本文關(guān)鍵詞：微管對心肌細胞線粒體功能及能量代謝與電生理影響研究　出處：《第三軍醫(yī)大學(xué)》2008年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 心肌細胞 微管 線粒 VDAC 電生理

【摘要】： 我們對“休克心”發(fā)生機理的長期研究中發(fā)現(xiàn),在心肌缺血缺氧早期,微管即發(fā)生顯著破壞,而微管在缺氧引起的一系列效應(yīng)中所起的作用及對缺氧所致的心肌細胞的能量代謝障礙的影響及其發(fā)生機制目前罕見報道。作為細胞能量供應(yīng)核心細胞器的線粒體,在缺氧條件下細胞狀態(tài)的改變中起至關(guān)重要的作用。線粒體是心肌細胞缺氧損害的核心靶細胞器,線粒體損害是“休克心”及全身性缺氧損害的最關(guān)鍵環(huán)節(jié)。線粒體在細胞缺氧性損害中起重要作用,不僅因其是細胞生物反應(yīng)過程重要的能量供應(yīng)者,他們還能通過其膜上的通透性轉(zhuǎn)換孔(mPTP)直接參與啟動細胞的壞死和凋亡。我們以往的研究發(fā)現(xiàn),mPTP的開放引起線粒體通透性轉(zhuǎn)換(MPT),導(dǎo)致線粒體基質(zhì)膨脹、外膜破裂,凋亡信號分子從內(nèi)外膜間釋放,引起細胞的壞死和凋亡;mPTP的開放導(dǎo)致線粒體的不可逆損傷是缺氧性心肌細胞損害的關(guān)鍵環(huán)節(jié)。 而微管與線粒體之間有密切的聯(lián)系。近年來,對細胞骨架的深入研究表明,微管除了作為胞內(nèi)的剛性物質(zhì),具有錨定亞細胞結(jié)構(gòu)如線粒體、高爾基體、細胞核等而對細胞起穩(wěn)定性作用外,還參與調(diào)節(jié)信號轉(zhuǎn)導(dǎo)、核轉(zhuǎn)錄及蛋白質(zhì)合成等。細胞骨架除了對線粒體胞內(nèi)定位及分布起一定作用外,還可能參與線粒體呼吸功能的調(diào)節(jié)過程。我們前期在乳鼠心肌細胞缺氧的研究中發(fā)現(xiàn),缺氧條件下微管破壞可導(dǎo)致線粒體通透性轉(zhuǎn)換孔MPTP的持續(xù)開放,進而使線粒體呼吸功能下降,提示微管對乳鼠心肌細胞線粒體具有重要的調(diào)節(jié)作用,而這一具體調(diào)控環(huán)節(jié)尚不清楚。由于VDAC是MPTP在線粒體外膜上的通道蛋白,故我們推測,微管可能是通過某種未知機制對VDAC的開放狀態(tài)產(chǎn)生影響。通過上述途徑改變,微管影響了細胞的產(chǎn)能,進一步使得心肌細胞的功能產(chǎn)生各種不同的影響。 作為心肌細胞,在功能研究中,最重要的是其電生理活性,與能量代謝息息相關(guān),破壞微管后,大鼠成體心肌細胞的電生理活性改變,罕見報道。 在心臟缺氧的實驗研究中,大鼠成體心肌細胞與乳鼠心肌細胞相比,存在顯著的差異,由于成體心肌細胞已經(jīng)分化成熟,其自我修復(fù)能力遠較乳鼠細胞差,其對缺氧的代償能力也較差。在活性及功能研究方面,成體細胞更加接近整體水平,其結(jié)果更具有說服力。對原代培養(yǎng)的成體心肌細胞而言,容易受外界各種環(huán)境影響,故培養(yǎng)的難度較大,但對缺氧及各種實驗施加因素更為敏感。另外原代培養(yǎng)的成體心肌細胞背景更加一致,試驗數(shù)據(jù)穩(wěn)定、可信度高。在其結(jié)構(gòu)、功能方面則區(qū)別更大,其線粒體的分布更加具有規(guī)律,各項電生理及收縮功能發(fā)育完善,有利于深入的機理研究�；谏鲜鎏攸c,本研究采用大鼠成體心肌細胞作為研究對象,并利用一定濃度的秋水仙堿固定時間處理細胞,模擬缺氧條件下的微管解聚狀態(tài),作為單一實驗處理因素,觀察大鼠成體心肌細胞的各種指標(biāo)變化,以分析單純微管解聚所引起的各種變化。 本研究假設(shè):微管破壞可能通過改變線粒體的亞細胞定位;通過某中間微管相互作用蛋白分子對VDAC進行調(diào)控,使VDAC開放增加,線粒體活性下降,兩條途徑加重心肌細胞能量代謝障礙,進而使其功能產(chǎn)生改變,細胞膜電生理活性下降。研究目的 應(yīng)用微管解聚劑模擬缺氧條件下微管的破壞,研究微管解聚對心肌細胞線粒體功能及能量代謝與電生理的影響,分析其可能機制,深入探討微管解聚在缺氧過程中的作用。 材料和方法 1、成年大鼠心肌細胞培養(yǎng) 2、利用8μM微管解聚劑秋水仙堿(colchicine)作用于大鼠成體心肌細胞,模擬缺氧引發(fā)的微管解聚狀態(tài)。實驗分組為正常對照組(N組)及紫杉醇微管解聚組(C組)。 3、利用免疫細胞化學(xué)染色觀察N組、C組心肌細胞聚合態(tài)微管、線粒體形態(tài)及分布變化規(guī)律,Western blot法檢測各組心肌細胞聚合態(tài)微管蛋白含量變化。 4、利用四甲基羅丹明乙酯(TMRE)檢測線粒體內(nèi)膜電位;使用免疫印記法檢測胞漿中細胞色素C含量變化;運用MTT法測定細胞活性;運用乳酸測定試劑盒檢測心肌細胞內(nèi)乳酸濃度。 5、高效液相色譜測定心肌細胞中ATP、ADP、AMP含量。 6、利用酵母雙雜交實驗系統(tǒng),以VDAC為誘餌在肝細胞文庫中篩選可能與其有相互作用的蛋白,對實驗結(jié)果進行酵母回轉(zhuǎn)驗證,并對篩選出的蛋白進行免疫組化細胞共定位研究;進一步實驗結(jié)果進行生物信息學(xué)研究。 7、應(yīng)用膜片鉗技術(shù)全細胞紀錄方法,紀錄心肌細胞膜電容、動作電位、鈉電流(INa)、鈣電流(ICa),并應(yīng)用Axon公司的pClamp8.1軟件中的Clampit進行數(shù)據(jù)分析。 主要結(jié)果 1、正常乳鼠心肌細胞微管圍繞核周呈放射狀排列,微管管狀結(jié)構(gòu)清晰。正常大鼠成體心肌細胞微管部分圍繞核周排列,其他呈線性沿細胞長軸方向平行排列。C組乳鼠心肌細胞細胞微管結(jié)構(gòu)遭受破壞,大鼠成體心肌細胞微管沿肌小節(jié)縱軸方向規(guī)律排列破壞,表現(xiàn)為免疫熒光強度減弱,微管結(jié)構(gòu)的連續(xù)性喪失,變得粗糙且不光滑,微管結(jié)構(gòu)不清晰,且呈特征性卷曲狀結(jié)構(gòu)。WB結(jié)果顯示:C組心肌細胞聚合態(tài)微管蛋白含量較N組明顯減少;成體心肌細胞減少程度較乳鼠有顯著增加。 2、正常成體心肌細胞線粒體呈橢圓或長桿狀,沿細胞長軸分布,與各肌束間呈線性均勻分布。大鼠成體心肌細胞微管呈線性管狀分布,與心肌纖維方向平行,VDAC顯示的線粒體呈顆粒裝分布,其分布方向與微管相同,并重疊其上,提示成體心肌細胞線粒體沿微管分布。C組線粒體的分布散亂,失去規(guī)律性。 3、C組線粒體內(nèi)膜電位較N組明顯降低,表現(xiàn)為線粒體熒光強度減弱;C組心肌細胞胞漿中細胞色素C含量較正常對照明顯增高。 4、微管解聚后心肌細胞與正常對照相比ATP含量下降、ADP、AMP含量上升,ADP / ATP明顯升高,能荷下降;細胞活性明顯降低;心肌細胞內(nèi)乳酸含量下降。 5、應(yīng)用酵母雙雜交技術(shù),在人肝臟文庫中篩選出VDAC的可能相互作用蛋白為DYNL1、PTPRH,經(jīng)酵母回轉(zhuǎn)驗證結(jié)果為陽性。生物信息學(xué)分析結(jié)果提示,本實驗研究發(fā)現(xiàn)VDAC-DYNL1,VDAC-PTPRH的兩對相互作用分子,目前未見報道,為新的可能存在的相互作用蛋白,DYNL1與微管有明確的相互作用。 6、與N組相比較,C組靜息電位(rest potential, RP)無顯著變化;而連續(xù)動作電位(action potential, AP)的形狀發(fā)生顯著改變,N組心肌細胞連續(xù)AP形態(tài)一致,動作電位振幅(action potential amplitude,AMP)峰值一致,復(fù)極化時動作電位持續(xù)時間(action potential duration,APD)APD時長一致,C組AP形態(tài)不穩(wěn)定,AMP峰值大小不一,APD時長明顯減小。微管解聚組APD20、APD50和APD90較對照組明顯縮短。 7、微管解聚后INa電流顯著增加;I/V曲線結(jié)果提示微管解聚組電流密度在-50～-20 mV的電壓范圍內(nèi)均明顯高于正常對照組。兩組Ica電流密度一電壓曲線均一致,幾乎重疊,微管解聚組與對照組組間無明顯差別。ICa有明顯的電壓依賴性,去極化電壓正于一40mV時ICa被激活,去極化電壓至-10mV時ICa最大。 討論與結(jié)論 1、微管與線粒體在成體心肌細胞內(nèi)分布方向一致。微管解聚后心肌細胞線粒體的排列分布規(guī)律紊亂。 2、微管解聚使大鼠成體心肌細胞活性顯著下降,推測為微管解聚使細胞內(nèi)能量生成單元崩解,降低了心肌細胞的能量供應(yīng)。 3、微管解聚使心肌細胞線粒體膜電位降低,細胞色素C漏出增加,表明微管對線粒體VDAC存在調(diào)控作用。 4、微管解聚抑制心肌細胞糖酵解。心肌細胞內(nèi)糖酵解酶依附于微管,按照一定比例及次序排列,構(gòu)成最佳的快速產(chǎn)能效應(yīng)。微管解聚后,這種規(guī)律排列遭到嚴重破壞,糖酵解產(chǎn)能效率受到抑制,故能量生成減少,相應(yīng)乳酸生成減少。 5、應(yīng)用酵母雙雜交技術(shù),在人肝臟文庫中篩選出VDAC的可能相互作用蛋白為DYNL1、PTPRH,經(jīng)酵母回轉(zhuǎn)驗證結(jié)果為陽性。生物信息學(xué)分析結(jié)果提示,本實驗研究發(fā)現(xiàn)VDAC-DYNL1,VDAC-PTPRH的兩對相互作用分子,目前未見報道,為新的可能存在的相互作用蛋白,DYNL1與微管有明確的相互作用。DYNL1可能為微管對線粒體VDAC進行調(diào)控作用的中間蛋白。PTPRH可能對線粒體VDAC具有調(diào)控作用,其信號傳導(dǎo)途徑可能為ERK-MAPK-PTPRH---VDAC 6、微管解聚使心肌細胞電生理發(fā)生改變,可能導(dǎo)致心律加快增加能量消耗及誘發(fā)心律失常。其機理可能為微管解聚使INa電流顯著增加所致。微管解聚可使游離態(tài)αtubulin,βtubulin二聚體增加,使得GTP信號激活,進而對細胞膜INa產(chǎn)生調(diào)節(jié)。微管解聚對L-鈣通道電流(ICa-L)無顯著影響,但由于APD縮短,總鈣離子內(nèi)流減少,使心肌收縮力下降。
[Abstract]:We found that the "long term study on pathogenesis of cardiac shock in early myocardial ischemia and hypoxia, which is significant microtubule damage, and the effect of energy metabolism plays a series of effects of microtubules in hypoxia induced in vitro and on myocardial cell induced by hypoxia and the vigorous system is rarely reported. As the cell energy the supply of core organelles mitochondria play a vital role in cells under hypoxic conditions change. Mitochondria are the core target organelles of myocardial hypoxia damage, mitochondrial damage is the key link of cardiac shock and systemic hypoxia damage. Mitochondria play an important role in cell hypoxia injury, not only because of its is an important energy supplier cell biological process, they will be able to use the membrane permeability transition pore (mPTP) directly involved in initiating cell necrosis and apoptosis. We Previous studies have found that mPTP caused the opening of mitochondrial permeability transition (MPT), leading to mitochondrial matrix swelling, membrane rupture, apoptotic signal molecules released from the inner and outer membrane, causing necrosis and apoptosis; irreversible injury induced mitochondrial mPTP open is a key link of myocardial hypoxia damage.
But there is a strong relationship between microtubules and mitochondria. In recent years, in-depth study of the cytoskeleton showed that microtubules except as rigid intracellular substances, has anchored subcellular structures such as mitochondria, Golgi, nucleus and the cell stability effect, but also involved in the regulation of signal transduction, transcription and protein synthesis. In addition to the cytoskeletal distribution and localization of intracellular mitochondria play a role, the adjustment process may also be involved in mitochondrial respiratory function. We found in the previous study of hypoxic myocardial cells of neonatal rats in hypoxia micro tube disruption can lead to continuous opening of mitochondrial permeability transition pore MPTP, and mitochondrial respiratory function decline, suggesting that microtubule has an important role in the regulation of mitochondria in rat myocardial cell, and the specific regulation is not clear. Because the VDAC is MPTP in the mitochondrial membrane channel Therefore, we speculate that microtubules may have an effect on the open state of VDAC through some unknown mechanism. Through these pathways, microtubules affect the production of cells, and further affect the function of cardiac myocytes.
As a cardiac muscle cell, the most important function is its electrophysiological activity, which is closely related to the energy metabolism. After destroying the microtubule, the electrophysiological activity of adult rat cardiomyocytes has been rarely reported.
In the experimental study of cardiac hypoxia in rat adult cardiomyocytes and cardiomyocytes were compared, there are significant differences, because adult cardiomyocytes had matured, the self repair ability than cells of neonatal rats, the hypoxia compensatory ability is poor. In the study of activity and function of adult the cell more close to the overall level, the result is more convincing. The myocardial cells into primary cultured, susceptible to external environmental impact, so the training is difficult, but on the hypoxia and various experimental factors applied more sensitive. The background of myocardial cell body into another primary culture more consistent, test data stable and reliable. In the structure, function of the difference is bigger, the distribution of mitochondria is more regular, the electrophysiological and contractile function of development, is conducive to the further study on the mechanism. Based on the above characteristics, the Study on the adult cardiomyocytes of rats as the research object, and using the fixed time of colchicine treated cells in certain concentration, state of microtubule depolymerization under simulated hypoxia, as single treatment factors, observe the changes of various indexes of myocardial cells of rats, changes in microtubule depolymerization caused by simple analysis.
The research hypothesis: the destruction of microtubules by subcellular localization changes of mitochondria; interaction through a middle molecular regulation of VDAC tubulin, VDAC increases, mitochondrial activity decreased, two ways to increase myocardial energy metabolic disorder, and thus the function change, decreased cell membrane electrophysiological activity. The purpose of the study
Microtubule depolymerization agent was used to simulate the destruction of microtubules under anoxic condition. The effects of microtubule depolymerization on mitochondrial function and energy metabolism and electrophysiology of cardiomyocytes were studied, and its possible mechanism was analyzed. The role of microtubule depolymerization in anoxia process was further discussed.
Materials and methods
1, adult rat cardiomyocyte culture
2, we used 8 micron M microtubule depolymerization agent, colchicine (colchicine), to treat adult rat cardiomyocytes, simulating the microtubule depolymerization state induced by hypoxia. The experiment was divided into normal control group (group N) and paclitaxel microtubule depolymerization group (group C).
3, immunocytochemical staining was used to observe the changes in the morphology and distribution of myocardial microtubules and mitochondria in group N and group C, and the changes of aggregated tubulin content in each group were detected by Western blot.
4, the mitochondrial potential was detected by four methyl Luo Danming ethyl ester (TMRE). The content of cytochrome C in cytoplasm was detected by immuno imprinting. Cell activity was detected by MTT assay, and lactate concentration in cardiac muscle cells was detected by lactate test kit.
5, high performance liquid chromatography (HPLC) was used to determine the content of ATP, ADP and AMP in cardiac myocytes.
6, using the yeast two hybrid experiment system, using VDAC as a bait in liver cDNA library screening and interacting proteins by yeast, rotary verification of the experimental results, and the screened protein of cells were co localization; further experimental results in bioinformatics research.
7, patch clamp technique was used to record myocardial cell membrane capacitance, action potential, sodium current (INa) and calcium current (ICa). The data were analyzed by Clampit in Axon's pClamp8.1 software.
Main results
1, the normal rat myocardial cell microtubules around the nucleus are arranged radially, microtubule tubular structure clear. Normal rats into myocardial cells arranged around the perinuclear microtubule body part, the other is linear parallel arranged along the long axis of the cell group.C rat myocardial cell microtubule structure damaged, rat myocardial cell microtubules along the longitudinal axis of the regular arrangement of sarcomere damage, showed decreased fluorescence intensity, continuous loss of microtubules, become rough and not smooth, microtubule structure is not clear, and a characteristic structure of curl.WB showed that myocardial cells of C group of polymeric tubulin content was significantly reduced compared with N group; adult cardiomyocytes was reduced significantly in neonatal rats.
2, normal myocardial mitochondria of body oval or long rod, distributed along the cell axis, a linear and uniform distribution of each muscle. The rat myocardial cell body into microtubules showed a linear tubular distribution, parallel with myocardial fiber orientation, VDAC display of the mitochondria in the particle loaded distribution, its distribution and the same direction with microtubules. The overlap, suggesting that into myocardial mitochondria along microtubules distribution group.C mitochondria scattered, lost regularity.
3, the mitochondrial potential of C group was significantly lower than that of N group, showing a decrease in mitochondrial fluorescence intensity. The cytochrome C content in cytoplasm of C group was significantly higher than that of normal control group.
4, after the microtubule depolymerization, the content of myocardial cells decreased compared with normal contrast. The content of ADP and AMP increased, ADP / ATP increased significantly, the energy load decreased, cell activity decreased significantly, and lactate content in myocardial cells decreased.
5, using the yeast two hybrid technique in human liver cDNA library, screened for proteins interacting with DYNL1, PTPRH VDAC, after the yeast rotary verification result is positive. Bioinformatics analysis showed that the experimental study found that two of VDAC-DYNL1, the interaction of VDAC-PTPRH molecules, there are no reports, for interacting proteins there are new possibilities, DYNL1 and microtubule interactions clear.
6, compared with the N group, C group (rest potential, RP of resting potential) showed no significant changes; and continuous action potentials (action potential, AP) the shape changed significantly, the myocardial cells of N group AP consistent shape, amplitude of action potential (action potential, amplitude, AMP) peak, repolarization of action potential duration (action potential, duration, APD) APD long C group, AP form is not stable, the peak value of AMP size, APD length was significantly reduced. The microtubule depolymerization groups APD20, APD50 and APD90 was significantly shorter than the control group.
7, INa current increased significantly after microtubule depolymerization; I/V curve showed that group of microtubule depolymerization current density in the control group -50 ~ -20 voltage range in mV were significantly higher than those in group Ica were consistent. Two, current density voltage curves almost overlap, microtubule depolymerization group and the control group had no significant difference between groups.ICa dependent obviously the voltage is a 40mV voltage depolarization when ICa is activated, depolarizing voltage to -10mV ICa.
Discussion and conclusion
1, the distribution of microtubules and mitochondria in the adult cardiomyocytes is the same. After the microtubule depolymerization, the distribution of mitochondria in cardiac myocytes is irregular.
2, microtubule depolymerization significantly reduced the activity of adult rat cardiomyocytes, suggesting that microtubule depolymerization can disintegrate energy generation units and reduce the energy supply of cardiomyocytes.
3, microtubule depolymerization made the mitochondrial membrane potential of myocardial cells decreased and the leakage of cytochrome C increased, indicating the regulation of microtubules on mitochondrial VDAC.
4, microtubule depolymerization inhibition of myocardial cell glycolysis. Intracellular glycolytic enzymes attached to microtubules, arranged according to a certain proportion and order, a capacity for rapid effects. After the regular arrangement of microtubule depolymerization, was severely damaged, the production efficiency of glycolysis is inhibited, so the energy production is reduced, the corresponding reduction of lactic acid.
5, using the yeast two hybrid technique in human liver cDNA library, screened for proteins interacting with DYNL1, PTPRH VDAC, after the yeast rotary verification result is positive. Bioinformatics analysis showed that the experimental study found that two of VDAC-DYNL1, the interaction of VDAC-PTPRH molecules, there are no reports, for interacting proteins there are new possibilities, DYNL1 and microtubule interacting.DYNL1 may regulate microtubule specific effects on mitochondrial VDAC intermediate protein.PTPRH may play a role in the regulation of mitochondrial VDAC and its signal transduction pathway may be ERK-MAPK-PTPRH---VDAC
6, the microtubule depolymerization in cardiomyocytes electrophysiological changes, may lead to increased energy consumption and heart rate induced arrhythmia. The possible mechanism is that microtubule depolymerization was significantly increased by INa current. The microtubule depolymerization free alpha tubulin beta two tubulin dimer increase, making GTP activation signal, and then produce the regulation on cell. INa on L- membrane microtubule depolymerization calcium current (ICa-L) had no significant effect, but due to the shortening of APD, the total decrease of calcium influx, which decreased myocardial contractility.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R363

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