本文選題：心肌 + 縫隙連接　； 參考：《第四軍醫(yī)大學(xué)》2016年博士論文

【摘要】：【背景】縫隙連接是位于心肌細(xì)胞之間閏盤部位的一種非選擇性物質(zhì)交換通道,允許小于1 k Da的小分子物質(zhì),例如離子、第二信使分子等通透,實(shí)現(xiàn)細(xì)胞漿的部分物質(zhì)交換與信息傳遞。近年來(lái)隨著對(duì)縫隙連接研究的深入,發(fā)現(xiàn)縫隙連接依賴其可以通透物質(zhì)的特性,參與了多種生理和病理過(guò)程,例如縫隙連接可以影響細(xì)胞的轉(zhuǎn)錄翻譯過(guò)程,改變蛋白表達(dá)水平與種類,促進(jìn)凋亡信號(hào)的擴(kuò)散,增加心肌梗死面積,影響細(xì)胞周期等。用小分子物質(zhì)的通透很難解釋縫隙連接的這些功能,因此,我們推測(cè)縫隙連接的通透特性不僅限于小于1 k Da的物質(zhì),此通道可能允許大分子物質(zhì)的通透。本課題采用多種實(shí)驗(yàn)方法,系統(tǒng)地研究了縫隙連接的通透特性,以及大分子物質(zhì)通過(guò)縫隙連接在細(xì)胞間交換的作用。【目的】(1)獲得大量具有功能性縫隙連接的連體心肌細(xì)胞。(2)顯微注射檢測(cè)心肌細(xì)胞縫隙連接的物質(zhì)通透性,以及通道孔徑。(3)研究縫隙連接對(duì)心肌細(xì)胞死亡信號(hào)和生長(zhǎng)信號(hào)傳遞的作用�！痉椒ā勘狙芯恐饕捎蔑@微注射的方法,將一系列分子量不同、體積不同的熒光分子注入連體心肌細(xì)胞內(nèi),觀察分子的擴(kuò)散過(guò)程,間接測(cè)定心肌縫隙連接的通透性及通道孔徑尺寸。隨后建立在體心肌梗死、離體心肌梗死模型,檢測(cè)縫隙連接在細(xì)胞序貫死亡中的作用;建立心臟肥大模型、新生大鼠原代心肌細(xì)胞肥大模型,檢測(cè)縫隙連接在細(xì)胞生長(zhǎng)中的作用;構(gòu)建病毒載體,感染心肌細(xì)胞,研究大分子物質(zhì)經(jīng)縫隙連接在培養(yǎng)細(xì)胞間的擴(kuò)散過(guò)程與作用�！窘Y(jié)果】(1)改進(jìn)實(shí)驗(yàn)方法,獲得具有功能性縫隙連接的連體心肌細(xì)胞酶消化法急性分離成年大鼠心肌細(xì)胞,可以獲得大量高質(zhì)量的單個(gè)長(zhǎng)桿狀心肌細(xì)胞,縫隙連接結(jié)構(gòu)與功能均遭到破壞,不能滿足本實(shí)驗(yàn)的要求。為了獲得與生理狀態(tài)基本一致的縫隙連接,本研究通過(guò)改變酶消化過(guò)程中液體Ca2+濃度,增加液體中Ca2+濃度,并在5~10μmol/L范圍內(nèi)微調(diào),同時(shí)全程維持液體的p H值(7.35~7.45)在恒定范圍,可以使部分閏盤在消化過(guò)程中保持完整,消化結(jié)束后獲得大量的連體心肌細(xì)胞。顯微注射熒光分子碘化丙啶(PI,分子量668 Da)和縫隙連接阻斷劑octanol孵育細(xì)胞,兩個(gè)實(shí)驗(yàn)證實(shí)連體心肌細(xì)胞間的縫隙連接功能正常,沒(méi)有受到酶消化處理的影響產(chǎn)生功能失調(diào),因此可以采用酶消化法獲得的連體心肌細(xì)胞,針對(duì)縫隙連接的結(jié)構(gòu)與功能開展下一步的研究。(2)縫隙連接可以通透大分子物質(zhì),但是對(duì)物質(zhì)的通透性受到多種因素的影響免疫熒光方法確定心室肌表達(dá)的縫隙連接蛋白(connexin)亞型包括Cx40、Cx43、Cx45,心室肌縫隙連接是由12個(gè)connexin亞基組成的異源復(fù)合體。在現(xiàn)有的實(shí)驗(yàn)方法不能直接檢測(cè)心肌細(xì)胞縫隙連接通透性的情況下,本課題采用顯微注射熒光分子的方法間接測(cè)定縫隙連接的通透性。分別向一個(gè)心肌細(xì)胞內(nèi)注射熒光分子10 k Da、40 k Da、70 k Da、150 k Da、250 k Da或2000 k Da的Dextran-FITC,熒光顯微鏡觀察熒光分子在連體細(xì)胞間的擴(kuò)散過(guò)程,證實(shí)縫隙連接可以通透小于150 k Da的Dextran-FITC,但是隨著分子量的增加,熒光分子的擴(kuò)散速度越來(lái)越慢,說(shuō)明分子量的大小影響物質(zhì)的通透性;為觀測(cè)蛋白分子的擴(kuò)散特性,顯微注射紅色熒光蛋白(RFP)進(jìn)入細(xì)胞,發(fā)現(xiàn)RFP也可以順利通過(guò)縫隙連接,但是擴(kuò)散速度遠(yuǎn)遠(yuǎn)慢于同等分子量的Dextran-FITC,說(shuō)明物質(zhì)的電荷數(shù)和空間構(gòu)象也會(huì)對(duì)物質(zhì)的通透性產(chǎn)生影響。根據(jù)已知熒光分子Dextran-FITC的直徑,可以估測(cè)出心肌縫隙連接的孔徑約為17 nm。(3)縫隙連接可以介導(dǎo)死亡信號(hào)和細(xì)胞生長(zhǎng)信號(hào)的傳導(dǎo)縫隙連接可以促進(jìn)死亡信號(hào)的擴(kuò)散。分別構(gòu)建在體、離體心肌梗死模型,本研究發(fā)現(xiàn)在心肌梗死危險(xiǎn)區(qū),縫隙連接保持開放,會(huì)導(dǎo)致死亡信號(hào)從壞死心肌細(xì)胞蔓延至存活心肌細(xì)胞,細(xì)胞結(jié)構(gòu)首先從一端開始破壞,出現(xiàn)肌節(jié)Z線附近電子密度降低,線粒體腫脹,肌纖維斷裂,逐漸向整個(gè)細(xì)胞擴(kuò)散,最終導(dǎo)致整個(gè)細(xì)胞壞死。再灌注損傷過(guò)程中多種因素(缺氧、鈣超載、酸中毒)會(huì)降低縫隙連接的通透性,促進(jìn)縫隙連接關(guān)閉,可以阻斷死亡信號(hào)的傳遞,使存活的心肌細(xì)胞得以保持活性,減少死亡細(xì)胞數(shù)量,降低心肌梗死面積。縫隙連接可以介導(dǎo)生長(zhǎng)信號(hào)的傳遞。建立心臟壓力后負(fù)荷模型-腹主動(dòng)脈縮窄,分別觀察1d、8W、16W的心肌肥厚程度,發(fā)現(xiàn)閏盤雙側(cè)的心肌細(xì)胞生長(zhǎng)速度保持一致;肥大因子ET-1刺激新生大鼠原代培養(yǎng)細(xì)胞,48 h測(cè)定細(xì)胞表面積,重點(diǎn)測(cè)定保持相互接觸的細(xì)胞面積,因?yàn)檫@類細(xì)胞之間存在縫隙連接,發(fā)現(xiàn)相互接觸的細(xì)胞表面積沒(méi)有差異,沒(méi)有相互接觸的細(xì)胞之間表面積差別很大;結(jié)合這兩個(gè)實(shí)驗(yàn)的結(jié)果證實(shí),縫隙連接可以介導(dǎo)生長(zhǎng)信號(hào)的傳遞,協(xié)調(diào)細(xì)胞之間的生長(zhǎng)。構(gòu)建慢病毒載體感染新生大鼠心肌細(xì)胞,發(fā)現(xiàn)蛋白分子EGFP從感染病毒的細(xì)胞擴(kuò)散至未被病毒感染的細(xì)胞,證實(shí)大分子物質(zhì)增強(qiáng)綠色熒光蛋白(EGFP)可以在相互接觸的細(xì)胞之間擴(kuò)散,進(jìn)一步提供了縫隙連接可以通透大分子物質(zhì)的證據(jù)。【結(jié)論】心肌細(xì)胞縫隙連接可以通透小于150 k Da的大分子物質(zhì),通道孔徑大約為17 nm。因?yàn)閷?duì)大分子物質(zhì)的通透特性,縫隙連接可以介導(dǎo)細(xì)胞死亡信號(hào)和生長(zhǎng)信號(hào)的傳導(dǎo),影響細(xì)胞的死亡數(shù)量,協(xié)調(diào)細(xì)胞之間的生長(zhǎng)過(guò)程。
[Abstract]:[background] gap junction is a non selective material exchange channel located in intercalated disc between cardiac myocytes, allowing small molecules less than 1 K Da, such as ions, second messenger molecules to permeate and realize partial material exchange and information transfer of cytoplasm. Depending on its ability to penetrate the substance, it participates in a variety of physiological and pathological processes, such as gap junctions can affect the process of transcription and translation of cells, change the level and types of protein expression, promote the diffusion of apoptosis signals, increase the area of myocardial infarction, and affect the cell cycle. It is difficult to explain the gap junction with the penetration of small molecules. Some functions, therefore, we speculate that the permeability of gap junction is not only limited to substances less than 1 K Da, and this channel may allow the permeability of large molecular substances. A large number of continuous myocardial cells with functional gap junctions were obtained. (2) microinjection was used to detect the permeability of gap junctions and the aperture of the channels. (3) the effect of gap junctions on the death signal and growth signal transduction of cardiac myocytes. [Methods] a series of microinjection methods were used to make a series of molecular weights. Different volumes of different fluorescent molecules were injected into the conjoined myocardial cells, observed the diffusion process of the molecules, indirectly measured the permeability of the gap junction and the size of the channel aperture. Then, it was established in vivo myocardial infarction, in vitro myocardial infarction model, to detect the role of gap junction in the sequential death of cells, and to establish a model of cardiac hypertrophy and a neonatal rat. Primary cardiomyocyte hypertrophy model, detecting the role of gap junction in cell growth, constructing viral vectors, infecting cardiac myocytes, and studying the diffusion process and effect of large molecular substances through gap junction in cultured cells. [results] (1) improve the experimental method and obtain the enzyme digestion method of functional crevice connected myocardial cells. A large number of high quality single long rod-like cardiomyocytes can be obtained by sexual separation of adult rat cardiomyocytes. The gap junction structure and function are destroyed, which can not meet the requirements of this experiment. In order to obtain the gap junction which is basically consistent with the physiological state, this study increased the concentration of liquid Ca2+ in the enzyme digestion process and increased the concentration of Ca2+ in the liquid. Degree, microtuning in the range of 5~10 mu mol/L, and maintaining the P H value (7.35~7.45) at a constant range, which can keep part of the intercalated disc intact during digestion, and obtain a large number of conjoined cardiomyocytes after digestion. Microinjection of fluoro molecular iodide (PI, molecular weight 668 Da) and gap junction blocker octanol to incubate cells, The two experiments confirmed that the gap junction function between the cells of the conjoined myocardium was normal and was not affected by the enzyme digestion. Therefore, it was possible to use the enzyme digestion method to obtain the continuous myocardial cells. (2) the gap junctions can permeate the macromolecules, but to the substance. The permeability of the gap connexin (connexin) subtypes of ventricular myocytes, including Cx40, Cx43, Cx45, is a heterologous complex composed of 12 connexin subunits. The present experimental methods can not directly detect the gap junction permeability of cardiac myocytes. The permeability of gap junction was indirectly measured by microinjection of fluoro molecules. The fluorescence molecules 10 K Da, 40 K Da, 70 K Da, 150 K Da, 250 K Da or 2000 K Da were injected into a cardiac myocyte, respectively. The fluorescence microscope was used to observe the diffusion process of the fluorescent molecules in the intercellular cell, which proved that the gap junction could be penetrable and small. At 150 K Da Dextran-FITC, but with the increase of molecular weight, the diffusion rate of the fluorescent molecules is becoming slower and slower, indicating that the size of the molecular weight affects the permeability of the substance, and the diffusion characteristics of the observed protein molecules are injected into the cells by microinjection of red fluorescent protein (RFP), and it is found that RFP can also be connected smoothly through the gap, but the diffusion rate is far away. Dextran-FITC, which is slower than the same molecular weight, indicates that the number of charge and the space conformation of the substance also affect the permeability of the material. According to the diameter of the known fluorescent molecule Dextran-FITC, it can be estimated that the aperture of the gap junction of the myocardium is about 17 nm. (3) gap junction can mediate the gap junction between the dead signal and the cell growth signal. It can promote the diffusion of the death signal. In vivo and in vitro myocardial infarction model respectively. This study found that the gap junctions remain open in the dangerous area of myocardial infarction, which will cause the death signal to spread from necrotic myocardial cells to surviving myocardial cells, and the cell structure first begins to destroy from one end, and the electron density decreases near the Z line of the sarcomere. In the process of reperfusion injury, a variety of factors (hypoxia, calcium overload, acid poisoning) can reduce the permeability of gap junction, promote the closure of gap junction, block the transmission of the death signal, keep the survival of the cardiac muscle cells to maintain the activity, reduce death, and reduce death. The number of dead cells decreased the area of myocardial infarction. The gap junction could mediate the transmission of the growth signal. The cardiac stress post load model, abdominal aorta coarctation, was established to observe the degree of myocardial hypertrophy of 1D, 8W, 16W, and the growth rate of the bilateral cardiac myocytes in the intercalated disc was consistent, and the mast factor ET-1 stimulated the primary cultured cells of neonatal rats, 48 h The cell surface area was measured and the cell area kept in contact with each other was measured, because there were gap junctions between these cells, and there was no difference in the surface area of the contact cells, and the surface area between the cells without contact was very different. The results of these two experiments confirmed that gap junction could mediate the transmission of the growth signal. The growth of the cells was coordinated. The lentivirus vector was constructed to infect the neonatal rat cardiomyocytes, and the protein molecule EGFP spread from the infected cells to the cells that were not infected by the virus. It was proved that the macromolecule substance enhanced green fluorescent protein (EGFP) could spread between the cells exposed to each other, and further provided the gap junction to be permeable. Evidence of large molecular substances. [Conclusion] the gap junction of cardiac myocytes can permeate large molecules less than 150 K Da, with a channel aperture of about 17 nm. because of the permeability to large molecular substances. Gap junctions can mediate cell death signal and growth signal conduction, affect the number of dead cells and coordinate the growth of cells. Cheng.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R54

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