATP)。结果:(1)在有100μmol/L氯化铝存在的情况下,ATP-激活电流(IATP)的失活时间常数(τdes)变小,而激活时间常数(τon)增大;(2)在表达有P2X4的非洲爪蟾卵母细胞,铝在浓度为3～1000μmol/L的范围内对IATP的抑制作用呈浓度依赖性,浓度越高,抑制作用越明显,其作用的IC50为16.2μmol/L;(3)铝使ATP最大效应降低,从而使IATP的量效曲线下移,但并不改变ATP作用的EC50值;(4)铝的这种抑制作用并不依赖于钳制电压,也并不改变IATP的翻转电位;(5)在有10μmol/L铝存在的情况下,IATP并没有在每间隔3分钟给药(ATP)一次的情况下出现逐渐减小的情况。并且,铝的这种抑制作用存在时间依赖性,60s的预加时间可使铝达到其最大抑制效应。结论:铝能抑制表达在非洲爪蟾卵母细胞上的P2X4受体介导的ATP-激活电流。铝的这种抑制作用是一种非竞争性抑制,其机制可能是铝通过与P2X4受体膜外结合位点结合,通过变构调节来实现的。更多还原

【Abstract】 PART 1AIM: To determine the feasibility of establishing the heterologous expression model of P2X4 receptor. METHODS: cRNA of P2X4 was transcribed from cDNA, which was cloned in the pcDNA3 vector. Each oocyte of mature xenopus laevis was injected with transcribed cDNA in vivo and incubated at 18℃for 30 hours. Recording the current induced by P2X4 receptor with voltage clamp technique tested the function of the expressed P2X4 receptor. RESULTS: ATP-activated inward current could be observed in most of the oocytes which were injected with the transcribed P2X4 cRNA, and the ATP induced current were concentration dependent. CONCLUSION:The results demonstrate that the rat P2X4 receptor could expressed in Xenopus Laevis oocytes successfully.PART 2AIM:To explore the characterization of ATP-activated current(IATP)mediated by rat P2X4 receptor expressed in Xenopus Oocytes. METHODS: Two-electrode whole-cell voltage clamp are used to record IATP mediated by rat P2X4 receptor expressed in Xenopus oocytes. RESULTS:(1) The rat P2X4 receptor can be efficiently expressed in Xenopus oocytes,.(2) The activation phase time constant (τon) of IATP are 2.58±0.60 s, 1.80±0.47 s and 1.05±0.24 s respectively, while the desensitizing phase time constant (τdes) of IATP are 9.54±1.56 s,6.52±1.24 s and 4.11±0.78 s respectively,when 1μmol/L,10μmol/L,and 100μmol/L ATP are appied correspondingly.(3) The Concentration-response relationship in ATP is measured as: 16.03±1.81μmol/L of EC50 to 1.02±0.09 of Hill coefficient.(4) PPADS and Suramin at a concentration of 100μmol/L have no effect on ATP-activated current mediated by rat P2X4 receptor.(5)IATP exhibits characteristics of both voltage-dependency and inward-rectification,and (6) IATP doesnot have a significant variation when applying ATP times are 20 s,30 s,45 s,and 60 s.CONCLUSION:The results in this study advance our understanding in the characteristics of ATP-activated current, and prepare for further investigation in the functions of P2X4 receptor and its inner modulating mechanism.PART 3AIM:To investigate the modulation of aluminum on rat P2X4 receptors function and its underlying mechannisms.METHODS: Rat P2X4 receptors were expressed in Xenopus oocytes and studied using the two-electrode voltage clamp.RESULTS(:1)In the presence of 100μmol/L aluminum ,the desensitization time constant(τdes) value of ATP-activated current(IATP) decreased significantly more than without aluminum,while the activation time canstant(τon) increased.(2)In oocytes expressing P2X4 receptors,aluminum cholride,in the concentration range of 3～1000μmol/L,reversibly inhibited current activated by 10μmol/L ATP.Aluminum produced 50% of maximal inhibition of IATP(10μmol/L) was 16.2μmol/L.(3)Aluminum caused the concentration-response curve for IATP a downward displacement by reducing the maximal response,but did not change the EC50 value.(4)The aluminum inhibition of IATP was independent of membrane potential between -80 mV and -20 mV and did not involve a shift in the reversal potential of the current.(5)This block did not involve a gradual decline in the presence of 10μmol/L aluminum at 3-min intervals.Moreever,aluminum modulation of IATP(10μmol/L) was time-dependent.The aluminum-induced inhibition reached its maximum within 60 s pretreatment.CONCLUSION:These results always suggest that aluminum inhibites ATP-activated current mediated by P2X4 receptor in a noncompetitive manner.It may antagonize P2X4 receptors by allosteric modulation,and possibly acts at the binding sites within extracellular regions of the channel.更多还原