肝癌细胞间隙连接蛋白Cx32,Cx43的流式细胞仪分析

细胞间隙连接（gspjunction）基因是最近发现的另一类非突变型抑癌基因”’。通过该基因表达的蛋白产物一间隙连接蛋白（co。exin，Cx）所构成的间隙连接通道，进行相邻细胞间能量和信息的传递，在细胞生长、分化控制中起重要的作用“’。同时，CX表达的降低与多种肿店的形成及恶性程度相关”’。我们对其中CX32，Cx43与肝癌细胞发生的关系进行了研究。l材料和方法1．l细胞系人肝癌细胞系HHCC，SMMC－7721及正常人肝细胞系QZG，由中国科学院上海细胞生物所提供。以含100ml／L热灭活胎牛血清（美国Gibco公司）、IXI0iii／L青霉素及…     

血管内皮生长因子及其受体在肝癌细胞中的表达及意义

目的 探讨人肝癌细胞株血管内皮生长因子（VEGF）及其受体的表达,进一步认识VEGF在肝癌血管形成中的作用机制,方法 以人脐静脉血管内皮细胞系ECV304和小鼠成纤维细胞系L929作为对照,采用免疫组化染色及RT-PCR,检测体外培养的人肝细胞肝癌细胞系SMMC7721、HHCC和HepG2中VEGF及其受体的表达。结果 SMMC7721、HHCC和HepG2细胞均有VEGF的表达。同时VEGF受体1（Flt-1)在SMMC7721细胞中也有表达;而HHCC和HepG2细胞则表达VEGF的受体2（KDR）。结论 在肝癌的血管形成中可能存在VEGF的自分泌机制。     

Connexin43基因在宫颈癌细胞系HeLa中的瞬时表达

目的 探讨细胞间隙连接基因connexin43在宫颈癌细胞系HeLa中的表达以及对该细胞生长的影响。方法 应用流式细胞术（ＦＣＭ）研究connexin43基因在ＨeLa细胞中的表达，以及对生长抑制及细胞增殖周期的影响。结果 connexin43基因表达阳性的细胞计数率由０．７％上升至２６．５％，细胞生长明显受抑。结论 connexin43基因可能是潜在的肿瘤抑制基因。     

间隙连接蛋白-43的表达调控及其功能

细胞间间隙连接通讯(G JIC)是相邻细胞之间的信息和能量物质交换的重要基础。作为G JIC重要成员之一的connexin43(Cx43)基因在转录和翻译水平受到机体的严密调控。相应地,除了调节G JIC外,Cx43也通过影响细胞增殖、分化和凋亡过程参与多种疾病尤其是肿瘤的发生和发展过程。     

不同转移潜能的人体横纹肌肉瘤细胞系细胞间通讯的研究

目的：研究细胞间通讯功能在3种不同转移潜能的人体横纹肌肉瘤细胞系间及其与培养的正常人肌母细胞的差别及其意义。方法：采用间接免疫荧光法,用激光扫描共聚焦显微镜定量测定与细胞间通讯有关的间隙连接蛋白（connexin43,CX43)的含量,并用荧光漂白后恢复技术检测间隙连接介导的细胞间通讯的功能。结果：CX43在正常肌母细胞中呈高表达,主要定位于细胞膜,有时定位于胞质,而在横纹肌肉瘤中表达下降;CX43阳性率及荧光强度均随着横纹肌肉瘤转移潜能的增加而降低（P＜0．05）,荧光漂白后恢复技术结果显示横纹肌肉瘤细胞荧光漂白后恢复率下降而且与横纹肌肉瘤细胞的转移潜能呈负相关（P＜0．05）,结论：不同程度细胞间通讯细胞的抑制可能与横纹肌肉瘤的转移潜能不同有关,其有可能成为判断横纹肌肉瘤恶性程度及预后的指标之一。     

肝癌细胞增殖受M-CSF胞内和胞外自分泌的双重调控

目的：研究胞内M-CSF及其受体在肝癌SMMC 7721细胞的表达与性质,探讨胞内M-CSF对SMMC 7721细胞增殖的影响及其机制。 方法： 以高表达M-CSF的人肝癌细胞系（SMMC 7721细胞）为模型，以免疫组化、流式细胞计数、反义技术与蛋白印迹等方法观测胞内M-CSF对SMMC 7721细胞增殖的影响及其机制。 结果： M-CSF 及其受体主要在SMMC 7721细胞的胞质、胞核中表达，胞内的M-CSF的相对分子量为20 000，M-CSFR的相对分子量为120 000；免疫共沉淀分析证明M-CSF在细胞内与M-CSFR以复合物的形式存在；M-CSF的单克隆抗体及其反义寡聚核苷酸能抑制SMMC 7721细胞的增殖、下调cyclinD1/E的表达和上调p16的表达，且M-CSF的单克隆抗体及其反义寡聚核苷酸的联合使用能进一步加强对SMMC 7721细胞抑制作用和增加下调cyclinD1/E和上调p16的表达幅度。 结论： SMMC 7721细胞受M-CSF胞外自分泌和胞内自分泌的双重调控。     

维生素D3受体mRNA在肝细胞增殖和肝癌发展中的作用

目的：探讨维生素D3受体mRNA在肝细胞增生和肝癌发展中的作用。方法：体外培养肝癌细胞株SMMC－7721和HCC－T细胞，培养时添加1000nmol/L、100nmol/L、10nmol/L 1，25-(OH)2D3作用1、3、6天后，用四唑盐比色试验（MTT）检测细胞的存活和生长；用反转录PCR（RT－PCR）检测维生素D3受体mRNA的表达。结果：1.25-(OH)2D3可以抑制维生素D3受体mRNA表达阳性的SMMC－7721细胞增生并且有剂量效应关系；对维生素D3受体mRNA表达阴性的HCC－T细胞没有抑制作用。9例肝癌组织标本维生素D3受体mRNA表达均为阳性。结论：1，25－（OH）2D3对于人肝癌细胞株SMMC－7721的增殖具有显著的抑制作用，其机械可能是通过维生素D3受体来实现的。     

CD133和CD34在肝细胞癌血管生成拟态形成中的表达及意义

背景：在恶性肿瘤中血管生成拟态的形成过程与肿瘤干细胞有密切联系。 目的：分析肝癌干细胞标志物CD133和CD34在肝细胞癌血管生成拟态形成中的表达及意义。 方法：建立肝癌细胞HCC97H、SMMC7721和正常肝细胞L02三维培养体系,结合激光捕获显微切割技术分离形成血管生成拟态的肝癌细胞,分别利用RT-PCR和Western blot技术检测CD133和CD34表达水平。 结果与结论：三维培养条件下,肝癌细胞HCC97H细胞形成血管生成拟态,肝癌细胞SMMC7721以及正常肝细胞L02未形成血管生成拟态。形成血管生成拟态的肝癌细胞HCC97H中CD133、CD34在mRNA及蛋白表达水平上均高于未形成血管生成拟态的肝癌细胞SMMC7721和正常肝细胞L02(P < 0.05)。表明高侵袭性肝癌细胞在三维培养下形成血管生成拟态,而低侵袭性肝癌细胞及正常肝细胞不能形成血管生成拟态;肝癌细胞形成血管生成拟态的过程中与表达肝癌干细胞有关。     

NNMT真核表达载体的构建并转染SMMC7721肝癌细胞株

目的构建peDNA3．1（-）／NNMT（尼克酰胺-N-甲基化酶）真核表达载体并将其稳定转染到肝癌细胞系SMMC7721中。方法采用PCR法从PGEX4T-1／NNMT重组质粒中克隆得到NNMT cDNA全长序列,并将扩增的eDNA片段与pMD19-T载体连接后亚克隆到真核表达载体pcDNA3．1（-）中。重组子经酶切分析及测序鉴定后,用脂质体转染技术将其导入到人肝癌细胞系SMMC7721,经G418筛选并建立稳定的转染细胞株,应用RT—PCR检测转染前后该细胞株NNMT基因的mRNA表达水平。结果真核表达载体构建成功．经RT-PCR检测,重组质粒转染株的NNMT基因mRNA表达水平高于对照组,证实NNMT基因已经稳定转染到SMMC7721细胞中并得到表达。结论成功建立了人基因NNMT的稳定转染细胞株,为进一步研究NNMT的功能奠定了基础。     

利用基因敲入技术在肝癌细胞系中稳定表达HBV的X抗原

目的利用基因诱捕载体整合到人类肝癌细胞系SMMC7721细胞的染色体基因中,建立稳定表达HBx蛋白的细胞系。方法通过电击转染将基因诱捕载体pU17导入人类肝癌细胞系SMMC7721细胞,经G418筛选,报告基因X-gal染色,PCR,Western印迹等方法检测HBxDNA的存在和蛋白质的表达。结果得到永久性高表达诱捕载体报告基因X-gal的阳性克隆;用Cre-LoxP置换系统,将构建好的HBx全长片段与诱捕载体的报告基因部分交换,HBx全长片段完整地整合在SMMC7721细胞的染色体基因中,并能从该细胞系中检测到HBx抗原。结论本实验提供了一种新的稳定表达蛋白的方法。该细胞系为制备、纯化X抗原和研究X基因调控提供了实验材料。     

Neoplastic reversal of human ovarian carcinoma cells transfected with connexin43

Gap junctional intercellular communication and expression of gap junction proteins (connexins) are decreased frequently in neoplastic cells including human ovarian carcinoma cells. In order to test the hypothesis that these changes contribute to the neoplastic phenotype of ovarian carcinoma cells, we transfected human ovarian carcinoma SKOV-3 cells with connexin43. Stable, connexin43-expressing transfectants were characterized for cell proliferation in vitro in normal, low-serum, and serum-free culture medium, for tumorigenicity in nude mice, and for sensitivity to adriamycin in vitro. Transfected clones expressed higher levels of connexin43 and gap junctional intercellular communication, reduced proliferation and greater dependence upon serum for growth in vitro, decreased tumor formation, increased sensitivity to adriamycin, and reduced expression of p-glycoprotein. These data suggest that gap junctional intercellular communication and/or connexin43 expression suppresses the neoplastic phenotype of ovarian carcinoma cells and their downregulation is involved in neoplastic transformation of ovarian epithelial cells. The increased sensitivity to adriamycin and elevated expression of p-glycoprotein by the transfected cells also suggest that gap junctional intercellular communication and connexin43 expression are involved in drug sensitivity and might be manipulated to enhance the clinical response.     

Expression of gap junction proteins connexin 26 and connexin 43 in normal human breast and in breast tumours

Gap junctional intercellular communication (GJIC) has been proposed as a cellular mechanism for tumour suppression and there is experimental evidence in support of this. If aberrant GJIC contributes to the formation of human breast tumours, one might expect that the connexins (gap junction proteins) expressed by epithelial cells in normal human breast would be down-regulated in tumour epithelial cells, or that tumour cells might show aberrant expression of other connexin family members. This study examines the immunocytochemical expression of connexins 26 (Cx26) and 43 (Cx43) in normal human breast, 11 benign breast lesions, two special-type carcinomas, and 27 invasive carcinomas of no special histological type (NST). Cx26 generally was not expressed at detectable levels in normal human breast, but punctate Cx43 immunostaining of the myoepithelial cells was found. Cx43 staining of the myoepithelium was also a feature of the benign lesions and ductal carcinoma in situ (DCIS). In general, the epithelial cells of benign lesions failed to stain for either connexin. Similarly, a lobular carcinoma did not express Cx26 or Cx43, but there was punctate Cx43 in the epithelial cells of a mucoid carcinoma. Cx26 was up-regulated in the carcinoma cells of 15 of the 27 invasive NST carcinomas, although the staining was usually cytoplasmic and heterogeneous. Cx43 was expressed by stromal cells, possibly myofibroblasts, in all NST carcinomas. Furthermore, there was heterogeneous Cx43 expression in the carcinoma cells of 14 of the 27 NST carcinomas and the staining was often intercellular and punctate, characteristic of functional connexins. Up-regulation of Cx26 and/or Cx43 in the carcinoma cells of over two-thirds of invasive lesions of NST is not necessarily inconsistent with a tumour suppressor role for GJIC. However, the role of gap junctions in the formation and progression of solid human tumours is likely to be more complex than indicated from experimental systems. © 1998 John Wiley & Sons, Ltd.     

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.     

Expressing connexin 43 in breast cancer cells reduces their metastasis to lungs

Recently the concept that gap junctions play a role in cancer cell metastasis has emerged. However, the mechanism by which this might occur is unknown. To examine this issue a metastatic breast cancer cell line, MDA-MB-435, was stably transfected with human Cx43 cDNA. Four clones of 435 transfectants (435/Cx43(+) c1, c6, c8, c14) and two clones of plasmid control (435/hy) were isolated and examined in this study. We found that expressing Cx43 in MDA-MB-435 cells decreased their expression of Cx32 but did not affect gap junctional intercellular communication, migration or invasion through Matrigel((R)). However, forced expression of Cx43 decreased the growth of MDA-MB-435 cells, decreased expression of N-cadherin, which is frequently associated with an aggressive phenotype, and increased MDA-MB-435 sensitivity to apoptosis. More importantly, there were fewer lung metastases in mice injected with 435/Cx43(+) cells relative to mice injected with 435/hy. These results suggest that expressing Cx43 in breast cancer cells decreases their metastatic potential through a mechanism independent of gap junctional communication but, rather, related to N-cadherin expression and apoptosis.     

Replacement of connexin 43 by connexin 32 in a knock-in mice model attenuates aortic endothelium-derived hyperpolarizing factor-mediated relaxation

Previous studies demonstrated that intercellular communication through endothelial, smooth muscle or myoendothelial connexin channels contributes to the control of vascular tone. At least four connexin types are present in the arterial wall. The aim of the present work was to assess the role played by connexin 43 (Cx43)-formed gap junctions on vessel function. Aortic reactivity to noradrenaline, acetylcholine and sodium nitroprusside, and endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxations, were analysed in a Cx43KI32 mouse model in which the coding region of Cx43 was replaced by that of connexin 32 (Cx32). Aortic rings were placed in organ baths containing a Krebs solution oxygenated at 37°C (pH 7.4). Confocal images of aortic rings confirmed connexin substitution in mutant mice. In control conditions, replacement of Cx43 by Cx32 in homozygous mutant mice did not modify endothelium-independent contractile responses to noradrenaline, or relaxations in response to sodium nitroprusside (endothelium independent) or acetylcholine (endothelium dependent). However, residual endothelium-dependent relaxations in response to acetylcholine after nitric oxide synthase and cyclooxygenase inhibition (EDHF type) were significantly reduced in homozygous Cx43KI32 mice (maximal effect values: 4.86 ± 0.37% of noradrenaline precontraction versus 7.06 ± 0.31% in wild-type, n = 8, P < 0.05). This attenuation was mimicked by treatment of rings from wild-type animals with the connexin-mimetic peptide ^37,43Gap27 (5 × 10⁻⁶ m ). In conclusion, replacement of Cx43 by Cx32 attenuates EDHF-mediated relaxations in mice aortic rings, suggesting that they are, at least in part, dependent on Cx43-formed gap junctions. In contrast, aortic responses to tested endothelium-independent agonists were not modified in knock-in animals.     

HAb18G/CD147拮抗肽对肝癌细胞表面抗原HAb18G的亲和性

目的：研究HAb18G／CDl47拮抗肽对肝癌细胞上HAb18G／CDl47抗原的亲和性。方法：利用流式细胞仪测定人肝癌细胞(HHCC，SMMC7721)上HAbl8G抗原的表达。分别在HAb18G／CDl47拮抗肽AP—1、AP—2和AP-6的N端标记生物素，用流式细胞仪和激光共聚焦显微镜测定AP—1、AP—2和AP-6与HHCC或SMMC7721的结合能力。结果：HAbl8G抗原在HHCC和SMMC7721细胞上均呈高表达。AP-6对HHCC的亲和力最强，AP—2次之，AP—1最弱。AP-6对与SMMC7721的亲和力也最强，AP—1次之，AP—2末检测到。结论：HAb18G／CDl47拮抗肽对肝癌细胞表面抗原HAb18G／CDl47具有较高的亲和性。     

Dilated bile canaliculi and attenuated decrease of nerve-dependent bile secretion in connexin32-deficient mouse liver

Gap junction channels in the rodent liver are composed of connexin26 (Cx26) and connexin32 (Cx32) proteins. Gap junctional intercellular communication in the mouse liver enhances the effects of hormonal or sympathetic stimulation of glucose release from glycogen stores. To determine whether contraction of bile canaliculi and bile secretion are dependent on the function of gap junction channels, we compared wild-type and connexin32-deficient mice. Confocal laser scanning microscopy of the wild-type mouse liver confirmed the close association of connexin26 and -32 proteins with the zona occludens-1 protein and actin filaments of the bile canaliculi. The decrease of bile flow after electrical stimulation of sympathetic nerves in the perfused liver was attenuated in the Cx32-deficient liver compared with wild-type controls. The amount of secreted bile, however, was similar in wild-type and Cx32-deficient livers. Furthermore, Cx32-deficient mice exhibited dilated bile canaliculi, suggesting that the contraction of bile canaliculi could be impaired in these animals.     

Gap junction communication between cells aggregated on a cellulose-coated polystyrene: influence of connexin 43 phosphorylation

The appropriate functioning of tissues and organ systems depends on intercellular communication such as gap junctions formed by connexin (Cx) protein channels between adjacent cells. We have previously shown that Swiss 3T3 cells aggregated on hydrophilic cellulose substratum Cuprophan (CU) establish short linear gap junctions composed of Cx 43 in cell surface plaques. This phenomenon seems to depend on the high intracellular cyclic AMP (cAMP) concentration triggered by attachment of the cells to CU. We have now used a cellulose-coated polystyrene inducing the same cell behaviour to analyse the gap junction communication between aggregated cells. The transfer of the dye Lucifer Yellow (LY) between cells showed that cells aggregated on cellulose substratum rapidly (within 90 min) establish functional gap junctions. Inhibitors of cAMP protein kinase (PKI) or protein kinase C (GF109203X) both inhibited the diffusion of LY between neighbouring cells. Western blot analysis showed that this change in permeability was correlated with a decrease in Cx 43 phosphorylation. Thus, cellulose substrata seem to induce cell-cell communication through Cx 43 phosphorylation modulated by PKA and PKC. To understand the mechanisms by which a substratum regulates gap junctional communication is critically important for the emerging fields of tissue engineering and biohybrid devices.