连接蛋白43、细胞连接通讯与心血管疾病

细胞连接通讯是动物体内细胞通讯的重要方式之一，其发挥细胞之间信息传递功能的结构基础是细胞间隙连接。细胞间隙的主要结构成份是连接蛋白。连接蛋白４３是心肌，血管平滑肌细胞间隙连接的主要组成成份。连接蛋白４３的表达与心律失常，缺血性心脏病，动脉粥样硬化，先天性心脏畸形等心血管疾病均有密切关系。     

心房颤动患者心房组织连接蛋白40和连接蛋白43基因转录表达的研究

目的：探讨心 房颤动（房颤）患者心房组织连接蛋白40（connexin40,Cx40)和连接蛋白43（Cx43)基因转录的变化，方法：39例风湿性心瓣膜病患者，心脏外科手术时取右心耳组织，通过逆转录－聚合酶链反应，以GAPDH为内参照，测量Cx40和Cx43的mRNA表达量。结果：阵发性房颤组和慢性房颤组心房组织Cx40的mRNA表达水平均显著高于窦性心律组（分别为P＜0．05，P＜0．01），而慢性房颤组心房组织Cx40的mRNA水平又明显高于阵发性房颤组（P＜0．01），窦性心律组，阵发性房颤组和慢性房颤组之间心房组织Cx43的mRNA水平无明显差异（均为P＞0．05），结论：心房Cx40基因转录上调是促进房颤发生和持续的重要因素。     

连接蛋白43表达的变化与心律失常的关系

近年研究表明,多种途径都可以改变连接蛋白43（connexin43,Cx43）的表达和分布,进而影响到心律失常的发生与进展。本文主要综述在房颤和骨骼肌成肌细胞心脏移植中,Cx43表达的变化,以及体外定向重离子流放射、缺血预处理、缝隙连接阻滞剂和阿片类物质对Cx43的影响,说明Cx43表达的变化与心律失常的关系。     

连接蛋白43在肺腺癌中表达的意义

目的检测肺腺癌组织及正常肺组织中缝隙连接蛋白43(Connexin 43)的表达。方法应用免疫组织化学法和原位杂交方法检测146例肺腺癌组织及20例正常肺组织中Connexin 43蛋白及mRNA的表达,并分析其表达与肺腺癌转移的关系。结果正常肺组织和肺腺癌组织中Connexin 43蛋白阳性表达率分别为95%(19/20)和39.9%(59/148);mRNA的阳性表达率分别为90%(18/20)和54.7%(81/148)。肺腺癌组织中Connexin 43蛋白及mRNA阳性表达率均低于正常肺组织(P0.05)。在高/中分化和低/未分化的肺腺癌组织中阳性表达率分别为48.7%和30.0%(P0.05);Cx43在无淋巴结转移和有淋巴结转移的肺腺癌组织中阳性表达率分别为56.3%和34.5%(P0.05);Connexin43在Ⅰ、Ⅱ期和Ⅲ、Ⅳ期肺腺癌组织中的阳性表达率分别为51.5%和34.0%(P0.05)。结论 Connexin 43蛋白和mRNA低表达与肺腺癌发生、发展及浸润、转移有关。     

泛素蛋白酶体途径对高糖刺激肾系膜细胞连接蛋白43表达和纤维连接蛋白分泌影响的初步观察

目的 探讨高糖对系膜细胞连接蛋白43(Cx43)表达和纤维连接蛋白(FN)分泌的影响,以及泛素蛋白酶体途径在其中的作用.方法 将培养的大鼠肾小球系膜细胞用高糖作为刺激因子,泛素蛋白酶体特异性抑制剂MG132作为阻断剂,甘露醇作为渗透压对照,观察各组细胞间Cx43的表达并检测细胞上清液中FN的含量.结果 (1)不同浓度的葡萄糖刺激系膜细胞48h后,Cx43表达较正常对照组显著降低,MG132可部分逆转高糖所致的Cx43的低表达(P＜0.05),甘露醇对Cx43表达无影响(P＞0.05).(2)高糖呈浓度依赖性促进系膜细胞 FN的分泌,MG132可部分抑制高糖所致的系膜细胞 FN的分泌(P均＜0.05).结论 高糖呈浓度依赖性抑制系膜细胞Cx43的表达和促进FN的分泌;泛素蛋白酶体途径参与了高糖所致Cx43的低表达和FN分泌.     

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。     

细胞连接蛋白基因在胃癌中的表达、诱导及突变研究

目的 研究人胃癌基因组中Ｃｘ基因的表达、探讨诱导剂作用后Ｃｘ基因的表达变化及Ｃｘ４３编码序列有无突变。方法 Ｎｏｒｔｈｅｒｎ杂交、ＲＴ－ＰＣＲ和ＰＣＲ－单链构象多肽分析。结果 １．发现了在人正常胃黏膜上皮、癌旁组织及胃癌中Ｃｘ基因的表达规律,明确了Ｃｘ基因在人胃癌基因组中的表达谱。２．维甲酸（ＲＡ）、二甲基亚砚（ＤＭＳＯ）对胃癌细胞基因组中Ｃｘ４３基因有诱导表达作用,而其本身表达的Ｃｘ４６在ＲＡ及     

间隙连接蛋白43对胰腺癌细胞株BxPC3凋亡的作用及其机制研究

目的 研究间隙连接蛋白43(Cx43)在胰腺癌细胞凋亡中的作用,并探讨其机制.方法 采用脂质体2000法将pcDNA-Cx43、pcDNA-Cx43N、空质粒peDNA3.0、siRNA-Cx43和对照siRNA-NC分别转染BxPC3细胞.Western blotting检测细胞Cx43蛋白表达、细胞色素C(Cyt C)含量;流式细胞仪检测细胞凋亡、线粒体膜电位;荧光分光光度计检测细胞Caspase-9和Caspase-3活性;染料传递法测定细胞间间隙连接(GJ).结果 Cx43转染BxPC3细胞后,Cx43蛋白表达明显上调,细胞凋亡从空质粒转染组的(6.35±0.43)%增加到(14.29±1.24)%,经H_2O_2处理后,从(20.34 ±2.47)%增加到(31.27±2.56)%(P<0.05),同时线粒体膜电位下降,Cyt C从线粒体释放增多,Caspase蛋白酶活性增加;而siRNA43干扰细胞后,细胞凋亡从(7.42±0.47)%减少到(5.19±1.37)%,经H_2O_2处理后,从(19.43±1.71)%减少到(11.67±1.97)%(P<0.05),线粒体膜电位去极化,Cyt C从线粒体释放减少,Caspase 酶活性下调.细胞间间隙连接指数在无GJ抑制剂B-GA情况下从对照组的14.52±0.57增加到peDNA-Cx 43转染组的23.05±3.84,在存在β-GA情况下从1.70±0.24增加到3.84±0.45(P<0.05),但细胞凋亡率改变无显著差异.结论 Cx43可通过线粒体凋亡途径促进BxPC3细胞凋亡,Cx43调节细胞凋亡存在间隙连接以外的机制.     

间隙连接蛋白43对胰腺癌细胞株BxPC3凋亡的作用及其机制研究

目的研究间隙连接蛋白43（Cx43）在胰腺癌细胞凋亡中的作用,并探讨其机制。方法采用脂质体2000法将pcDNA—Cx43、pcDNA—Cx43N、空质粒pcDNA3．0、siRNA—Cx43和对照siRNA-NC分别转染BxPC3细胞。Western blotting检测细胞Cx43蛋白表达、细胞色素C（Cyt C）含量;流式细胞仪检测细胞凋亡、线粒体膜电位;荧光分光光度计检测细胞Caspase-9和Caspase-3活性;染料传递法测定细胞间间隙连接（GJ）。结果Cx43转染BxPC3细胞后,Cx43蛋白表达明显上调,细胞凋亡从空质粒转染组的（6．35±0．43）％增加到（14．29±1．24）％,经H2O2处理后,从（20．34±2．47）％增加到（31．27±2．56）％（P〈0．05）,同时线粒体膜电位下降,CytC从线粒体释放增多,Caspase蛋白酶活性增加;而siRNA43干扰细胞后,细胞凋亡从（7．42±0．47）％减少到（5．19±1．37）％,经H2O2处理后,从（19．43±1．71）％减少到（11．67±1．97）％（P〈0．05）,线粒体膜电位去极化,CytC从线粒体释放减少,Caspase酶活性下调。细胞间间隙连接指数在无GJ抑制剂B—GA情况下从对照组的14．52±0．57增加到pcDNA—Cx43转染组的23．05±3．84,在存在β—GA情况下从1．70±0．24增加到3．84±0．45（P〈0．05）,但细胞凋亡率改变无显著差异。结论Cx43可通过线粒体凋亡途径促进BxPC3细胞凋亡,Cx43调节细胞凋亡存在间隙连接以外的机制。     

钙通道阻滞剂对梗死心肌中不同部位心肌连接蛋白43表达的影响

目的:研究L和L/T型钙通道阻制剂对梗死心脏不同部位(坏死区域、肥厚区域等)心肌组织中连接蛋白43（Cx43）的影响。方法: 随机将大鼠48只分为假手术组、心肌梗死(MI)组、阿莫地平(L型钙通道阻滞剂)组和米贝拉地尔(L/T型钙通道阻滞剂)组(每组n=12只)。通过结扎大鼠左冠状动脉建立MI模型,术前7 d,上述4个组分别用安慰剂、L型钙通道阻滞剂阿莫地平4 mg/(kg·d)和L/T型钙通道阻滞剂米贝拉地尔10 mg/(kg·d)。术后1、3、7 d,分别检测左心室游离壁（LVFW,梗死区）、心室间隔（IS,肥厚区）和右心室壁（RV）,正常心肌组织中Cx43蛋白的表达。术后7 d显微直视下测LVFW处MI病灶的大小、IS的厚度及左心室的大小。结果: IS中Cx43蛋白表达于术后1、3、7 d呈逐渐增加的趋势;LVFW中Cx43蛋白的表达于术后1、3、7 d时均处于低水平,与对照组相比差异显著（P<0.05）。RV中Cx43蛋白的表达于术后1、3、7 d无显著差异,与对照组相比也无显著性差异。米贝拉地尔能明显地抑制LVFW心肌组织中Cx43表达的下调,缩小MI病灶;阿莫地平则抑制肥厚心肌中Cx43蛋白的表达,明显抑制IS的肥厚。结论: MI病理过程中,梗死病灶内Cx43的表达下调,肥厚组织中Cx43的表达上调。L和L/T型钙通道阻滞剂均能减轻心肌重构与选择性地调节心肌组织中Cx43的表达有关。     

卡维地洛对大鼠心肌间隙连接通讯的影响及作用机制研究

目的研究卡维地洛对大鼠心肌缺血再灌注损伤、心肌间隙连接通讯（GJIC）及连接蛋白43（CX43）的影响,验证卡维地洛可通过改变CX43磷酸化状态抑制GJIC起到防止心肌再灌注损伤的假设.方法将大鼠随机分为假手术组、缺血再灌注组和卡维地洛组.结扎左冠状动脉前降支致缺血30 min后复灌4 h,建立心肌缺血再灌注损伤模型,于再灌注4 h末测定心肌酶及心肌梗死范围变化;制作Langendorf心脏灌流模型,随机分为假手术组、缺血再灌注组、卡维地洛组和庚醇组,于整体缺血30 min末期用改良的划痕标记染料示踪技术测定GJIC;用Western blot技术检测缺血30 min CX43磷酸化状态的改变.结果与假手术组相比,缺血再灌注组心肌酶及心肌梗死范围明显增加,GJIC无明显变化,非磷酸化CX43升高.与缺血再灌注组比较,卡维地洛组心肌酶及心肌梗死范围显著减少,同间隙连接抑制剂庚醇作用相似,GJIC也明显受到抑制,伴非磷酸化CX43水平明显上升.结论卡维地洛具有使CX43去磷酸化进而抑制GJIC防止心肌再灌注损伤的作用.     

Role of connexin 43 in ischemic preconditioning does not involve intercellular communication through gap junctions

Connexin 43 (Cx 43) has recently been implicated in protection of ischemic preconditioning. Cx 43 colocalization with protein kinase C and p38 mitogen-activated protein kinase is increased in preconditioned myocardium, Cx 43 phosphorylation is preserved in preconditioned myocardium, and hearts from Cx 43-deficient mice cannot be preconditioned. It is, however, unclear whether the important role of Cx 43 relates to intercellular communication through gap junctions or its function in volume homeostasis. To address this issue, we used isolated cardiomyocytes, which no longer-form gap junctions, from wild-type (n = 5) and heterozygous Cx 43-deficient mice (n = 8) and subjected them to 2 h simulated ischemia (hypoxia, acidosis) and an additional challenge by extracellular hypo-osmolarity (from 310 to 250 mOsm/l). Viability (trypan blue exclusion) was well maintained in normoxic wild-type cardiomyocytes (54 +/- 5% at baseline vs. 46 +/- 4 (mean +/- S.D.) % at 2 h). With simulated ischemia, viability was reduced to 17 +/- 5%. Preconditioning by a preceding exposure to 10 min simulated ischemia and 15 min reoxygenation preserved viability after 2 h simulated ischemia (36 +/- 1%, P < 0.001 vs. simulated ischemia). In Cx 43-deficient cardiomyocytes, viability was also well maintained in normoxia (56 +/- 10% vs. 44 +/- 10%). Viability was also reduced to 17 +/- 6% with 2 h simulated ischemia. In contrast to wild-type cells, preconditioning did not prevent the reduction in viability (18 +/- 8%). In conclusion, Cx 43 is essential for preconditioning in the absence of gap junctions, supporting its function through improved volume regulation.     

细胞间隙连接通讯及其通路蛋白与大肠癌发生关系的研究进展

细胞间隙连接通讯(gap junctional intercellular communication,GJIC)是多细胞生物体内普遍存在的一种通讯方式,他参与离子和其他小分子信号物质的转运.GJIC对细胞的生长、增殖和分化起重要的调控作用,他的改变与肿瘤的发生密切相关.大肠癌是人类常见的恶性肿瘤之一,目前研究表明,大肠癌的发生是一个多步骤、多基因、多阶段的过程,包括癌基因激活、抑癌基因失活、DNA转录表达失控、DNA损伤等.不论何种原因的细胞转化,其最终表现为细胞周期失控、细胞无限增殖.本文就GJIC及其通路蛋白、细胞因子与大肠癌的发生的研究进展作一综述.     

Age-related changes in gap junctional protein of the rat heart

OBJECTIVE:

Connexin 43 (Cx43), a membrane protein involved in the control of cell-to-cell communication, is thought to play a role in physiological processes such as tissue homeostasis, growth regulation and development. The aim of the present study was to investigate the change of Cx43 expression in aged myocardium.

METHODS AND RESULTS:

Sixteen male Sprague-Dawley rats (adult: 10 weeks old, n=8; aged: two years old, n=8) were used in the present study. In an isolated rat heart Langendorff model, hearts were perfused for 10 min with a modified Krebs-Henseleit bicarbonate buffer. Contractile functions were measured and all hearts were stained with anti-Cx43 antibody for fluorescence microscopic examinations. There were no significant differences observed in heart rate (234±8.2 beats/min versus 231±15.6 beats/min), left ventricular developed pressure (112.5±6.3 mmHg versus 107.2±2.5 mmHg), first derivative of the left ventricular pressure (1450.4±165.1 mmHg/s versus 1384.6±95.4 mmHg/s) and coronary flow (17.4±0.7 mL/min versus 21.3±1.8 mL/min) between adult and aged rats, respectively. However, significant differences were observed in left ventricular weight (adult versus aged; 0.639±0.108 g versus 1.124±0.257 g, P=0.04) and in fluorescence examinations where there was reduced distribution of Cx43 in aged myocardium compared with adult myocardium.

CONCLUSIONS:

These results demonstrated that the role of Cx43 may be more important than previously reported, and that this protein is partially responsible for the maintenance of cellular structure in myocardial development.     

Luteinizing and human chorionic gonadotropin hormones increase intercellular communication and gap junctions in cultured mouse leydig cells

The effect of luteinizing (LH) and human chorionic gonadotropin (hCG) hormones on gap junctions (Gjs) and intercellular communication (ic) was evaluated in Leydig (interstitial) cells from mouse testes. Cell cultures enriched in Leydig cells were studied under control conditions and when maintained in the presence of 100 ng/mL LH, 10 ng/mL hCG, or 1 mM dibutiryl-cAMP (db-cAMP), for 8, 24, and 36 h. To monitor the extent of ic, Lucifer yellow (LY) was injected through a patch pipet into one cell of-small cell aggregates (6–10), and its transfer was evaluated using fluorescent microscopy. The expression of GJs was monitored using immunofluorescent (IF) labeling of connexin 43 (Cx43) with a specific antibody. Testosterone secretion was determined by radioimmunoassay. At all culture times, testosterone levels in the medium were higher in treated than in control cell cultures. In cell cultures of 8 h, LY transferred to most of the neighboring cells (93%) and cell membrane appositions showed abundant Cx43; no difference was found between control and treated cells. In contrast, in control cell cultures of 24 and 36 h, LY transferred to a reduced fraction of neighboring cells (46 and 21%, respectively) and Cx43 labeling was markedly decreased. Addition of LH, hCG, or db-cAMP, to cell cultures for 24 and 36 h completely prevented the decrease in ic and Cx43 expression. Immunoblot studies, from total protein homogenates of cell cultures of 36 h, showed that relative levels of 40- and 43-kDa bands, characteristic of Cx43, were higher in treated than in control cells. These results demonstrate that the expression of Cx43 and ic in Leydig cells is modulated by LH and hCG, and suggest that their effect is mediated by the second messenger of these hormones, cAMP.     

Up-regulation of gap junctional intercellular communication and connexin43 expression by retinoic acid in human endometrial stromal cells

CONTEXT: Gap junctions, made up of connexins (Cxs), play fundamental roles in coordinating a number of cellular processes through their ability to directly regulate cell-cell communication. Cx43 is the most widely expressed Cx in the endometrium and is known to be important in a variety of physiological and pathological processes in this tissue. OBJECTIVE: In this study, we investigated the ability of the retinoid, all-trans-retinoic acid (RA), to regulate Cx43 expression in human endometrial stromal cells. DESIGN: Primary endometrial stromal cells obtained from patients undegoing surgery for infertility workup were treated in vitro with RA and control compounds for different time periods, up to 48 h. Cx43 mRNA and protein levels, protein phosphorylation, and gap junctional intercellular communication (GJIC) were analyzed. RESULTS: Treatment of the cells with RA showed a dose-dependent increase in Cx43 expression at both the mRNA and protein levels. In addition, RA induced a relative decrease in the phosphorylated species of Cx43 while causing a corresponding increase in the nonphosphorylated form. Concomitant with these changes, RA-treated cells demonstrated up to a 250% enhancement of GJIC as assessed by dye transfer experiments. Augmentation of GJIC and alterations of Cx43 expression were observed over the same range of RA concentrations. Treatment of cells with the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate increased the phosphorylated species of Cx43 and correspondingly inhibited GJIC. CONCLUSIONS: Phosphorylation of Cx43 is inversely related to GJIC in endometrial stromal cells. Retinoids increase GJIC in endomentrial stromal cells through upregulation of Cx43 expression while inducing a decrease in the phosphorylated species of the protein. The data suggest a novel mechanism by which retinoids can influence endometrial cell biology.     

Connexin40 and connexin43 determine gating properties of atrial gap junction channels

While ventricular gap junctions contain only Cx43, atrial gap junctions contain both Cx40 and Cx43; yet the functional consequences of this co-expression remain poorly understood. We quantitated the expression of Cx40 and Cx43 and their contributions to atrial gap junctional conductance (g_j). Neonatal murine atrial myocytes showed similar abundances of Cx40 and Cx43 proteins, while ventricular myocytes contained at least 20 times more Cx43 than Cx40. Since Cx40 gap junction channels are blocked by 2 mM spermine while Cx43 channels are unaffected, we used spermine block as a functional dual whole cell patch clamp assay to determine Cx40 contributions to cardiac g_j. Slightly more than half of atrial g_j and ≤ 20% of ventricular g_j were inhibited. In myocytes from Cx40 null mice, the inhibition of ventricular g_j was completely abolished, and the block of atrial g_j was reduced to < 20%. Compared to ventricular gap junctions, the transjunctional voltage (V_j)-dependent inactivation of atrial g_j was reduced and kinetically slowed, while the V_j-dependence of fast and slow inactivation was unchanged. We conclude that Cx40 and Cx43 are equally abundant in atrium and make similar contributions to atrial g_j. Co-expression of Cx40 accounts for most, but not all, of the differences in the V_j-dependent gating properties between atrium and ventricle that may play a role in the genesis of slow myocardial conduction and arrhythmias.