脑缺血后星形胶质细胞连接蛋白43表达及功能改变的研究进展

正脑缺血是以脑循环血流量减少为特征的脑血管疾病,发病率占卒中的70%～80%。星形胶质细胞是中枢神经系统中数量最多的细胞,缝隙连接蛋白43(connexin 43,Cx43)构成了星形胶质细胞间的半通道及缝隙连接~([1-3])。脑缺血后,Cx43的表达及功能都会发生变化,有研究表明,使用缝隙连接阻断剂     

缝隙连接蛋白43与心律失常的研究进展

正缝隙连接是介导相邻细胞间离子和小分子信号物质直接交换的跨膜通道,心室肌细胞中主要表达为连接蛋白43(connexin43,Cx43),缝隙连接蛋白在维持心肌细胞的连接通讯功能,电信号传导和正常的节律性的收缩中起重要作用;Cx43的空间分布、数量、结构的异常均能影响缝隙连接电荷耦联和代谢耦联的功能,导致心律失常的发生,提示Cx43可能成为心律失常治疗的新靶点~([1])。本文就缝隙连接蛋白43与心律失常的关系做一综述。     

Wnt-3a对Connexin43及相关基因的表达调控

目的 探讨信号分子Wnt3a蛋白对缝隙连接蛋白43(connexin43)的表达调控作用,以期揭示Wnt3a与connexin43在心脏发育过程中的特定联系与作用.方法 以本室自行构建pCD-NA3.1/wnt-3a真核表达载体转染大鼠心肌细胞系H9c2,在细胞水平通过RT-PCR及Western-blot实验方法研究Wnt-3a对其中Cx43表达及心脏发育相关基因的影响.结果 经转染wnt-3a后,H9c2细胞系中connexin43的表达明显上调,同时与心脏发育相关的GATA4,Nkx2.5基因表达也明显上调.结论 Wnt3a信号通路对connexin43表达存在影响;并可能对心脏发育存在调控作用.     

血管紧张素Ⅱ对心肌细胞肥大和缝隙连接蛋白43表达的影响

人们已经发现心肌肥大时心肌细胞间的缝隙连接重构参与心肌电生理特性的失稳态变化,而缝隙连接蛋白43(connexin 43,Cx43)是心肌细胞缝隙连接的主要蛋白成分,目前人们尚不清楚心肌肥大时Cx43变化的机制和规律,血管紧张素Ⅱ(angiotensin Ⅱ,AngⅡ)作为最强大的促心肌肥大因子对缝隙连接蛋白重构的影响至今也还鲜见报道,本组观察了AngⅡ对心肌cx43基因和蛋白表达及细胞周期影响,探讨心肌肥大过程中Cx43的变化及AngⅡ对缝隙连接蛋白重构的机制.     

基因重组质粒pBudCE4.1_Cx43转染骨髓间充质干细胞及其表达和功能的测定

目的用重组质粒pBudCE4.1_Cx43转染大鼠骨髓间充质干细胞(BMSCs),观察缝隙连接蛋白43(connexin43,Cx43)的mRNA和蛋白的表达及细胞间隙连接通讯功能的变化。方法用脂质体转染的方法将重组表达质粒pBudCE4.1_Cx43转染细胞;用逆转录聚合酶链式反应(RT-PCR)检测Cx43mRNA表达,免疫细胞化学法检测Cx43蛋白的表达,划痕染料示踪法检测细胞间通讯功能。结果转染Cx43基因后的细胞在形态学、生长能力等方面的特征与未转染的细胞比较没有明显变化;Cx43mRNA与蛋白的相对表达量和对照组相比差异显著(P<0.01);转染组细胞传递的荧光强度与对照组相比亦差异显著(P<0.01)。结论转染Cx43基因的BMSCs能表达有生物学活性的基因产物,改善细胞间通讯功能,能为细胞性心肌成形术提供较理想的基因工程细胞。     

抑制细胞间通讯功能可加速人成纤维细胞的衰老

目的研究间隙连接蛋白connexin43在人成纤维细胞衰老过程中的调节作用。方法设计并合成针对人connexin43的双链RNA(siRNA),抑制人二倍体成纤维细胞WI-38细胞的connexin43表达和细胞间通讯功能,观察对其衰老相关β-半乳糖苷酶(SA-β-gal)染色阳性率、细胞增殖能力、细胞周期调节蛋白P27、P21表达水平等衰老表型的影响。结果我们合成的siRNA-cx43可高效、特异的抑制connexin43 mRNA和蛋白质表达及细胞间通讯功能。转染WI-38细胞后,细胞增殖能力降低、细胞SA-β-gal染色阳性率(75.32±5.17)较对照组(32.48±3.94)和转染siRNA-con组(37.81±4.1 2)细胞升高(P<0.05),P27、P21表达增加,转染siRNA-cx43的WI-38细胞增殖能力显著降低,细胞变扁平、肥大,细胞SA-β-gal染色阳性率达100%,细胞提前出现衰老。结论间隙连接蛋白connexin43对WI-38细胞的衰老进程有重要的调节作用,其表达减少和细胞间通讯功能降低,可以促进WI-38细胞衰老进程。     

下调间隙连接蛋白43表达促进人胰腺癌细胞的侵袭和血管形成

背景：细胞间缝隙连接由间隙连接蛋白（Cx）构成,在肿瘤侵袭和血管形成过程中起重要作用。目的：以RNA干扰技术下调人胰腺癌细胞Cx43表达,观察Cx43表达改变对胰腺癌细胞缝隙连接的影响,探讨其在胰腺癌细胞侵袭和血管形成中的作用。方法：设计靶向Cx43的小干扰RNA（siRNA）并转染人胰腺癌细胞株BxPC3,以实时定量逆转录聚合酶链反应（RT-PCR）和蛋白质印迹法检测Cx43siRNA的干扰效果。染料传递实验检测细胞间缝隙连接,体外血管形成实验检测与BxPC3细胞共培养的人脐静脉内皮细胞（HUVEC）的血管生成能力,体外侵袭实验检测BxPC3细胞的侵袭能力。结果：Cx43siRNA转染BxPC3细胞后,细胞Cx43mRNA和蛋白表达明显下调。Cx43表达下调后,BxPC3细胞间、BxPC3细胞与HUVEC间缝隙连接显著减少;与BxPC3细胞共培养的HUVEC血管生成增多,每视野血管节点数显著高于对照组（15.69±1.09对5.24±1.32,P〈0.05）;BxPC3细胞的侵袭能力亦明显增强,每视野侵袭细胞数显著多于对照组（76.0±3.0对48.0±2.0,P〈0.05）。结论：下调Cx43表达可能通过减少细胞间缝隙连接,促进人胰腺癌细胞的侵袭和血管形成。     

缝隙连接蛋白43与心肌缺血再灌注心律失常的研究进展

近年来,心肌缺血再灌注损伤(myocardial ischemic-reperfusion injury,MIRI)的治疗和预防是心血管领域新兴的研究课题之一。缝隙连接(gap junction,GJ)是心脏电生理的基本结构。缝隙连接蛋白(connexin,Cx)是缝隙连接的基本单位。缝隙连接蛋白43(connexin 43,Cx43)是心脏Cx家族中最丰富的成员,Cx43的正常表达对于心脏发育、电耦联的心肌细胞活性和心肌功能的协调至关重要。Cx43与MIRI之间的关系已成为当前研究的重点。该文就Cx43与心肌缺血再灌注心律失常的关系作一综述。     

慢病毒载体介导人缝隙连接蛋白43基因在大鼠骨髓间充质干细胞中的表达

目的构建带有缝隙连接蛋白43基因的慢病毒载体,并实现该基因在大鼠骨髓间质干细胞中的表达。方法通过逆转录聚合酶链反应获得人缝隙连接蛋白43基因,利用infusion技术重组构建慢病毒载体质粒FUGW-缝隙连接蛋白43,在脂质体介导下与包装质粒和包膜质粒VSVG共转染293T细胞包装生产慢病毒。所获慢病毒感染大鼠骨髓间充质干细胞后,在荧光显微镜下观察缝隙连接蛋白43蛋白表达情况。结果所获缝隙连接蛋白43基因经测序后与GeneBank报道序列完全一致;重组慢病毒载体质粒FUGW-缝隙连接蛋白43经鉴定正确;三质粒共转染293T细胞成功,收集、浓缩病毒后测定其滴度为1×1011TU/L;感染大鼠骨髓间充质干细胞后荧光显微镜观察到胞膜位置点线状荧光分布。结论成功构建带有缝隙连接蛋白43基因的慢病毒载体并实现在大鼠骨髓间充质干细胞中的表达,为间充质干细胞移植治疗心肌梗死的应用奠定了基础。     

缝隙连接蛋白与心律失常的相关性研究进展

以往认为心律失常的发生主要与心肌细胞兴奋性异常有关但近年来的研究表明细胞间电耦联障碍是心律失常的另一个重要原因之一,其所起的致心律失常作用甚至大于兴奋性异常。由心肌缝隙连接蛋白(connexin,Cx)构成的缝隙连接(gapjunction,GJ)通道是心肌细胞之间的一种特殊连接通道,     

Signal transduction of gap junctional genes,connexin32, connexin43 in human hepatocarcinogenesis

AIM: To investigate gap junctional intercellular communication (GJIC) in hepatocellular carcinoma cell lines, and signal transduction mechanism of gap junction genes connexin32(cx32),connexin43(cx43) in human hepatocarcinogenesis. METHODS: Scarped loading and dye transfer (SLDT) was employed with Lucifer Yellow (LY) to detect GJIC function in hepatocellular carcinoma cell lines HHCC, SMMC-7721 and normal control liver cell line QZG. After Fluo-3AM loading, laser scanning confocal microscope (LSCM) was used to measure concentrations of intracellular calcium (Ca(2+))i in the cells. The phosphorylation on tyrosine of connexin proteins was examined by immunoblot. RESULTS: SLDT showed that ability of GJIC function was higher in QZG cell than that in HHCC and SMMC-7721 cell lines. By laser scanning confocal microscopy, concentrations of intracellular free calcium (Ca(2+))i was much higher in QZG cell line (108.37 nmol/L) than those in HHCC (35.13 nmol/L) and SMMC-7721 (47.08 nmol/L) cells. Western blot suggested that only QZG cells had unphosphorylated tyrosine in Cx32 protein of 32 ku and Cx43 protein of 43 ku; SMMC-7721 cells showed phosphorylated tyrosine Cx43 protein. CONCLUSION: The results indicated that carcinogenesis and development of human hepatocellular carcinoma related with the abnormal expression of cx genes and disorder of its signal transduction pathway, such as decrease of (Ca(2+))i, post-translation phosphorylation on tyrosine of Cx proteins which led to a dramatic disruption of GJIC.     

Defective gap junctional intercellular communication in lung cancer: loss of an important mediator of tissue homeostasis and phenotypic regulation

Gap junctions provide direct pathways for the exchange of molecules and ions between neighboring cells, a process known as gap junctional intercellular communication (GJIC). This GJIC is important for homeostasis and regulation of mitosis, differentiation, and apoptosis. Gap junctions are present in lung airway and alveolar epithelial cells and, in addition to the above roles, might coordinate ciliary beating and surfactant secretion. GJIC is decreased in human and mouse lung carcinoma cells because of reduced expression of the gap junction protein, connexin43 (Cx43), and defects in signal transduction pathways that mediate Cx43 function. This reduced GJIC is important in the behavior of lung carcinoma cells because forced expression of Cx43 in lung carcinoma cells inhibits their growth and tumorigenicity. In this report, we summarize our studies on the role of GJIC in lung neoplasia.     

Inorganic arsenic trioxide induces gap junction loss in association with the downregulation of connexin43 and E-cadherin in rat hepatic “stem-like” cells

Chronic exposure to inorganic arsenic trioxide causes tumors of the skin, urinary bladder, lung, and liver. Several cancer initiators and promoters have been shown to alter cell–cell signaling by interference with gap junction intercellular communication (GJIC) and/or modulation of cell adhesion molecules, such as connexin43 (Cx43), E-cadherin, and β-catenin. The aim of this study was to determine whether the disruption of cell–cell interactions occurs in liver epithelial cells after exposure to arsenic trioxide. WB-F344 cells were treated with arsenic trioxide (6.25–50 μM) for up to 8 hours, and gap junction function was analyzed using the scrape-load/dye transfer assay. In addition, the changes in mRNA and protein levels of Cx43, E-cadherin, and β-catenin were determined. A significant dose- and time-dependent decrease in GJIC was observed when WB-F344 cells were exposed to arsenic trioxide (p < 0.05). Consistent with the inhibition of GJIC, cells' exposure to arsenic trioxide resulted in dose- and time-dependent decreases in Cx43 and E-cadherin mRNA expression and protein levels. However, arsenic trioxide did not alter the mRNA or protein levels of β-catenin. In an immunofluorescence study, nuclei were heavily stained with anti-β-catenin antibody, indicating significant nuclear translocation. In this study, we also demonstrated that arsenic trioxide-induced GJIC loss was a reversible process. Taken together, these data support the hypothesis that disruption of cell–cell communication may contribute to the tumor-promoting effect of inorganic arsenic trioxide.     

Connexins in leukocytes: shuttling messages?

Gap junctions, formed by the connexin (Cx) protein family, are intercellular channels that permit the cytoplasmic exchange of ions and small metabolites between neighboring cells, a process called gap junction intercellular communication (GJIC). These channels possess unique properties, including distinctive permeabilities for various signaling molecules, which depend on the connexin member(s) that form them. Importantly, GJIC must be properly controlled as its misregulation might contribute to diseases. Morphological and functional studies have revealed 'gap junction-like' structures and cell-to-cell communication involving cells of the immune system. The connexins involved in such contacts have been partially identified in recent years. This review focuses on the potential physiological roles of gap junctions in the development and recruitment of leukocytes as well as in the regulation of the immune response. Furthermore, the importance of GJIC in immuno-inflammatory pathologies is illustrated in atherosclerosis.     

From the Cover: Connexin 43 as a signaling platform for increasing the volume and spatial distribution of regenerated tissue

Gap junction intercellular communication (GJIC) is ubiquitous in the majority of vertebrate cells and is required for the proper development of most tissues. The loss of gap junction-mediated cell-to-cell communication leads to compromised development in many tissues and organs. Because cells constantly interact through gap junctions to coordinate tissue functions and homeostasis, we hypothesized that increasing cell-to-cell communication, via genetically engineering cells to overexpress gap junction proteins, could enhance cell differentiation in the interior regions of 3D tissue equivalents, thereby increasing the ability to regenerate larger and more uniform volumes of tissue. To test this hypothesis, we used bone as a model tissue because of the difficulty in achieving spatially uniform bone regeneration in 3D. In bone marrow stromal cells (BMSC), GJIC and osteogenic differentiation were compromised in 3D cultures relative to 2D monolayers and in the core of 3D cultures relative to the surface. Overexpression of connexin 43 (Cx43) via transduction of BMSCs with a lentivirus overcame this problem, enhancing both the magnitude and spatial distribution of GJIC and osteogenic differentiation markers throughout 3D constructs. Transplantation of cells overexpressing Cx43 resulted in an increased volume fraction and spatial uniformity of bone in vivo, relative to nontransduced BMSCs. Increased GJIC also enhanced the effect of a potent osteoinductive agent (BMP-7), suggesting a synergism between the soluble factor and GJIC. These findings present a platform to improve cell-to-cell communication in 3D and to achieve uniformly distributed tissue regeneration in 3D.     

Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins.

In gene therapy to treat cancer, typically only a fraction of the tumor cells can be successfully transfected with a gene. However, in the case of brain tumor therapy with the thymidine kinase gene from herpes simplex virus (HSV-tk), not only the cells transfected with the gene but also neighboring others can be killed in the presence of ganciclovir. Such a "bystander" effect is reminiscent of our previous observation that the effect of certain therapeutic agents may be enhanced by their diffusion through gap junctional intercellular communication (GJIC). Herein, we present the evidence, from in vitro studies, that gap junctions could indeed be responsible for such a gene therapy bystander effect. We used HeLa cells for this purpose, since they show very little, if any, ability to communicate through gap junctions. When HeLa cells were transfected with HSV-tk gene and cocultured with nontransfected cells, only HSV-tk-transfected HeLa cells (tk+) were killed by ganciclovir. However, when HeLa cells transfected with a gene encoding for the gap junction protein, connexin 43 (Cx43), were used, not only tk+ cells, but also tk- cells were killed, presumably due to the transfer, via Cx43-mediated GJIC, of toxic ganciclovir molecules phosphorylated by HSV-tk to the tk- cells. Such bystander effect was not observed when tk+ and tk- cells were cocultured without direct cell-cell contact between those two types of cells. Thus, our results give strong evidence that the bystander effect seen in HSV-tk gene therapy may be due to Cx-mediated GJIC.     

Localization of connexin26 and connexin32 in putative CO(2)-chemosensitive brainstem regions in rat.

Recent studies have suggested that cell-to-cell coupling, which occurs via gap junctions, may play a role in CO(2) chemoreception. Here, we used immunoblot and immunohistochemical analyses to investigate the presence, distribution, and cellular localization of the gap junction proteins connexin26 (Cx26) and connexin32 (Cx32) in putative CO(2)-chemosensitive brainstem regions in both neonatal and adult rats. Immunoblot analyses revealed that both Cx subtypes were expressed in putative CO(2)-chemosensitive brainstem regions; however, regional differences in expression were observed. Immunohistochemical experiments confirmed Cx expression in each of the putative CO(2)-chemosensitive brainstem regions, and further demonstrated that Cx26 and Cx32 were found in neurons and Cx26 was also found in astrocytes in these regions. Thus, our findings suggest the potential for gap junctional communication in these regions in both neonatal and adult rats. We propose that the gap junction proteins Cx26 and Cx32, at least in part, form the neuroanatomical substrate for this gap junctional communication, which is hypothesized to play a role in central CO(2) chemoreception.     

Development of gap junctional channels and intercellular communication in rat liver during ontogenesis

BACKGROUND/AIMS: We investigated the expression of connexin (Cx) 32 and 26 subunit proteins of the gap junction (GJ) in the rat liver during ontogenesis to clarify their roles in control of growth and differentiation, and observed their channels in association with development of gap junctional intercellular communication (GJIC). METHODS: The expression of Cx32 and 26 in prenatal and postnatal livers was examined by Western blot and immunofluorescence. GJ channels were investigated not only by double immunofluorescence study but also by immunogold electron microscopy. The spread of lucifer yellow 5 min after its microinjection was examined in the cultured liver tissues. RESULTS: 1) Western blot showed the expression of both Cx from the late stage of gestation and their peak a week after birth. 2) Cx32- or 26-positive plaques were scattered on hepatocytes of the fetal liver and some of them were colocalized; both were increased just after birth. On day 7 after birth, Cx32-positive plaques were present on all hepatocytes within a lobule, and Cx26-positive plaques were distributed in the periportal area. 3) Double-immunogold electron microscopy just after birth showed that most GJ channels were homotypic type of Cx32 or 26, and that few were heterotypic. On day 7 after birth, most channels had the homotypic type of type of Cx32 in the middle and pericentral areas, and there was a heterotypic type of Cx32 and 26 in the periportal area. 4) The dye transfer of lucifer yellow showed a wider spread in the liver tissues on day 7 after birth than on day 1. CONCLUSION: Increased GJ formation and compatibility or incompatibility of GJ channels are closely associated with development of GJIC, and GJIC may develop at cytodifferentiation during ontogenesis.     

Heterocellular contact at the myoendothelial junction influences gap junction organization

Heterocellular communication between vascular smooth muscle cells (VSMC) and endothelial cells (EC) at the myoendothelial junction (MEJ) is a critical part of control of the arteriolar wall. We have developed an in vitro model of the MEJ composed of primary cultures of murine EC and VSMC. Immunoctytochemistry and immunoblots demonstrated Cx37 and Cx43 in both cell types, whereas Cx40 was found only in EC. Cx37 was excluded from the MEJ in both EC and VSMC. Connexin composition as well as functionality of the gap junctions at the MEJ was assessed by measuring diffusional transfer of biocytin and Cy3. Using connexin-specific blockers and manipulations of expression of individual connexin proteins, we confirmed that Cx37 is not a part of EC-VSMC coupling, and we demonstrated that heterotypic gap junctions are functional at the MEJ. We speculate that specific gap junction organization may be a vital component of EC-VSMC contact at the MEJ.