本文選題：大電導(dǎo)鈣激活鉀通道　切入點：Ca~(2+)載體　出處：《河北大學(xué)》2017年碩士論文

【摘要】：為了探索中華絨螯蟹(Eriocheir sinensis)(簡稱河蟹)精子大電導(dǎo)鈣激活鉀通道(large-conductance calcium-activated potassium channel,簡稱BK通道)功能和頂體反應(yīng)的關(guān)系,本研究采用副性腺蛋白溶解法獲得游離中華絨螯蟹精子,以精子為試驗對象,開展了以下研究,首先以Ca~(2+)載體A23187進行精子頂體反應(yīng)的體外誘導(dǎo),并通過Fluo-4 AM熒光標記檢測精子頂體反應(yīng)前后胞內(nèi)游離Ca~(2+)濃度的變化情況。其次,采用全細胞膜片鉗技術(shù)記錄精子的全細胞鉀電流并結(jié)合阻斷劑分析鉀通道的電生理特性及其類型,并模擬頂體反應(yīng)時Ca~(2+)濃度變化,觀察對中華絨螯蟹精子鉀電流的影響,探討鉀通道在中華絨螯蟹精子頂體反應(yīng)過程中的作用規(guī)律。主要研究結(jié)果如下:1.以不同濃度的Ca~(2+)載體A23187,分別采用ASW(artificial sea water)和Ca~(2+)-FASW(Calcium-free artificial sea water)配制,對精子進行頂體反應(yīng)誘導(dǎo),發(fā)現(xiàn)Ca~(2+)載體A23187可顯著提高精子的頂體反應(yīng)率,且發(fā)生頂體反應(yīng)的精子多處于III~IV期,最佳誘導(dǎo)濃度為80μg/m L,誘導(dǎo)時間為30 min。同時說明,Ca~(2+)在中華絨螯蟹精子頂體反應(yīng)中具有主導(dǎo)作用。2.以Ca~(2+)載體A23187誘導(dǎo)中華絨螯蟹精子發(fā)生頂體反應(yīng),同時采用鈣熒光染料Fluo-4AM進行標記,以激光共聚焦技術(shù)追蹤單個精子頂體反應(yīng)發(fā)生前后胞內(nèi)Ca~(2+)濃度的變化。研究發(fā)現(xiàn),中華絨螯蟹精子未發(fā)生頂體反應(yīng)前,Ca~(2+)主要分布于細胞質(zhì)和頂體囊的頂體管上,誘發(fā)精子發(fā)生頂體反應(yīng)的瞬間,精子內(nèi)Ca~(2+)濃度明顯升高,主要分布于細胞質(zhì)和延長的頂體管前端。此外,在無鈣緩沖液中,精子自身熒光并無顯著增強,提示頂體反應(yīng)的發(fā)生主要依賴于精子外Ca~(2+)的內(nèi)流。3.以全細胞膜片鉗技術(shù)記錄精子全細胞鉀電流,發(fā)現(xiàn)該電流由快速IA樣電流和延遲IK樣電流組成。在無鈣內(nèi)液條件下,加入BK通道阻斷劑IbTX(Iberiotoxin,10 nmol/L)后,IA樣和IK樣電流鉀電流均被顯著抑制,抑制幅度分別為17.74%和21.51%。提示中華絨螯蟹精子存在BK通道,部分通道可以被膜去極化激活。4.在內(nèi)液中加入Ca~(2+)(50μmol/L)模擬頂體反應(yīng)時Ca~(2+)內(nèi)流,發(fā)現(xiàn)精子IA樣和IK樣鉀電流顯著提高,提高幅度分別為61.49%和44.11%。說明Ca~(2+)可以增強全細胞外向鉀電流,加入IbTX后,IA樣和IK樣電流被顯著抑制,抑制率分別為27.17%和22.65%。說明BK通道在精子外向鉀電流中具有重要作用,而且對Ca~(2+)具有依賴性。以上研究結(jié)果表明,以Ca~(2+)載體A23187體外誘導(dǎo)中華絨螯蟹精子發(fā)生頂體反應(yīng)時,內(nèi)流的Ca~(2+)可激活BK通道,造成K+大量外流,改變精子膜電位,引發(fā)精子發(fā)生頂體反應(yīng)。說明BK通道在中華絨螯蟹精子頂體反應(yīng)中具有重要作用。
[Abstract]:In order to explore the Chinese Mitten Crab (Eriocheir sinensis) (the crab) of large conductance calcium activated potassium channels in sperm (large-conductance calcium-activated potassium channel, referred to as BK channel) function and acrosome reaction, this study uses accessory gland protein dissolution method to obtain free Eriocheir sinensis sperm, the sperm as test object, carry out the following research, firstly the Ca~ (2+) A23187 vector induced sperm acrosome reaction in vitro, and by detection of Fluo-4 AM fluorescence labeled sperm acrosome reaction and intracellular free Ca~ (2+) of concentration. Secondly, using the whole cell potassium current in the whole cell patch clamp techniques combined with electrophysiological blocking sperm analysis of characteristics of agent potassium channel and its types, and to simulate the acrosome reaction of Ca~ (2+) concentration changes, observe the effect of Eriocheir sinensis sperm potassium current, on potassium channels in Eriocheir sinensis sperm The top sub rule body in the reaction process. The main results are as follows: 1. with different concentrations of Ca~ (2+) vector A23187, respectively using ASW (artificial sea water) and Ca~ (2+) -FASW (Calcium-free artificial sea water) with the sperm acrosome reaction induced by Ca~ (2+) vector A23187 can significantly improve the acrosome reaction rate and acrosome reaction sperm at III~IV phase, the best induced concentration is 80 g/m L, the induction time was 30 min. at the same time, Ca~ (2+) in Eriocheir sinensis sperm acrosome reaction in the leading role of.2. in the Ca~ (2+) Eriocheir sinensis sperm acrosome reaction induced by plasmid A23187 were labeled by using calcium fluorescent dye Fluo-4AM, single sperm acrosome reaction and intracellular Ca~ by confocal laser tracking technology (2+) concentration. The study found that Eriocheir sinensis sperm acrosome reaction did not occur before Ca~ (2+), mainly distributed in the cytoplasm and acrosome acrosomal vesicle tube, induced sperm acrosome reaction of the sperm in the moment, Ca~ (2+) concentration increased significantly, mainly distributed in the cytoplasm and acrosome extended at the front end of the pipe. In addition, in the absence of calcium buffer, sperm autofluorescence was significant enhanced prompt acrosome reaction depends mainly on sperm Ca~ (2+) by whole cell potassium whole cell patch clamp recording current flow in sperm.3., found that the current by fast IA like current and delayed IK like current. In the absence of calcium in the liquid condition, adding BK channel blocker (IbTX Iberiotoxin, 10 nmol/L), IA like and IK like current potassium currents were significantly inhibited and the inhibition rate were 17.74% and 21.51%. showed that the BK channel in the presence of Eriocheir sinensis sperm, part of the channel can be activated with Ca~ membrane depolarization (2+),.4. solution (50 mol/L) to simulate the acrosome reaction at Ca ~ (2+) influx, found in sperm IA like and IK like potassium currents increased significantly, increased by 61.49% and 44.11%. Ca~ (2+) can enhance the whole cell outward potassium current, after joining IbTX, IA like and IK like current was inhibited, the inhibition rate was 27.17% and 22.65%. respectively shows that the BK channel has an important role in sperm outward potassium current, and the Ca~ (2+) has the dependence. The above results indicated that Ca~ (2+) of Eriocheir sinensis sperm acrosome reaction induced by plasmid A23187 in vitro, the flow within the Ca~ (2+) can activate BK channels, resulting in a massive outflow of K+, change the sperm membrane potential induced sperm acrosome reaction. It plays an important role in BK channel acrosome reaction in Eriocheir sinensis sperm.

【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q954.43

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