同种异体骨髓间充质干细胞移植上调急性心肌梗死大鼠Cx43的表达

目的研究同种异体骨髓间充质干细胞(MSCs)移植心肌梗死(MI)大鼠后缝隙连接蛋白43(Cx43)在不同时期的动态变化。方法建立大鼠MI模型。将同种异体MSCs用5-氮胞苷诱导成心肌样细胞并行荧光标记,经二次开胸注射入MI大鼠梗死区和梗死边缘区。各亚组分别于移植后4、8和12周在荧光显微镜下跟踪MSCs移植情况。同时用免疫组化分析Cx43表达与缝隙连接(GJ)分布。结果MSCs体外诱导可分化为自发搏动的心肌样细胞,表达心肌特异性肌钙蛋白T(cTnT)和形成肌丝结构。MSCs移植后可长期存活并在4、8和12周,并有效上调缺血区Cx43的表达,改善GJ分布紊乱状态。在梗死区Cx43无特殊改变。结论MSCs具有分化为心肌样细胞的可塑性,移植后上调MI后缺血区Cx43表达,改善GJ分布紊乱。     

急性心肌缺血时连接蛋白43降解对传导速度影响的研究

目的：探讨急性短时间缺血时心肌细胞连接蛋白43(Cx43)的降解对缺血心肌电传导速度的影响。方法：16只犬随机分为正常对照组(n=4)和缺血组(n=12), 缺血组结扎冠状动脉1h造成急性心肌缺血, 测定缺血区心肌电传导速度, 应用激光共聚焦显微镜技术和荧光免疫组织化学方法对缺血心肌Cx43含量进行定量检测。结果：(1)急性心肌缺血时Cx43迅速降解, 心肌电传导速度明显下降；(2)缺血区各局部传导速度与该部位Cx43像素密度呈明显正相关；(3)出现持久传导阻滞的区域其Cx43降解程度均大于50%。结论： 急性短时间(1 h)缺血时Cx43的降解已经开始对心肌传导速度产生明显的影响, 而局部心肌Cx43的严重降解将导致该区域出现持久传导阻滞。     

银杏酮酯预处理对心肌缺血再灌注大鼠心肌的保护作用及可能机制

目的探讨银杏酮酯预处理对心肌缺血再灌注大鼠心肌的保护作用及可能机制。方法以36只雄性Wistar大鼠为研究对象,将实验动物随机分为3组:假手术组、心肌缺血再灌注模型组与银杏酮酯组。采用结扎大鼠冠状动脉左前降支的方法,建立大鼠心肌缺血再灌注损伤模型。假手术组穿线后不结扎,模型组于缺血前30 min腹腔注射生理盐水,银杏酮酯组大鼠于缺血前30 min腹腔注射银杏酮酯(100mg/kg)。造模24h后处死大鼠,HE染色观察心肌组织变化,ELISA法测定血清肌酸激酶-同工酶(CK-MB)和肌钙蛋白(cTnI)水平以及炎性因子白介素1 (IL-1)、白介素6 (IL-6)与肿瘤坏死因子-α(TNF-α)水平,Western blot方法检测心肌组织缝隙连接蛋白(Cx43)蛋白表达。结果假手术组大鼠心肌纤维排列整齐,模型组心肌纤维排列紊乱,心肌间质出现炎性细胞浸润,银杏酮酯组心肌组织较模型组有明显改善。与假手术组相比,模型组大鼠血清CK-MB、cTnI、IL-1、IL-6与TNF-α水平显著上升,心肌组织Cx43蛋白表达显著降低;与模型组相比,银杏酮酯组大鼠血清CK-MB、cTnI、IL-1、IL-6与TNF-α水平显著降低,心肌组织Cx43蛋白表达显著升高。结论银杏酮酯预处理可降低缺血-再灌注大鼠心肌损伤程度及炎性反应。     

大鼠心肌成纤维细胞通过增加基质金属蛋白酶2活性抑制心肌细胞缝隙连接功能

目的：探讨缺氧复氧（hypoxia/reoxygenation, H/R）后大鼠心肌成纤维细胞（cardiac fibroblasts, CFs条件培养液对心肌细胞中缝隙连接蛋白43(connexin 43, Cx43)表达和缝隙连接功能的影响及其分子机制。方法：（1）将H9c2细胞随机分为5组：对照（control）组、常氧（normal）组、基质金属蛋白酶2抑制剂ARP-100(ARP)组、H/R组和H/R+ARP组。荧光划痕技术评价缝隙连接功能,Western blot实验检测Cx43的表达及磷酸化水平,明胶酶谱法测定条件培养液中基质金属蛋白酶的活性。（2）将SD大鼠随机分为control组、ARP组、缺血再灌注（I/R）组和I/R+ARP组,每组8只。微电极阵列技术采集心律失常发生类型和持续时间,免疫组织化学实验检测心肌组织中Cx43蛋白的表达及分布,Western blot实验检测Cx43的表达及磷酸化水平。结果：与control组相比,H/R组Cx43蛋白表达显著下降（P<0.01）,磷酸化水平下调（P<0.01）,荧光扩散范围变窄（P<0.01）,MMP...     

急性缺血心肌的血流量再分布

利用放射性生物微球技术,研究了犬在急性心肌缺血时,侧支循环血流量的再分布,在结扎左前降支冠脉后,中心缺血区血流量明显减少。近边缘区血流量大于中心缺血区血流量,小于远边缘区和非缺血区的血流量。中心缺血区和近边缘区的心内膜下层心肌血流量低于心外膜下层心肌血流量,两者之比小于1.00。随着缺血时间的延长(1—6小时),中心缺血区血流量逐渐减小,而非缺血区的血流量逐渐增加。     

小鼠心脏发育过程中Cx43表达的时空变化

目的：探讨小鼠心脏发育过程中连接蛋白43（connexin43，Cx43）表达的时空变化。方法：应用SP免疫组化和图像分析方法，观测胎小鼠、出生1d、6d、2月和14月小鼠心肌细胞Cx43的表达。结果：1、胎小鼠和出生1d小鼠心肌细胞Cx43呈斑点状遍布于心肌细胞侧-侧连接和细胞质内，闰盘处极少。2、出生6d时Cx43开始向端闰盘处聚集，2月和14月小鼠心室肌细胞Cx43已经典型分布于闰盘处，而心房仍分布于细胞侧面连接处和细胞质。3、分布密度总规律是：胎小鼠〈出生1d〈6d〉2月〉14月。结论：小鼠心肌细胞Cx43分布随心脏发育逐渐向闰盘处重排，其密度逐渐下降。心房肌Cx43的表达与分布具有一定的幼稚性。     

星形胶质细胞的缝隙连接蛋白43参与冷冻伤后脑水肿的形成

目的:探讨脑水肿后星形胶质细胞缝隙连接蛋白43(Cx43)的表达及其在脑水肿的发生发展过程中所起的作用。方法:采用颅骨外液氮冷冻法建立大鼠右侧顶叶皮层脑水肿模型。实验分为假手术组、脑水肿模型组和脑水肿模型+缝隙连接阻断剂(carbenoxolone或octanol)干预组。干湿重法测定冷冻伤后的脑含水量;甲酰胺法测定大鼠血脑屏障通透性的改变;HE染色观察冷冻伤脑组织的病理变化;Western blot法和免疫组化检测Cx43蛋白的表达情况。结果:冷冻伤可引起大鼠损伤脑皮层区的含水量增加,在冷冻伤后24 h脑水肿发展到高峰。冷冻伤引起损伤脑皮层区域的血脑屏障通透性增加,范围大于直接冷冻损伤区。HE染色观察显示冷冻伤中心区细胞坏死明显,而冷冻伤周围区域出现水肿。脑冷冻伤引起冷冻伤周围皮层区域的Cx43蛋白表达增加,但冷冻伤中心区的Cx43蛋白表达降低。Carbenoxolone或octanol阻断Cx43的功能,降低了冷冻伤皮层区的含水量和血屏障通透性。结论:脑水肿时星形胶质细胞上的Cx43表达上调,功能增强;阻断Cx43的功能可在一定程度上减轻脑水肿。     

心房纤颤患者心房肌Cx43的蛋白表达

目的：研究房颤患者心房肌细胞连接蛋白43(Cx43)的蛋白表达。方法：应用SP免疫组化方法，比较正常和房颤患者心房肌Cx43的表达。结果：(1)正常心房肌细胞Cx43呈斑点状或带状，大量分布于细胞侧连接处。部分位于闰盘处。(2)房颤患者心房肌细胞Cx43颗粒发生重排，杂乱地分布于心肌的各个部位。房颤时间长者，部分Cx43移入细胞质中，表现为某些区域极度增加或减少。(3)房颤患者心房肌细胞Cx43的蛋白表达量未发生显著变化。结论：Cx43在房颤患者心房肌细胞发生重排，呈无序分布，可能是心房纤颤发病的原因之一；并提示房颤仅对Cx43的表达形式产生影响。     

发育大鼠心肌细胞间隙连接蛋白43表达特性

目的：探讨大鼠心肌细胞间隙连接蛋白43(Cx43)表达的发育特性。方法：应用SP免疫组化和图像分析方法,观察胎大鼠、出生1 d、5 d、10 d和25 d大鼠心肌细胞Cx43的蛋白表达。结果：(1)胎鼠心肌细胞Cx43呈斑点状遍布于心肌细胞侧—侧连接、细胞质内和闰盘处。(2)出生后1 d大鼠心肌细胞的Cx43分布与胎鼠相似,5 d和10 d组逐渐向闰盘处聚集。25 d时部分已聚集于端闰盘处,其余仍呈斑点状散在分布。(3)出生前后心室肌细胞Cx43分布有差异。分布密度总规律是：胎大鼠＞出生1 d＞5 d＞10 d＞25 d。结论：出生前后大鼠心肌细胞Cx43分布随心脏发育逐渐向闰盘处重排,其分布密度呈逐渐下降趋势。     

丹参、美托洛尔对大鼠心肌缺血坏死区干细胞修复的影响

目的:探讨丹参(danshen)、美托洛尔(metoprolol)药物干预对异丙肾上腺素诱导大鼠心缺血坏死区干细胞修复的影响.方法:健康SD大鼠40只,按4mg/kg的剂量连续腹腔注射盐酸异丙肾上腺素(ISO)7 d制备大鼠心肌缺血模型,将造模成功的32只大鼠随机分为4组,异丙肾上腺素对照组(ISO组),丹参干预组(Danshen组),美托洛尔干预组(Meto组),丹参+美托洛尔联合干预组(Danshen+-Meto组).H-E染色比较各组心室肌缺血坏死区大小、免疫荧光观察缺血坏死区c-kit、CD34干细胞标记物及心肌早期转录因子GATA-4和心肌缝隙连接蛋白connexin 43的表达情况.结果:与ISO组比较,其他3组大鼠心肌组织H-E染色心室内膜下缺血面积减小,免疫荧光观察显示缺血区及缺血周边表面标记物c-kit、CD34阳性干细胞增多,缺血区可观察到GATA-4及connexin 43的表达.结论:丹参、美托洛尔能促进心肌缺血坏死区干细胞的迁移和增殖,并最终形成具心肌细胞特征的“心肌样细胞”.     

脂联素对大鼠缺血再灌注心肌缝隙连接蛋白43表达的影响

目的: 观察脂联素(APN)对大鼠缺血再灌注心肌缝隙连接蛋白43(Cx43)表达的影响,进一步探讨其抗心律失常的可能机制。方法: 将48只雄性Wistar大鼠随机分成:(1)假手术 (SM)组;(2)缺血再灌注(I/R)组:结扎冠状动脉左前降支,30 min后松开结扎线,再灌注120 min;(3)脂联素+缺血再灌注组1(I/R+APN1):先阻断血流30 min,于再灌注120 min开始时给予3.5 μg/kg APN;(4)脂联素+缺血再灌注组2(I/R+APN2):缺血前10 min给予3.5 μg/kg APN,余同I/R组。观察各组心律失常的发生情况;应用RT-PCR观察各组心室肌细胞 Cx43 基因表达;用免疫组织化学方法观察Cx43分布的变化;应用硫代巴比妥酸(TBA)法和黄嘌呤氧化酶法测各组动物血清中丙二醛(MDA)的含量和超氧化物歧化酶(SOD)的活性;用RT-PCR及Western blotting法分析内皮型一氧化氮合酶(eNOS)mRNA及蛋白的表达,用电镜观察各组心室肌超微结构的改变。结果: (1)I/R组与SM组比较,心律失常评分和血清MDA含量显著增高(P<0.01),SOD活性降低(P<0.01);心室肌Cx43表达明显减少,差异有统计学意义(P<0.01),Cx43分布紊乱,失去正常的规律性;心肌细胞超微结构有明显损伤;心室肌eNOS mRNA及蛋白的表达明显降低,差异有统计学意义(P<0.01)。(2)无论缺血前还是缺血后使用脂联素处理,与I/R组相比,心律失常评分显著降低(P<0.01);Cx43及eNOS表达增高(P<0.01),Cx43分布紊乱的程度减轻;心肌细胞超微结构损伤明显改善。结论: APN可能通过氧化应激调节Cx43的功能,从而发挥抗缺血/再灌注心律失常的作用。     

High-pressure freezing and freeze substitution of rat myocardium for immunogold labeling of connexin 43

The value of high-pressure freezing (HPF) and freeze substitution (FS) for immunoelectron microscopy (immuno-EM) of the heart was investigated in bioptic specimens taken from isolated hearts of 0-, 5-, and 14-day-old rats at baseline and at 15, 30, 45, and 60 min after induction of ischemia. The target antigen chosen here was the gap junction protein connexin 43 (Cx43). After HPF and FS, immunogold labeling was applied for detection of Cx43. Gold particles associated with gap junction areas, free plasma membrane, and annular gap junctions (AGJs) were counted and distributions compared by contingency table analysis. HPF and FS resulted in excellent preservation of antigenicity for Cx43. The mostly good preservation of the ultrastructure was limited by mechanical damage at the border and by ice crystal formation in the center of the tissue blocks. In normal myocardium of newborns, gold particles associated with free plasma membrane were frequently observed, with AGJs only seldom. In older rats, the opposite relation was found. During ischemia, no distribution changes occurred in newborn or 14-day-old rats. In 5-day-old rats, however, ischemia induced a shift of Cx43 from gap junction plaques to AGJs. In conclusion, HPF and FS are an ideal alternative to chemical fixation for immuno-EM as the excellent preservation of antigenicity is combined with a well-preserved ultrastructure. The results indicate that the process of degradation of gap junctions via AGJs gradually increases during postnatal rat heart development, a process that may be accelerated by ischemia in an early developmental state.     

Gap junctional coupling with cardiomyocytes is necessary but not sufficient for cardiomyogenic differentiation of cocultured human mesenchymal stem cells

Gap junctional coupling is important for functional integration of transplanted cells with host myocardium. However, the role of gap junctions in cardiomyogenic differentiation of transplanted cells has not been directly investigated. The objective of this work is to study the role of connexin43 (Cx43) in cardiomyogenic differentiation of human mesenchymal stem cells (hMSCs). Knockdown of Cx43 gene expression (Cx43↓) was established in naturally Cx43-rich fetal amniotic membrane (AM) hMSCs, while Cx43 was overexpressed (Cx43↑) in inherently Cx43-poor adult adipose tissue (AT) hMSCs. The hMSCs were exposed to cardiomyogenic stimuli by coincubation with neonatal rat ventricular cardiomyocytes (nrCMCs) for 10 days. Differentiation was assessed by immunostaining and whole-cell current clamping. To establish whether the effects of Cx43 knockdown could be rescued, Cx45 was overexpressed in Cx43↓ fetal AM hMSCs. Ten days after coincubation, not a single Cx43↓ fetal AM hMSC, control adult AT MSC, or Cx43↑ adult AT mesenchymal stem cell (MSC) expressed α-actinin, while control fetal AM hMSCs did (2.2% ± 0.4%, n = 5,000). Moreover, functional cardiomyogenic differentiation, based on action potential recordings, occurred only in control fetal AM hMSCs. Of interest, Cx45 overexpression in Cx43↓ fetal AM hMSCs restored their ability to undergo cardiomyogenesis (1.6% ± 0.4%, n = 2,500) in coculture with nrCMCs. Gap junctional coupling is required for differentiation of fetal AM hMSCs into functional CMCs after coincubation with nrCMCs. Heterocellular gap junctional coupling thus plays an important role in the transfer of cardiomyogenic signals from nrCMCs to fetal hMSCs but is not sufficient to induce cardiomyogenic differentiation in adult AT hMSCs.     

抑制小鼠HL-1心肌细胞桥粒斑蛋白基因表达对缝隙连接蛋白43结构和功能的影响

 目的：采用基因沉默技术抑制小鼠HL-1心肌细胞桥粒斑蛋白（DSP）基因表达以明确DSP与缝隙连接蛋白43(Cx43)的结构和功能关系。方法：用基因沉默技术抑制DSP基因的表达,Western blotting和流式细胞术检测HL-1细胞DSP和Cx43蛋白的表达,用双免疫荧光方法检测DSP与Cx43蛋白的表达与定位情况,并用划痕标记染料示踪技术检测细胞缝隙连接通讯状况。结果：与空白组和对照组相比,siRNA-DSP组的DSP和Cx43蛋白表达量降低（P＜0.05）。免疫荧光检测发现空白组和对照组DSP与Cx43蛋白存在共定位情况,而siRNA-DSP组DSP和Cx43蛋白共定位遭到破坏,Cx43蛋白出现再分布,在细胞内检测到Cx43蛋白,并且划痕标记染料示踪技术检测发现siRNA-DSP组Lucifer yellow通过HL-1细胞缝隙连接的传输功能降低。结论：DSP表达抑制不仅使Cx43出现再分布,而且影响缝隙连接传导功能。     

Cx31 and Cx43 double-deficient mice reveal independent functions in murine placental and skin development.

The overlapping expression of gap junctional connexins in tissues has indicated that the channels may compensate for each other. During development, Cx31 and Cx43 are coexpressed in preimplantation embryos, in the spongiotrophoblast of the placenta and in the epidermis. This study shows that Cx31/Cx43 double-deficient mice exhibit the known phenotypes of the single-knockout strains but no combined effects. Thus, Cx43, coexpressed with Cx31 at midgestation in the spongiotrophoblast of the placenta, cannot be responsible for a partial rescue of the lethal Cx31 knockout phenotype, as assumed before (Plum et al. [2001] Dev Biol 231:334-337). It follows that both connexins have unique functions in placental development. Despite an altered expression of other epidermal connexin mRNAs, epidermal differentiation and physiology was unaltered by the absence of Cx31 and Cx43. Therefore, in epidermal and preimplantation development, gap junctional communication can probably be compensated by other isoforms coexpressed with Cx31 and Cx43.     

Modulation of connexin 43 in rotenone-induced model of Parkinson's disease

Gap junctional communication plays an important role in various models of brain pathology, but the changes of gap junctions in Parkinsonism are still not understood. In this study, we show that a major gap junctional protein, connexin43 (Cx43), in astrocytes is enhanced both in a rat Parkinson's disease (PD) model induced with rotenone, a widely used pesticide that inhibits mitochondrial complex I, and in vitro in cultured astrocytes stimulated with rotenone. Enhancement of Cx43 protein levels in rotenone-treated cultured astrocytes occurred in parallel with an increase in gap junctional intercellular communication, but was not accompanied with an increase in Cx43 mRNA levels. Furthermore, the rotenone-induced increase of Cx43 protein levels both in vitro and in vivo was associated with increased levels of phosphorylated Cx43, which is required for gap junctional intercellular communication. In our rat PD model, phosphorylated Cx43 was selectively enhanced in the basal ganglia regions, which contain DA neurons or their terminal areas. The increase of Cx43 levels was lower in the substantia nigra pars compacta and the striatum than in the substantia nigra pars reticulata and the globus pallidus. Our findings indicate that modulation of Cx43 protein, and consequently gap junctional cellular communication, in astrocytes may play an important role in PD pathology.     

连接蛋白43在病理性瘢痕成纤维细胞中的表达

目的:探讨连接蛋白43(Cx43)及其构成的缝隙连接细胞间通讯(GJIC)在病理性瘢痕中的调控作用.方法:选择临床上不同病理分类的瘢痕组织(包括瘢痕疙瘩、增生性瘢痕)和正常修复组织,以正常皮肤为对照,应用免疫组织化学检测Cx43在成纤维细胞中的表达.结果:Cx43在增生性瘢痕和瘢痕疙瘩成纤维细胞中的表达明显少于正常修复组织及正常皮肤.结论:成纤维细胞Cx43的表达下调可能是造成病理性瘢痕组织中成纤维细胞间GJIC异常,从而导致病理性瘢痕发生的因素之一.     

Temporal profile of connexin 43 expression after photothrombotic lesion in rat brain

Following focal ischemic injury, several mechanisms lead to secondary expansion of the affected area and therefore increase the initial damage. We thoroughly investigated the expression of astrocytic connexin 43 (Cx43) after photothrombosis in rat brain. The temporal profile of Cx43 mRNA as well as protein expression was studied in remote, structurally uninjured cortical and hippocampal areas. The hippocampal formation revealed an increased number of Cx43 mRNA positive astrocytes and an up-regulated protein expression exclusively in the ipsilateral stratum oriens. We assume a participation of this region in glia scar formation. While Cx43 mRNA positive cells were transiently increased, immunoreactivity was reduced in the somatosensory cortex of injured hemispheres. The observed decrease of Cx43 protein in the post-ischemic cerebral cortex implies an impairment of gap junctional intercellular communication which might be detrimental to the brain.     

Connexin 43 expression in rat aortic smooth muscle after ovariectomy and hormonal replacement

The purpose of this study was to determine the effects of ovariectomy and long-term combined sexual hormone replacement on the gap junctional protein, connexin 43 (Cx43) of aortic medial smooth muscle cells in rats. Twenty non-pregnant mature Wistar female rats were divided into five groups (four animals in each group). Group A underwent ovariectomy, Group B underwent ovariectomy and received estradiol propionate, Group C underwent ovariectomy and received medroxyprogesterone acetate and Group D underwent ovariectomy and received both hormones. Group E was sham-operated and used as control. After 15 weeks of treatment, thoracic aortas were removed and immunohistochemistry was carried out using a specific fluorescent antibody against Cx43. Tissue sections were examined by confocal laser scanning microscopy and analysed by the Scion Image program. All five different groups had the same distribution and extent of Cx43 in the aorta. Neither the ovariectomy nor the hormone replacement had any effect on the Cx43 expression of aortic smooth muscle cells in rats as compared to control animals. These results indicate that sexual steroids do not influence the gap junctional protein Cx43 of the medial layer of aorta in rats. They may suggest that the beneficial effects of estrogen are not mediated via gap junctions in the human aorta either.