【作者】 张莉；

【导师】 肖杭；

【作者基本信息】 南京医科大学 ， 卫生毒理学， 2003， 硕士

【摘要】 第一部分：小鼠粗线期精母细胞T型Ca²⁺通道特性的研究 在影响精子生理活动的众多离子中，Ca²⁺的作用倍受人们关注，在精子发生、成熟、获能、顶体反应等生理过程中，胞内Ca²⁺均起重要作用。电压依赖性钙通道（voltage dependent CalCium Channel, VDCC）是控制Ca²⁺跨膜内流的主要途径之一，现已清楚精母细胞及精子上主要存在T型Ca²⁺通道。但以前的研究多采用酶解分离细胞的方法，而现在清楚酶解可改变细胞通道的功能，而且操作时间长，不适合药理学和毒理学研究快速的要求，因此我们采用机械分离、未经酶消化的小鼠粗线期精母细胞，应用全细胞膜片钳技术研究了Ca²⁺通道的特性。结果显示：小鼠精母细胞Ca²⁺电流在-60mV时激活，-30mV时达到最大值，翻转电位约为48mV；峰电流时间和失活时间常数呈电压依赖性变化，常数改变e倍，电压变化分别为13.11mV、9.72mV；半数激活电压-49.43±0.62mV，激活斜率因子6.05±0.55mV，在-65mV到-10mV通道开放；半数失活电压-60.79±0.24mV，失活斜率因子4.71±0.22mV，在-90mV到-40mV通道失活；二价无机阳离子对Ca²⁺电流的抑制作用Ni²⁺＞Cd²⁺，符合T型Ca²⁺通道的特征。但与Darszon等研究结果比较，通道失活时间减慢，开放时 南京医科大学硕士学位论文间延长，分析可能是未经酶消化的细胞更接近生理状态。第H部分：氰戊菊酯对小鼠粗线期精母细胞T型Ca卜通道的影响 精子与卵子结合前必须完成一些重要的生理过程，包括获能和顶体反应。顶体反应是精卵融合的前提，只有完成顶体反应的精子才能和卵子结合。T型时”通道介导的Ca’”内流是顶体反应中的关键环节。本研究采用全细胞膜片钳枝术观察了氰戊菊酯仰，Fen)对小鼠粗线期精母细胞T型Ca》通道的作用。结果表明氰戊菊酯对 T型 Cay电流有明显的抑制作用，呈现浓度依赖性关系，1卜molL”‘氰戊菊酯抑制作用最大，继续升高浓度并不能增强抑制作用。氰戊菊酯对CaZ“通道的作用缓慢，前3分钟观察不到影响，约94 分钟抑制作用最大，冲洗后难以恢复。这与氰戊菊酯对神经细胞Na“通道的作用相似，分析其原因，可能是＊型拟除虫菊酯高脂溶性，与细胞膜脂质层结合紧密，分离速度慢，缓慢到达作用靶点。氰戊菊酯对Ca‘”通道的激活和失活均有影响：通道激活阈值降低，失活离子通道数目增加。激活曲线和失活曲线均向超级化方向移动约 14mV，提示通道的开放时间没有发生大的变化。氰戊菊酯可能是在静息状态影响 T型 Ca》通道，使 T型 CaD通道开放数目减少，Ca卜通透性降低。生精细胞T型Cah通道调节细胞分化与成熟，氰戊菊酯的抑制作用提示其影响生精过程。T型CaZ”通道也存在于成熟精子，氰戊菊酯对 T型 Ca‘”电流的抑制，使进入精子细胞内的 Ca‘”减少，抑制精子顶体反应的友生，这与我们以前的研究结果一致。第三部分 氰戊菊酯对小鼠粗线期精母细胞钙调蛋白的作用 虽然对哺乳动物精母细胞T型Cah通道的药理、生理特性已经研究的较清楚，但对其调节机制还了解不多、资料表明钙调蛋白一一个胞内受体蛋白，参与钙通道的反馈调节；而氰戊菊酯神经毒性的研究显示其可抑制钙调蛋白…，C洲)的活性。鉴于此我们在小鼠精母细胞通道水平上观察了氰戊菊酯对钙调蛋白的作用。实验显示将10卜mol·L”’钙调蛋白通过电极内液注入细胞，氰戊菊酯对 南京医科大学硕士学位论文T型Ca’”通道的抑制作用减弱，提示氰戊菊酯可抑制钙调蛋白的活性；改变钙调蛋白对 T型 Cah通道的调节作用，降低 CaZ”通道的通透性，使进入胞内Ca’”减少，从而影响精子生理过程。更多还原

【Abstract】 Part One: Properties of T-type Ca2+ currents in mouse pachytene spermatocytes using patch clampCa2+ is an essential element in all ions influenced physiological processes for mammal sperm. Intracellular Ca2+ is the key to the aspects including spermatogenesis, mature, capacitation and acrosome reaction. The voltage dependent calcium channels is one of main paths to controlling Ca2+ influx and T-type Ca2+ channel is the main Ca2+ current carrier in mouse spermatocyte and sperm. Previous study on T-type Ca2+ channel in mouse spermatogenic cells adopted the method of enzyme digestion which we have known could affect the channel function in cells and it would take too much time for the test to meet the requirement of quickness in pharmacology and toxicology. So properties of Ca2+ channels in acutely dissociated mouse spermatogenic cells devoid of enzyme digestion were studied using whole-cell patch clamp technique. Ca2+ currents were obtained by stepping membrane potential to voltage between -80 and +10mV, in 10mV increments from a holding potential of -90mV. The threshold for Ca2+ current activation was at about -60mV, thepeak amplitude occurred at -30mV and the reversal potential about was 48mV. The constant of both the peak time and the time of inactivation is voltage dependent, kinetics changing with e-fold and the voltage change is 13.11 and 9.72mV, respectively. Half-activation value of -49.43+0.62 mV and steepness parameter of 6.05+055mV were obtained from the data, and activation occurs between -65 and -10mV. Half-inactivation value and steepness parameter averaged -60.79+0.24mV, 4.71+0.22mV, and inactivation occurs from -90 to -40mV. Kinetics of activation and inactivation show that Ca2+ channels presented in mouse spermatogenic cells belong to T-type channels. Accordingly, NiCl2(100umol/L and CdCl2(100umol/L) reduced current amplitude by 62.48% and 20.03%, respectively. Compared with the results reported by Darszon, time constant of inactivation was significant elongation resulted from its good behavior.Part Two Potent mechanism of fenvalerate on T-type Ca2+ channels in mouse pachytene spermatocytesSperm must undergo some crucial physiological courses before fertilization, including capacitation and acrosome reaction. Only acrosome-reacted sperm can penetrate the egg and fertilization. During the course of increase of intracellular Ca2+ plays an important role in acrosome reaction and the main path of Ca2+ influx is through T-type Ca2+ channels. In the study, the effect of fenvalerate on T-type Ca2+ channel in mouse pachytene spermatocytes, was observed using patch clamp technique. The results showed that fenvalerate had a significant inhibitory effect on the currents presented in mouse pachytene spermatocytes in a dose-dependent manner. The maximum inhibitory rate of the currents was reached in the presence of 1 micromolar fenvalerate. It took 3 min to appear any sign of effects and 9-1 Omin reached maximum inhibition after addition of fenvalerate to the bath, which suggests that the slow rate offenvalerate action was controlled by the rate at which fenvalerate moves and unbinds from membrane lipid phase. And the current was irreversible after the drug removal. Analysis of the kinetics of Ca2+ channels indicated that fenvalerate could affect both the activation and inactivation of Ca2+ channel. The curves of activation and inactivation were shifted in hyperpolarizing direction for about 14 mV, suggesting the open time of Ca2+ channel was unchanged. It was indicated the change of permeability of the channel to ions accounts for the inhibition of fenvalerate on Ca2+ current. Fenvalerate may block the course of spermatogensis through inhibiting T-type Ca2+ current, which could mediate differentiation and maturation. These Ca2+ channels are also likely to be present in these cells, where they may contribute to the Ca2+ influx required to trigger the acrosome reaction. Thus the reduction of Ca2+ influx by fenvalerate resulted in the suppression of acrosome reaction.Part Three The eff更多还原