【作者】 康喜讯；

【导师】 孔维佳；

【作者基本信息】 华中科技大学 ， 耳鼻咽喉科学， 2008， 博士

【摘要】 第一部分光学倒置相差显微镜下前庭囊斑两型毛细胞的形态学鉴别目的:探索电生理实验中,光学倒置相差显微镜下较为可靠的前庭椭圆囊斑和球囊斑所分离的两型单离毛细胞的辨别标准,为合理分析所记录到的电流打下比较可靠的基础。方法:对于豚鼠椭圆囊斑和球囊斑分离所得的单离前庭毛细胞,分别在光学倒置相差显微镜下对细胞胞体的长度、宽度以及表皮板纤毛的长度进行显微测量,并对长和宽的比例值以及细胞体的长度和纤毛长度的比例值即胞长比进行分析。结果:经过极低浓度胶原酶配合轻微机械吹打得到豚鼠椭圆囊斑和球囊斑的两型单离的前庭毛细胞。这两种毛细胞经过显微测量后相比,两种细胞之间细胞的宽度和纤毛的长度统计学比较没有显著性差异。有显著统计学差别的是和囊斑II型毛细胞相比,囊斑I型毛细胞的长度明显较长,长宽比比较大,纤毛胞长比比较小。结论:在既往以是否具有明显狭窄的颈部来区分两型前庭毛细胞的基础上,结合分析囊斑细胞的长宽比以及纤毛胞长比进一步进行辨别,有助于更准确地区分两型毛细胞,不失为区别囊斑两型毛细胞的有效辅助方法。第二部分前庭椭圆囊I型毛细胞的钾电流目的:通过应用电生理的方法来研究豚鼠椭圆囊I型毛细胞存在的通道电流及其特性,并对所记录的电流进行初步鉴别。方法:利用膜片钳全细胞和单通道两种方法,在标准外液中,对单离的豚鼠椭圆囊I型毛细胞记录到的电流进行记录和分析,并应用钾通道抑制剂TEA进行鉴别。结果:标准细胞外液中测得的全细胞电流含有内向电流成分和外向整流成分,应用TEA阻滞后电流完全消失,提示为钾电流。单通道贴附式和内面向外两种模式下电流均为较大的外向电流,被TEA阻滞后所记录的电流特性不同,提示记录到的为大的外向钾电流,并且被TEA阻滞时贴附式和内面向外两种模片上经过不同的阻滞途径。贴附式时TEA清除后电流不再出现,内面向外情况下TEA清除后电流完全恢复。结论:在标准细胞外液中豚鼠椭圆囊I型毛细胞表面存在较大的钾通道电流,可以被TEA阻滞,膜片钳单通道贴附式情况下阻滞不可逆,内面向外情况下阻滞可逆。第三部分钙和乙酰胆碱对前庭椭圆囊I型毛细胞钾电流的调节目的:研究钙离子和乙酰胆碱对单离的豚鼠椭圆囊I型毛细胞所记录到的大电导的钾电流是否有调节作用,期望从电生理学方面为豚鼠椭圆囊I型毛细胞表面可能存在乙酰胆碱受体提供佐证。方法:将细胞外液分为三种:标准外液、高钙外液、含有乙酰胆碱的高钙外液。并把标准外液组定位对照组。分别用膜片钳单通道的方法对单离的椭圆囊I型毛细胞外向的钾电流进行记录。组间应用两两比较的方法对结果进行统计学分析。结果:在标准外液中引出可靠钾电流后,钙离子终浓度为2.0mM的高钙外液使原来在标准外液中记录到的钾通道电流明显增加,电流开放的幅值和开放概率均发生明显变化。但也有部分钾电流表现不同:钙离子虽然也提高了电流的幅值,却降低了通道的开放概率。钙离子联合应用乙酰胆碱除可以明显提高钾电流的幅值外,还恢复了通道的开放概率。在标准外液中应用乙酰胆碱无效。结论:钙离子可以通过提高钾电导而减少通道开放次数起到稳定细胞膜的作用。加用适量的乙酰胆碱可以提高钾通道的兴奋性,这个过程在离体的细胞环境下需要高浓度的钙离子参与。更多还原

【Abstract】 PART1 Comparative morphology of two type’s hair cells from saccule and utricle under inverted phase contrast light microscopeObjective: To explore more reliable standards of vestibular hair cells of saccule and utricle from guinea pig, prepared in studies with patch clamp technique under inverted microscope, and found a more reliable base for analyzing the currents recorded in the coming research.Methods: Under inverted phase contrast light microscope, two type’s hair cell’s length, width and length of cilia were measured. Ratios of length, width and length of cilia were calculated and all numbers were analyzed statistically.Results: We found that width and length of cilia of two type’s hair cells in saccule and utricle from guinea pig are same. Because the length of type I hair cell is longer than that of type II, against type II hair cell, the ratio between length and width is larger and the ratio of the length between cilia and cell body is smaller.Conclusions: Two type’s vestibular hair cells of saccule and utricle from guinea pig may be distinguished through the ratio of cell body’s length and width ,even the ratio of the length between cilia and cell body, in addition to the standards before. PART 2 Identification of K+ channels in utricular type I hair cellsObjective: To study the characteristics of currents recorded in type I utricular hair cells with different patch clamp techniques, and the channels observed to be primarily identified.Methods: Currents were recorded with whole cell recording and single channel recording in standard external solution. Both currents with different methods were analyzed and identified with TEA.Results: Whole cell currents in standard external solution have both inward part and outward rectified part. All currents disappeared with TEA, which indicated that they were potassium currents. Different results were recorded under cell attached and inside out mode with TEA, which showed they played the different mechanics in different modes. After washout TEA, currents weren’t refounded in cell attached mode, while all currents recovered absolutely in inside out mode.Conclusions: Potassium channels are distributed in guinea pig type I utricular hair cells, with large conductance, and can be obstructed by TEA. The process can be reversed in inside out mode but not in cell attached mode. PART 3 Potassium currents modulated by calcium and Ach in utricular type I hair cellsObjective: To explore whether Ach or Ca can modulate the potassium currents with large conductance recorded in type I utricular hair cells. And hope to find verifications of AchR in type I utricular hair cells with patch clamp technique.Methods: Single channel recording were used to record currents from type I utricular hair cells in 3 different solutions, standard external solution, high-calcium solution, and high-calcium solution with Ach. Currents recorded in standard external solution were control. Both of three were analyzed statistically.Results: After potassium currents were recorded in standard external solution, 2.0mMCa2+ can increase both amplitude and the probability of opening of the currents. Amplitude of single channel currents increased obviously with 2.0mMCa2+ or with both 2.0mMCa2+ and 0.5mMAch in external solution. But not always, in some cells only increasing Ca2+ without Ach decreased the probability of opening (Po), which would increase obviously after 0.5mMAch were used.Conclusions: Ca2+ can stabilized membrane through increasing Amplitude of single channel currents and decreasing probability of opening (Po). 0.5mMAch can enhance the excitability of potassium channels in type I utricular hair cells, but the process need high concentration of calcium under the circumstance of unitary cell in vitro.更多还原