腺苷三磷酸对人横纹肌肉瘤细胞系P21和c-myc蛋白表达的影响

为进一步研究腺苷三磷酸抑制癌细胞增殖诱导分化的作用机理，用ＡＴＰ作用于人横纹肌肉瘤单细胞克隆亚系ＲＤＬ６细胞，用免疫荧光细胞化学方法观察ＡＴＰ作用后的ＲＤＬ６细胞核内Ｐ＾２１蛋白表达增强，ｃ－ｍｙｃ蛋白表达降低，膜下胞质内微丝束－应力纤维组改善，粘着斑内的主要粘附蛋白－纽蛋白（ｖｉｎｃｕｌｉｎ）表达增强，正常小鼠成肌细胞Ｃ２Ｃ１２细胞核内Ｐ＾２１蛋白高表达，ｃ－ｍｙｃ蛋白不表达。结果表明：ＲＤＬ６     

免疫荧光和激光共聚焦显微镜对人横纹肌肉瘤细胞系的观察

细胞质内骨架包括微丝、微管及中间丝。现今可采用多种技术如各种电镜技术、免疫荧光技术、流式细胞术来研究细胞骨架在细胞生命活动中的作用。近年来发展起来的激光扫描共聚焦显微镜 (ｌａｓｅｒｓｃａｎｎｉｎｇｃｏｎｆｏｃａｌｍｉｃｒｏｓｃｏｐｅ,ＬＳＣＭ)可以显示细胞骨架的结构及分布 ,并能进行定量分析。我们采用直接与间接免疫荧光法 ,运用ＬＳＣＭ观察细胞骨架 ,比较人体的横纹肌肉瘤细胞系与培养的正常人横纹肌母细胞的差别。一、材料与方法1.细胞来源 :ＲＤ和Ａ673是人胚胎性横纹肌肉瘤细胞系 ,引自上海细胞库 ,其中ＲＤ软琼…     

分化诱导剂对人单核样白血病J6－2细胞凝集素结合及［~（125）I］UdR掺入的影响

将Ｊ６－２细胞分别用分化诱导剂ＧＭ＿３（５０μｍｏｌ／Ｌ）、ＤＭＳＯ（１．２％）处理６天、ＴＰＡ（１０ｎｇ／ｍｌ）处理３天，观察它们对１１种凝集素结合反应的影响。结果表明，经ＧＭ＿３处理后，Ｊ６－２细胞对凝集素结合反应增强的只有ＵＥＡ（－至＋），变弱的只有ＢＳＡ（＋＋至－），其余的都无变化。经ＤＭＳＯ处理后，结合反应变强的有ＵＥＡ（－至＋）及ＰＮＡ（－至＋＋），变弱的有ＢＳＡ（＋＋至－）及ＰＳＡ（＋＋至＋）。经ＴＰＡ处理后，结合反应变弱的有ＢＳＡ（＋＋至＋）、ＷＧＡ（＋＋至－）及ＲＣＡ（＋＋至＋），变强的有ＵＥＡ（－至＋）、ＳＢＡ（－至＋）及ＳＪＡ（－至＋＋）。对３种分化诱导剂处理后都无变化的有ＤＢＡ（－）、ＬＣＡ（＋＋）及ＣｏｎＡ（＋＋）。本文还观察到３种分化诱导剂都能抑制［￣１２５Ｉ］ＵｄＲ向Ｊ６－２细胞的掺入。     

IL—6信号转导与转录激活子活性研究

目的　探讨 Ｉ Ｌ６ 生物效应与胞内磷酸化蛋白和转录激活子活性变化之间的关系。方法　应用原位杂交和 Ａ Ｐ Ａ Ａ Ｐ 方法检测细胞内 Ｉ Ｌ６ Ｒ ｍ Ｒ Ｎ Ａ 和蛋白的表达;采用 Ｄ Ｎ Ａ 结合蛋白凝胶阻滞电泳分析 Ｉ Ｌ６ 信号转导与 Ａ Ｐ Ｒ Ｆ 活性的相关性;用免疫沉淀和 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 观察 Ｉ Ｌ６ 信号转导中磷酸化蛋白的变化情况。结果　（１） Ｉ Ｌ６ 受体（ Ｉ Ｌ６ Ｒ） ｍ Ｒ Ｎ Ａ 和蛋白表达阳性的人骨髓瘤细胞系（ Ｓ Ｋ Ｏ００７） 体外对 Ｉ Ｌ６ 有增殖反应;（２） 胞内转录激活子活性与 Ｉ Ｌ６ 诱导时间和剂量有相关性,抗ｇｐ１３０ 单克隆抗体和酪氨酸蛋白激酶抑制剂均可特异性抑制转录激活子活性;（３） Ｉ Ｌ６ 诱导 Ｓ Ｋ Ｏ００７细胞后,胞浆内出现一组相对分子质量为（１３０ 、９０ 、５４ 、３６） ×１０３ 磷酸化蛋白,它们的磷酸化活性与转录激活子活性一样,也具有时间和剂量相关性。结论　结果提示,转录激活子和这组相对分子质量不同的酪氨酸磷酸化蛋白参与 Ｉ Ｌ６ 信号转导调节。     

P6A和RGDS对离体大鼠缺血／再灌注心肌的保护作用

本工作观察两种新型抗凝剂Ｐ６Ａ和ＲＧＤＳ对缺血／再灌注心肌的保护作用。结果表明：再灌注期给予１０＾－５ｍｏｌ／Ｌ的Ｐ６Ａ可显著降低再灌注心律失常的发生率，减轻再灌心肌的脂质过氧化和钙超载；１０＾－６ｍｏｌ／Ｌ的ＲＧＤＳ具有相似的作用，但二者对心肌局部血管紧张素Ⅱ的生成无明显影响，提示Ｐ６Ａ和ＲＧＤＳ可能是通过调节心肌细胞内的钙稳态来拮抗钙超载的发生，发挥其心肌保护作用。     

视黄酸对人外周血淋巴细胞CD25分子表达影响的研究

本实验采用分子杂交及流式细胞分析技术研究了视黄酸（ｒｅｔｉｎｏｉｃａｃｉｄ，ＲＡ）对人外周血淋巴细胞ＣＤ２５分子表达和细胞功能的影响，结果显示，大剂量（２．５×１０－４ｍｏｌ／Ｌ）ＲＡ处理ＰＨＡ活化４８ｈ淋巴细胞，其ＣＤ２５ｍＲＮＡ含量、ＣＤ２５阳性细胞百分率和膜表面ＣＤ２５分子数量均显著降低，淋巴细胞大部分受阻于Ｇ０／Ｇ１期，Ｇ２＋Ｍ期细胞仅占７．６％。而适量（２．５×１０－６～２．５×１０－７ｍｏｌ／Ｌ）ＲＡ作用后ＣＤ２５分子表达和Ｇ２＋Ｍ期细胞明显增加。该研究提示ＲＡ对ＣＤ２５分子表达和淋巴细胞功能的影响有一定的剂量依赖性。     

自身免疫病患者T淋巴细胞表型分析

本文报告利用流式细胞仪检测了１０例系统性红斑狼疮（ＳＬＥ）患者、１８例干燥综合症（ＳＳ）患者、３７例风湿性关节炎（ＲＡ）患者和１６例人外周血Ｔ淋巴细胞表型，发现ＳＬＥ患者ＣＤ３＾＋Ｔ细胞明显降低，ＳＳ和ＲＡ患者正常；ＳＬＥ患者ＣＤ４＾＋／ＣＤ８＾＋Ｔ细胞比值倒置，ＳＳ和ＲＡ正常，但ＳＳ和ＲＡ ＣＤ８＾＋Ｔ细胞明显减少。文中对这三种疾病发病机理进行了初步讨论。     

分化诱导剂对人单核样白血病J6—2细胞凝集素结合及[^125I…

将Ｊ６－２细胞分别分化诱导剂ＧＭ３（５０μｍｏｌ／Ｌ、ＤＭＳＯ８１．２％）处理６天、ＴＰＡ（１００ｎｇ／ｍｌ）处理３天，观察它们对１１种凝集素结合反应的影响。结果表明，经ＧＭ３处理后，Ｊ６－２细胞对凝集素结全反应增强的只有ＵＥＡ（－至＋，变弱的只有ＢＡＳ（＋＋至－），其余的都无变化。     

低密度脂蛋白诱导肾小管上皮细胞增殖的信号通路

为观察低密度脂蛋白（ＬＤＬ）对肾上管上皮细胞的影响及涉及的相关信号传导机制，以人近曲行小管上皮细胞系（ＨＫ－２）为对象，采用３Ｈ－ＴｄＲ掺入与细胞计数测定增殖；特异性底物磷酸化法测定细胞外信号调节激酶（Ｅｎｇ）活性；凝胶迁移率法检测转录活化因于（ＡＰ－１）ＤＮＡ结合活性。发现ＬＤＬ１００～５００ｕｇ／ｍＬ刺激ＨＫ－２细胞７２ｈ可促进其增殖：加入ＬＤＬ２５０ｕｇ／ｍＬ后２～６０ｍｉｎ内可使该细胞ＥＲＫ活性明显增加，１０ｍｉｎ时达高峰；不同剂量（５０～５００ｕｇ／ｍＬ）刺激时，ＥＲＫ活性分别较对照组增加１．５６～２．１９倍。ＬＤＬ促使核内转录因子ＡＰ－１结合活性增加。ＥＩＵＬ特异性阻断剂ＰＤ（５０ｕｍｏｌ／Ｌ）可以分别阻断ＬＤＬ刺激的ＨＫ－２细胞ＥＲＫ活性和ＤＮＡ合成。ＲＭＡ阻断ＰＫＣ信号途径后，ＬＤＬ对于ＥＲＫ的活化效应明显减弱。     

人可溶性IL-6R基因在COS7细胞中的表达及活性分析

目的在ＣＯＳ７细胞中表达具有生物学功能的人可溶性ＩＬ－６Ｒ（ｓＩＬ－６Ｒ），作为研究ｓＩＬ－６Ｒ结构与功能关系的基础。方法首先利用ＰＣＲ技术扩增出人可溶性ＩＬ－６Ｒ（ｈｓＩＬ－６Ｒ）编码基因片段，并重组入克隆载体ｐＡＬＴＥＲ－１。通过基因序列分析确定了目的基因的核苷酸序列，并进一步构建了由ＳＶ４０晚期启动子和ＨＣＭＶ早期启动子控制的表达质粒ｐＳＶＬ６Ｒ和ｐＣＭＶ６Ｒ。用脂质体介导的方法将表达质粒转染ＣＯＳ７细胞，并分别在ｍＲＮＡ水平（斑点杂交）和蛋白水平（ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ－ｂｌｏｔ）检测ｓＩＬ－６Ｒ基因在ＣＯＳ７细胞中的表达。在７ＴＤ１，ＬＴ１２两种ＩＬ－６反应细胞系上检测转染细胞上清（含ｓＩＬ－６Ｒ）的生物学活性。结果在ｍＲＮＡ水平和蛋白水平分别检测到ｓＩＬ－６Ｒ基因在ＣＯＳ７细胞中的表达，表达产物分子量约为５００００。表达产物在７ＴＤ１，ＬＴ１２细胞系上检测到明显的生物学活性。结论天然ｓＩＬ－６Ｒ基因在ＣＯＳ７细胞中的成功表达为进一步制备ｓＩＬ－６Ｒ突变体及其结构与功能关系的研究奠定了基础     

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.     

肝癌细胞中connexin32、connexin43的表达及其与间隙连接通讯功能的相关性

目的：研究肝细胞肝癌和正常肝细胞间隙连接蛋白connexin32（Cx32)、connexin43(Cx43）的表达，及其对间隙连接通讯功能（GJIC）的影响。方法：应用应用培养及流式细胞分析技术（FCM），研究肝癌细胞系HHCC、SMMC－7721和正常肝系QZG细胞中Cx32和Cx43的表达。结合Ｌucifer Yellow划痕标记荧光传输技术（SLDT），检测上述细胞的间隙连接通讯功能。结果：流式细胞仪分析证实，Cx32蛋白在肝癌细胞系HHCC、SMMC－7721和正常肝细胞系QZG细胞中表达的阳性率分别为1．9％、0．7％和99．0％；Cx43蛋白在HHCC、SMMC－7721和QZG细胞中表达的阳性率分别为7．3％、26．5％和99．1％。SLDT检测发现肝癌细胞HHCC，SMMC－7721的间隙连接通讯功能较正常肝细胞QZG明显减弱。结论；Cx3、Cx43蛋白在正常肝细胞中具有较高水平的表达，在肝癌细胞中表达水平显著降低，肝癌细胞的间隙连接通讯功能较正常肝细胞亦明显减弱。Cx32、Cx43表达调控异常引起的间隙连接通讯障碍可能与肝癌的发生密切相关。     

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.     

Connexin 43 reverses malignant phenotypes of glioma stem cells by modulating E-cadherin

Malfunctioned gap junctional intercellular communication (GJIC) has been thought associated with malignant transformation of normal cells. However, the role of GJIC-related proteins such as connexins in sustaining the malignant behavior of cancer stem cells remains unclear. In this study, we obtained tumorspheres formed by glioma stem cells (GSCs) and adherent GSCs and then examined their GJIC. All GSCs showed reduced GJIC, and differentiated glioma cells had more gap junction-like structures than GSCs. GSCs expressed very low level of connexins, Cx43 in particular, which are key components of gap junction. We observed hypermethylation in the promoter of gap junction protein α1, which encodes Cx43 in GSCs. Reconstitution of Cx43 in GSCs inhibited their capacity of self-renewal, invasiveness, and tumorigenicity via influencing E-cadherin and its coding protein, which leads to changes in the expression of Wnt/β-catenin targeting genes. Our results suggest that GSCs require the low expression of Cx43 for maintaining their malignant phenotype, and upregulation of Cx43 might be a potential strategy for treatment of malignant glioma.     

Connexin expression and functional analysis of gap junctional communication in mouse embryonic stem cells

Gap junctional intercellular communication (GJIC) has been suggested to be necessary for cellular proliferation and differentiation. We wanted to investigate the function of GJIC in mouse embryonic stem (ES) cells using pharmacological inhibitors or a genetic approach to inhibit the expression of connexins, that is, the subunit proteins of gap junction channels. For this purpose, we have analyzed all known connexin genes in mouse ES cells but found only three of them, Cx31, Cx43, and Cx45, to be expressed as proteins. We have demonstrated by coimmunoprecipitation that Cx31 and Cx43, as well as Cx43 and Cx45, probably form heteromeric gap junction channels, whereas Cx31 and Cx45 do not. The pharmacological inhibitors reduced GJIC between ES cells to approximately 3% and initiated apoptosis, suggesting an antiapoptotic effect of GJIC. In contrast to these results, reduction of GJIC to approximately 5% by decreased expression of Cx31 or Cx45 via RNA interference in homozygous Cx43-deficient ES cells did not lead to apoptosis. Additional studies suggested that apoptotic death of ES cells and adult stem cells reported in the literature is likely due to a cytotoxic side effect of the inhibitors and not due to a decrease of GJIC. Using the connexin expression pattern in mouse ES cells, as determined in this study, multiple connexin-deficient ES cells can now be genetically engineered in which the level of GJIC is further decreased, to clarify whether the differentiation of ES cells is qualitatively or quantitatively compromised.     

The metabolic inhibitor antimycin A can disrupt cell-to-cell communication by an ATP- and Ca2+-independent mechanism

In cardiac myocytes of new-born rats, the degree of intercellular communication through gap junctional channels closely depends on the metabolic state of the cells. In contrast, in stably transfected HeLa cells expressing rat cardiac connexin43 (Cx43, the main channel-forming protein present in ventricular myocytes), a major part of junctional communication persisted in ATP-depleted conditions, in the presence of a metabolic inhibitor (KCN) or of a broad spectrum inhibitor of protein kinases (H7). However, another metabolic inhibitor, antimycin A, which like cyanide inhibits electron transfer in the respiratory chain, totally interrupted cell-to-cell communication between Cx43-HeLa cells, even in whole-cell conditions, when ATP (5 mM) was present. Antimycin A caused a modest increase in cytosolic calcium concentration; however, junctional uncoupling still occurred when this rise was prevented. Conditions of ischemic insult (e.g. ischemia or chemical hypoxia) frequently cause the activation of protein kinases, particularly of Src and MAP kinases, and such activations are known to markedly disrupt gap junctional communication. Antimycin-induced junctional uncoupling occurred even in the presence of inhibitors of these kinases. Antimycin A appears able to cause junctional uncoupling either through the ATP depletion it induces as a metabolic poison or via a direct action on gap junction constituents.     

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.     

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.     

Impaired gap junction connexin43 in Sertoli cells of patients with secretory azoospermia: a marker of undifferentiated Sertoli cells

Gap junctions are intercellular channels formed of connexins (Cx) at appositional plasma membranes between adjacent cells that have been involved in the control of cell proliferation and differentiation. Altered Cx expression is implicated consistently in several human diseases and in tumorigenesis. Although Cx43 plays a critical role in Sertoli cell control of spermatogenesis, there is no evidence of its altered expression in human testicular pathologies. We show here that Cx43 mRNA expression was significantly reduced in testes of infertile patients with secretory azoospermia (p < 0.05) compared with testes displaying normal spermatogenesis (excretory azoospermic patients). In Sertoli cell-only syndrome, in situ hybridization and immunohistochemistry analyses indicated that Cx43 mRNA and protein were undetectable in Sertoli cells but were still present in the interstitial compartment. In a rat model of Sertoli cell-only syndrome, the lack of Cx43 in Sertoli cells was associated with an impairment of gap junction intercellular communication between adjacent Sertoli cells. These results reveal that Cx43 mRNA and protein expression are markedly impaired in Sertoli cells of infertile patients. This defect could be a new functional marker of undifferentiated Sertoli cells and could be related to the increased risk of testicular cancer recently described in the population of infertile men.