心肌细胞缝隙连接传递大分子物质及其功能

发布时间：2018-07-03 21:34

本文选题：心肌 + 缝隙连接　；参考：《第四军医大学》2016年博士论文

【摘要】：【背景】缝隙连接是位于心肌细胞之间闰盘部位的一种非选择性物质交换通道,允许小于1 k Da的小分子物质,例如离子、第二信使分子等通透,实现细胞浆的部分物质交换与信息传递。近年来随着对缝隙连接研究的深入,发现缝隙连接依赖其可以通透物质的特性,参与了多种生理和病理过程,例如缝隙连接可以影响细胞的转录翻译过程,改变蛋白表达水平与种类,促进凋亡信号的扩散,增加心肌梗死面积,影响细胞周期等。用小分子物质的通透很难解释缝隙连接的这些功能,因此,我们推测缝隙连接的通透特性不仅限于小于1 k Da的物质,此通道可能允许大分子物质的通透。本课题采用多种实验方法,系统地研究了缝隙连接的通透特性,以及大分子物质通过缝隙连接在细胞间交换的作用。【目的】(1)获得大量具有功能性缝隙连接的连体心肌细胞。(2)显微注射检测心肌细胞缝隙连接的物质通透性,以及通道孔径。(3)研究缝隙连接对心肌细胞死亡信号和生长信号传递的作用。【方法】本研究主要采用显微注射的方法,将一系列分子量不同、体积不同的荧光分子注入连体心肌细胞内,观察分子的扩散过程,间接测定心肌缝隙连接的通透性及通道孔径尺寸。随后建立在体心肌梗死、离体心肌梗死模型,检测缝隙连接在细胞序贯死亡中的作用;建立心脏肥大模型、新生大鼠原代心肌细胞肥大模型,检测缝隙连接在细胞生长中的作用;构建病毒载体,感染心肌细胞,研究大分子物质经缝隙连接在培养细胞间的扩散过程与作用。【结果】(1)改进实验方法,获得具有功能性缝隙连接的连体心肌细胞酶消化法急性分离成年大鼠心肌细胞,可以获得大量高质量的单个长杆状心肌细胞,缝隙连接结构与功能均遭到破坏,不能满足本实验的要求。为了获得与生理状态基本一致的缝隙连接,本研究通过改变酶消化过程中液体Ca2+浓度,增加液体中Ca2+浓度,并在5~10μmol/L范围内微调,同时全程维持液体的p H值(7.35~7.45)在恒定范围,可以使部分闰盘在消化过程中保持完整,消化结束后获得大量的连体心肌细胞。显微注射荧光分子碘化丙啶(PI,分子量668 Da)和缝隙连接阻断剂octanol孵育细胞,两个实验证实连体心肌细胞间的缝隙连接功能正常,没有受到酶消化处理的影响产生功能失调,因此可以采用酶消化法获得的连体心肌细胞,针对缝隙连接的结构与功能开展下一步的研究。(2)缝隙连接可以通透大分子物质,但是对物质的通透性受到多种因素的影响免疫荧光方法确定心室肌表达的缝隙连接蛋白(connexin)亚型包括Cx40、Cx43、Cx45,心室肌缝隙连接是由12个connexin亚基组成的异源复合体。在现有的实验方法不能直接检测心肌细胞缝隙连接通透性的情况下,本课题采用显微注射荧光分子的方法间接测定缝隙连接的通透性。分别向一个心肌细胞内注射荧光分子10 k Da、40 k Da、70 k Da、150 k Da、250 k Da或2000 k Da的Dextran-FITC,荧光显微镜观察荧光分子在连体细胞间的扩散过程,证实缝隙连接可以通透小于150 k Da的Dextran-FITC,但是随着分子量的增加,荧光分子的扩散速度越来越慢,说明分子量的大小影响物质的通透性;为观测蛋白分子的扩散特性,显微注射红色荧光蛋白(RFP)进入细胞,发现RFP也可以顺利通过缝隙连接,但是扩散速度远远慢于同等分子量的Dextran-FITC,说明物质的电荷数和空间构象也会对物质的通透性产生影响。根据已知荧光分子Dextran-FITC的直径,可以估测出心肌缝隙连接的孔径约为17 nm。(3)缝隙连接可以介导死亡信号和细胞生长信号的传导缝隙连接可以促进死亡信号的扩散。分别构建在体、离体心肌梗死模型,本研究发现在心肌梗死危险区,缝隙连接保持开放,会导致死亡信号从坏死心肌细胞蔓延至存活心肌细胞,细胞结构首先从一端开始破坏,出现肌节Z线附近电子密度降低,线粒体肿胀,肌纤维断裂,逐渐向整个细胞扩散,最终导致整个细胞坏死。再灌注损伤过程中多种因素(缺氧、钙超载、酸中毒)会降低缝隙连接的通透性,促进缝隙连接关闭,可以阻断死亡信号的传递,使存活的心肌细胞得以保持活性,减少死亡细胞数量,降低心肌梗死面积。缝隙连接可以介导生长信号的传递。建立心脏压力后负荷模型-腹主动脉缩窄,分别观察1d、8W、16W的心肌肥厚程度,发现闰盘双侧的心肌细胞生长速度保持一致;肥大因子ET-1刺激新生大鼠原代培养细胞,48 h测定细胞表面积,重点测定保持相互接触的细胞面积,因为这类细胞之间存在缝隙连接,发现相互接触的细胞表面积没有差异,没有相互接触的细胞之间表面积差别很大;结合这两个实验的结果证实,缝隙连接可以介导生长信号的传递,协调细胞之间的生长。构建慢病毒载体感染新生大鼠心肌细胞,发现蛋白分子EGFP从感染病毒的细胞扩散至未被病毒感染的细胞,证实大分子物质增强绿色荧光蛋白(EGFP)可以在相互接触的细胞之间扩散,进一步提供了缝隙连接可以通透大分子物质的证据。【结论】心肌细胞缝隙连接可以通透小于150 k Da的大分子物质,通道孔径大约为17 nm。因为对大分子物质的通透特性,缝隙连接可以介导细胞死亡信号和生长信号的传导,影响细胞的死亡数量,协调细胞之间的生长过程。
[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.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R54

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