前列腺癌细胞连接蛋白43表达和细胞间隙连接交流状况

目的　观察前列腺癌 (PCa)细胞连接蛋白 (Connexin ,Cx)表达和细胞间隙连接交流(GJIC)状况 ,探讨胸苷激酶 (TK)自杀基因治疗前列腺癌时旁观者效应不够强大的原因以及GJIC与前列腺癌发生的关系。　方法　分别采用逆转录 聚合酶链式反应 (RT PCR)、链霉亲和素免疫组化法 (SABC法 )和划痕标记染料示踪技术 (SLDT)检测前列腺癌细胞系PC 3m的连接蛋白 4 3(Cx4 3)mRNA、蛋白表达和GJIC功能状况 ,并观察Cx4 3蛋白在正常前列腺和前列腺癌组织中的表达。　结果　PC 3m有Cx4 3mRNA和蛋白表达 ,但其表达较弱 ,且Cx4 3蛋白大多异常定位于细胞浆中而非细胞膜上 ;组织切片显示前列腺癌组织Cx4 3蛋白异常定位且表达水平较正常前列腺组织明显减弱 ,与病理分级呈负相关关系 (χ2 =4 0 2 5 ,P <0 0 5 ) ,高分化和中等分化、低分化腺癌的表达率分别为5 2 9%及 7 1%。此外 ,PC 3m细胞GJIC功能低下 ,半定量为 ( )或 (- )。　结论　前列腺癌细胞GJIC功能低下 ,Cx4 3基因下调表达和蛋白异常定位均可能为导致这一现象的原因。GJIC功能缺陷可能是引起单纯疱疹病毒 胸苷激酶 /更昔洛韦系统杀伤前列腺癌细胞时旁观者效应不够强大的原因 ,亦可能是前列腺癌发生、发展中的分子事件。     

血管紧张素Ⅱ下调肥厚心肌细胞缝隙连接蛋白Cx43的表达

目的 研究血管紧张素Ⅱ诱导心肌细胞肥大后连接蛋白43(Cx43)表达的变化及与细胞周期分布的关系.方法 分离培养大鼠心肌细胞,用血管紧张素Ⅱ诱导心肌细胞肥大,72 h后用RT-PCR,Western-blot和免疫荧光方法 观察心肌细胞Cx43基因和蛋白表达,用流式细胞仪测定法观察心肌细胞周期分布变化以及与Cx43表达量的关系.结果 血管紧张素Ⅱ处理后的心肌细胞表现细胞肥大且细胞活力增强,S期、G2-M期细胞百分比增加,细胞内G2-M(二倍体)DNA含量降低,Cx43蛋白表达明显低于正常对照组,呈浓度依赖性下调,Cx43 mRNA表达水平显著下调,Cx43蛋白表达下调与细胞周期分布的改变相关.结论 血管紧张素Ⅱ诱导心肌细胞肥大后Cx43基因及Cx43蛋白表达出现浓度依赖性下调,这一改变与心肌肥大过程中的细胞周期变化有关,提示血管紧张素Ⅱ可能通过调控Cx43基因的表达而参与缝隙连接重构过程,而Cx43表达的变化可能与心肌肥大的机制有关.     

黄芩苷对肝癌细胞GJIC及Cx26、Cx43表达的影响

目的:探讨黄芩苷对肝癌细胞缝隙连接细胞间通讯(gap junction intercellular communication,GJIC)及细胞间隙连接蛋白26(connexion26,Cx26)、细胞间隙连接蛋白43(connexion43,Cx43)表达的影响.方法:人肝癌细胞株SMMC-7721分为黄芩苷10、20、40mg/L组和对照组.划痕染料示踪技术检测GJIC的变化.用RT-PCR法检测肝癌细胞Cx26、Cx43基因表达,用Westernblot法检测Cx26蛋白表达,用免疫细胞化学检测Cx43蛋白表达.结果:对照组细胞的染料局限于划痕两侧的细胞内,无明显的荧光染料传输现象.随着黄芩苷浓度的增强,LY染料传输范围逐渐增大.黄芩苷10、20、40mg/L各浓度组Cx26mRNA表达水平与对照组比较显著提高(0.148±0.111,10.253±0.222,17.283±0.024vs0.138±0.111;P<0.05).Cx26蛋白表达量与对照组比较明显增加(0.516±0.029,0.759±0.020,1.019±0.076vs0.367±0.029;P<0.05).黄芩苷各浓度组Cx43mRNA表达量与对照组比较无显著变化.而Cx43蛋白的表达水平与对照组比较明显增加(5.512±0.003,5.844±0.046,6.216±0.015vs4.316±0.032;P<0.05).结论:黄芩苷促进肝癌细胞Cx26及Cx43表达,导致肝癌细胞GJIC功能的恢复,这很可能是黄芩苷抑制肿瘤生长的分子机制之一.     

Cx43连接蛋白与心脏发育

缝隙连接蛋白43(Cx43)是哺乳动物心脏中最主要的连接蛋白,研究表明其对心脏的正常分化和发育发挥着重要作用。Cx43的表达异常可引起多种心血管疾病以及心脏先天性畸形的发生。本文就Cx43的结构、表达及其与心脏发育的最新研究进展予以综述。     

生肌调节因子和连接蛋白43基因诱导大鼠成纤维细胞分化为肌细胞实验研究

目的 探讨转染生肌调节因子(myoblast determination,MyoD)基因和连接蛋白43(Connexin,Cx43)基因的大鼠真皮成纤维细胞(dermal fibroblast,DFs)生物学功能的变化及MyoD和Cx43基因转染DFs细胞治疗心力衰竭的可行性.方法 采用Gateway技术,构建真核质粒表达载体,利用慢病毒(LV)表达系统,将大鼠MyoD cDNA和Cx43 eDNA转入大鼠成纤维细胞中,经Blasticidin筛选培养,通过RT-PCR,Western印迹法等方法检测MyoD及Cx43蛋白及mRNA表达情况,并且通过显微镜观察转染后生长情况,膜片钳技术检测离子电流变化.结果 RT-PCR,Western印迹法检测出MyoD及Cx43蛋白及相应mRNA表达,膜片钳检测到转染后钙离子电流,显微镜观察到基因转染筛选后培养1 W细胞的融合现象,并有多核肌管形成.结论 MyoD和Cx43基因转染使DFs分化为成肌细胞,为进一步研究基因治疗心力衰竭奠定基础.     

连接蛋白43与胃肠运动

连接蛋白（Cx）43是构成缝隙连接（GJ）最重要的Cx,GJ是介导胃肠平滑肌细胞（SMC）和Caja1间质细胞（ICC）间电化学信息交流,保证肌肉活动的协调性和同步性的特殊通道。Cx43广泛存在于胃肠Caja1间质细胞与平滑肌细胞之间,在胃肠运动中发挥重要作用。Cx43的异常表达与分布可导致胃肠运动障碍。     

连接蛋白43与胃肠运动

连接蛋白（Cx）43是构成缝隙连接（GJ）最重要的Cx,GJ是介导胃肠平滑肌细胞（SMC）和Caja1间质细胞（ICC）间电化学信息交流,保证肌肉活动的协调性和同步性的特殊通道。Cx43广泛存在于胃肠Caja1间质细胞与平滑肌细胞之间,在胃肠运动中发挥重要作用。Cx43的异常表达与分布可导致胃肠运动障碍。     

血管紧张素Ⅱ下调肥厚心肌细胞缝隙连接蛋白Cx43的表达

目的研究血管紧张素Ⅱ诱导心肌细胞肥大后连接蛋白43（Cx43）表达的变化及与细胞周期分布的关系。方法分离培养大鼠心肌细胞，用血管紧张素Ⅱ诱导心肌细胞肥大，72h后用RT-PCR，Western-blot和免疫荧光方法观察心肌细胞Cx43基因和蛋白表达，用流式细胞仪测定法观察心肌细胞周期分布变化以及与Cx43表达量的关系。结果血管紧张素Ⅱ处理后的心肌细胞表现细胞肥大且细胞活力增强，S期、G2-M期细胞百分比增加，细胞内G2-M（二倍体）DNA含量降低，Cx43蛋白表达明显低于正常对照组，呈浓度依赖性下调，Cx43mRNA表达水平显著下调，Cx43蛋白表达下调与细胞周期分布的改变相关。结论血管紧张素Ⅱ诱导心肌细胞肥大后Cx43基因及Cx43蛋白表达出现浓度依赖性下调，这一改变与心肌肥大过程中的细胞周期变化有关，提示血管紧张素Ⅱ可能通过调控Cx43基因的表达而参与缝隙连接重构过程，而Cx43表达的变化可能与心肌肥大的机制有关。     

病毒性心肌炎连接蛋白的变化

目的观察小鼠急性病毒性心肌炎时连接蛋白的变化，了解连接蛋白与病毒性心肌炎的关系。方法Balb／c4周龄雄性小鼠26只，随机分为模型组（20只）和正常对照组（6只），模型组予腹腔接种柯萨奇病毒B组3型（CVB3）建立小鼠急性病毒性心肌炎模型。在接种病毒后第14天处死所有存活小鼠，观察心肌形态学改变，并采用聚合酶链式反应（PCR）及免疫组织化学技术，检测各组小鼠心肌组织中connexin43（Cx43）、connexin40（Cx40）和connexin45（Cx45）基因水平及蛋白水平的变化。结果模型组小鼠心肌HE染色可见明显的炎性细胞浸润和心肌坏死等炎症改变；与正常对照组相比，Cx43和Cx45mRNA表达明显减少（分别为0．11±0．02vs0．34±0．05，0．21±0．02vs0．41±0．06）。Cx40mRNA表达较对照组无统计学差异，三者蛋白水平的变化与基因水平变化相一致。结论急性病毒性心肌炎小鼠心肌中connexin43和connexin45表达明显减少，上述连接蛋白的变化与病毒性心肌炎致心律失常的潜在作用有关。     

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。     

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.     

Switch in gap junction protein expression is associated with selective changes in junctional permeability during keratinocyte differentiation.

Gap junctional communication provides a mechanism for regulating multicellular activities by allowing the exchange of small diffusible molecules between neighboring cells. The diversity of gap junction proteins may exist to form channels that have different permeability properties. We report here that induction of terminal differentiation in mouse primary keratinocytes by calcium results in a specific switch in gap junction protein expression. Expression of alpha 1 (connexin 43) and beta 2 (connexin 26) gap junction proteins is down-modulated, whereas that of beta 3 (connexin 31) and beta 4 (connexin 31.1) proteins is induced. Although both proliferating and differentiating keratinocytes are electrically coupled, there are significant changes in the permeability properties of the junctions to small molecules. In parallel with the changes in gap junction protein expression during differentiation, the intercellular transfer of the small dyes neurobiotin, carboxyfluorescein, and Lucifer yellow is significantly reduced, whereas that of small metabolites, such as nucleotides and amino acids, proceeds unimpeded. Thus, a switch in gap junction protein expression in differentiating keratinocytes is accompanied by selective changes in junctional permeability that may play an important role in the coordinate control of the differentiation process.     

Role of connexin-related signalling in hepatic homeostasis and its relevance for liver-based in vitro modelling

Direct intercellular communication mediated by gap junctions constitutes a major regulatory platform in the control of hepatic homeostasis.Hepatocellular gap junctions are composed of two hemichannels of adjacent cells which are built up by connexin proteins,in casu Cx32.Mathieu Vinken,Pofessor at the Department of Toxicology of the Free University BrusselsBelgium,was one of the first investigators to demonstrate that hepatic connexin expression is controlled by epigenetic mechanisms.In particular,he found that inhibitors of histone deacetylase enzymes enhance Cx32 production and gap junction activity in cultures of primary hepatocytes,a finding that is of importance for liver-based in vitro modelling.Professor Dr.Mathieu Vinken’s recent work is focussed on the elucidation of the role of connexin proteins and their channels in the hepatocyte life cycle.Specific attention is paid to apoptosis in this context,whereby it has been found that Cx32 hemichannels control the termination of induced cell death in cultures of primary hepatocytes. Overall,Professor Dr.Mathieu Vinken’s research can be considered as an important contribution to the field of hepatic connexin physiology.     

Aberrant expression,function and localization of connexins in human esophageal carcinoma cell lines with different degrees of tumorigenicity

We have analyzed the level of mRNA expression and protein localization of the gap-junction protein connexins (Cx) 26, 32, and 43, as well as gap-junctional intercellular communication (GJIC) in seven human esophageal careinoma cell lines (TE series). These cell lines exhibited various degrees of tumorigenicity in nude mice; two (TE-1 and TE-8) formed progressively growing tumors, four (TE-2. TE-3, TE-9, and TE-13) developed non-progressing tumors and one (TE-10) showed no tumorigenicity. We found that normal human esophageal tissue expressed both Cx26 and Cx43 and that most of the cell lines expressed lower amounts of Cx26 and Cx43 mRNAs than normal human esophageal tissues or none at all. The co-expression of Cx26 and Cx43 mRNAs and proteins was observed only in two cell lines (TE-3 and TE-9) that showed a high level of GJIC and non-progressive tumor development. However, the non-tumorigenic cell line TE-10 did not express either connexin. A possible regulator of GJIC, E-cadherin, was expressed in all cell lines. These results suggest that aberrant expression and function of connexins are common among human esophageal carcinoma cell lines, but there is no quantitative relationship between connexin expression and tumorigenic properties of these cell lines.Abbreviations Cx connexin - GJIC gap-junctional intercellular communication     

Intercellular communication via gap junctions in activated rat hepatic stellate cells

BACKGROUND AND AIMS: Gap junctional communication was studied in quiescent and activated hepatic stellate cells. METHODS: Connexin expression and intercellular dye transfer were studied in rat hepatic stellate cells in culture and in vivo. RESULTS: Protein expression of connexin 43 was up-regulated in activated hepatic stellate cells in vivo and in vitro and was mainly localized on the cell surface, whereas connexin 26 was found intracellularly. In contrast to hepatocytes, hepatic stellate cells do not express connexin 32. Confluent hepatic stellate cells in culture communicate via gap junctions, resulting in lucifer yellow transfer and propagation of intracellular calcium signals. Phorbol ester induces a protein kinase C-dependent hyperphosphorylation and degradation of connexin 43 and inhibits intercellular communication on a short-term time scale. At the long-term level, vitamin D(3) , lipopolysaccharide, thyroid hormone T(3), dexamethasone, platelet-derived growth factor, endothelin 1, and interleukin 1beta up-regulate connexin 43 protein and messenger RNA expression and enhance intercellular communication. Slight down-regulation of connexin 43 is observed in response to vitamin A. Connexin 43 induction by endothelin 1 is inhibited by both endothelin A and endothelin B receptor antagonists. In coculture systems, hepatic stellate cells communicate with each other, which is suggestive of a syncytial organization, but no communication was found between hepatic stellate cells and other liver cell types. As shown by immunohistochemistry and electron microscopy, gap junctions are formed between activated hepatic stellate cells in vivo. CONCLUSIONS: Gap junctional communication occurs between hepatic stellate cells, is enhanced after activation, and underlies complex regulation by cytokines, hormones, and vitamins.     

Gap junction messenger RNA expression by vascular wall cells.

Gap junctions between vessel wall cells provide a pathway for the intercellular exchange of ions and small molecules. Pure cultures of microvascular and macrovascular endothelial and smooth muscle cells, vascular pericytes, and several nonvascular cell lines were tested for junctional communication by fluorescent dye transfer. All of the vascular wall cells were capable of dye transfer. Since gap junctions are formed by a family of related proteins (connexins) whose unique domains may confer physiological regulatory properties, we tested total RNA from these cultures by Northern blot analysis for expression of the currently available, characterized, and cloned mammalian gap junction proteins: connexin26, connexin32, and connexin43. All of the vascular wall cells expressed connexin43 messenger RNA. Connexin43 was expressed in vascular cells from bovine, porcine, rat, and human sources. Several nonvascular cell lines of mesenchymal origin also expressed connexin43 messenger RNA. When high stringency Northern blots were used, messenger RNAs for connexin32 or connexin26 were not detected in any of the vascular wall cells but were expressed in several cell lines of epithelial origin. Freshly isolated and purified aortic endothelial and smooth muscle RNA preparations similarly contained only connexin43 messenger RNA, excluding the possibility of culture-induced alterations in gene expression. The expression of connexin43 by all vascular wall cells may provide a mechanism for the functional integration of the vessel wall by gap junctions.     

硝苯吡啶对血管平滑肌细胞连接通讯的影响

为了解硝苯吡啶对血管平滑肌细胞连接通讯的影响，采用荧光黄划痕示踪法，测定硝苯吡啶作用的培养的大鼠平滑肌细胞连接通讯功能，并通过显微荧光光度计进行定量分析。此外，采用免疫组织化学S－P法了解硝苯吡啶对该类细胞连接蛋白43表达的影响，通过计算机图像分析进行定量研究。结果显示，硝苯吡啶促进细胞连接蛋白43的表达和细胞连接通讯。提示硝苯吡啶通过促进细胞连接蛋白43的表达和细胞连接通讯来抑制平滑肌细胞增殖，是抗动脉粥样硬化作用的重要机制之一。