干扰素膜蛋白Tet-on系统稳定细胞系的建立及对CA16病毒的抑制作用

通过Tet-on调控系统,构建受多西环素诱导表达干扰素诱导的跨膜蛋白(interferon-induced transmembrane proteins 1/2/3,IFITM1/2/3)基因的HeLa细胞系,并初步探索了IFITM蛋白对柯萨奇病毒A16(CA16)的抑制作用.首先将调控质粒pTet-on转染进入HeLa细胞,通过G418筛选出阳性克隆细胞系,在此细胞系基础上共同转染反应质粒pTRE2-IFITM1/2/3和伴侣质粒pTK-Hyg,通过潮霉素筛选出单克隆细胞系,加入多西环素后利用Western印迹筛选出可诱导表达IFITM1/2/3蛋白的单克隆细胞系.使用实时荧光定量PCR(RT-qPCR)检测发现,多西环素诱导表达的IFITM蛋白对不同感染复数(multiplicity of infection,MOI)的CA16具有明显的抑制作用,其中IFITM 3对CA16的抑制效果最为明显.Tet调控IFITM1/2/3基因表达HeLa细胞系的成功建立,为进一步研究IFITM基因的功能及其抗病毒机理提供了一个理想的细胞模型.     

Cx43 在调节胃肠运动障碍机制中的研究进展

胃肠运动功能障碍是许多胃肠道疾病及其他疾病的重要临床表现,其发病率高达胃肠道疾病的70%以上。缝隙连接蛋白43(connexin 43,Cx43)是细胞间隙连接通讯中最重要的间隙连接蛋白,对胃肠道动力的形成和调节起着关键性作用。中西医治疗胃肠道疾病临床疗效显著,但其起效的分子机制尚未阐释清楚。本文从Cx43的细胞间隙连接通讯的角度,对Cx43在调节胃肠运动障碍机制中的研究进展作一综述,为进一步探究中西医调节胃肠运动障碍的机制研究奠定基础。     

癌细胞逆转过程中N-cadherin与间隙连接蛋白Cx43功能表达的关系

钙粘合蛋白 (N cadherin ,N cad)在人肺癌细胞的表达明显低于正常人肺细胞 .肺癌细胞的间隙连接通讯功能缺陷 ,连接蛋白Cx43表达抑制 .Cx43cDNA转染肺癌细胞的 4个阳性克隆其Cx43蛋白表达升高水平相近 ,但通讯功能有差别 ,与各克隆N cad的表达水平有正相关性 .N cad表达高的克隆Cx43在膜间隙连接的分布和通讯功能最明显 ,细胞分化改善 ,在裸鼠体内生长抑制 (抑制率 75 % )有显著性 .反之N cad表达低的克隆Cx43在膜间隙连接不明显 ,细胞通讯功能弱 ,恶性表型无逆转 .提示N cad与Cx43转录后表达过程的调节密切相关 ,两者介导的粘合和通讯功能有协同促进肺癌细胞逆转的作用 .     

小梅山猪骨髓间充质干细胞Cx43基因修饰

通过在小梅山猪骨髓间充质干细胞(BMSCs)中过表达缝隙连接蛋白43基因(Cx43),探究Cx43对小梅山猪BMSCs增殖及凋亡的影响,为构建转基因动物模型奠定基础。利用分子技术构建Cx43真核表达载体p EGFP-Cx43,Nucleofector TM法转染P3代BMSCs,检测细胞转染效率,经RT-PCR、免疫荧光和Western blotting鉴定Cx43的表达,流式细胞技术分析BMSCs的增殖能力和凋亡情况。酶切鉴定和测序结果表明,p EGFP-Cx43真核表达载体构建成功,转染BMSCs后,EGFP阳性细胞约占60%。转基因BMSCs中Cx43蛋白表达显著增加,细胞增殖能力显著提高、凋亡率显著下降。结果表明,在小梅山猪BMSCs中过表达Cx43不仅能促进细胞增殖而且抑制其凋亡,为下一步在体研究奠定了基础。     

用荧光图像示踪不同分化的食管癌细胞Cx43的空间分布

研究不同分化的食管癌细胞Cx43蛋白的表达和空间定位及其对食管癌细胞的生物学行为的影响。应用合适的荧光探针和激光扫描共聚焦显微镜,通过单粒子跟踪图像分析,检测Cx43蛋白荧光团在食管癌细胞中的表达和分布。Cx43在高分化食管鳞癌EC9706中的分布与EC109细胞相似,特别是细胞膜呈现明显的荧光团;近细胞膜Cx43蛋白含量相对细胞质增高,具有在细胞膜构建细胞通信的结构基础,预示着细胞向良性发展的趋势。低分化食管鳞癌KY150细胞和食管癌SHEEC细胞中Cx43蛋白的荧光团主要分布在近核膜的细胞质中,较少分布在细胞膜附近;近核膜的Cx43蛋白含量比近细胞膜的含量高,说明Cx43蛋白在细胞质中的滞留状态。研究显示SPT图像可以直观地示踪Cx43蛋白荧光团在食管癌细胞中的分布,从而确定Cx43蛋白在不同分化的食管癌细胞中表达及定位的痕迹。     

缝隙连接在同型半胱氨酸介导的自发性高血压大鼠血管平滑肌细胞增殖中的作用

目的:探讨缝隙连接(GJ)是否参与同型半胱氨酸(Hcy)介导的自发性高血压(SHR)大鼠血管平滑肌细胞(VSMCs)增殖及可能的分子机制。方法:原代培养SHR VSMCs,细胞分四组:①对照组,②Hcy组,③缝隙连接阻断剂(18α-GA)组和④Hcy+18α-GA组。MTT法及流式细胞仪检测细胞的增殖活性,免疫荧光技术观察细胞中Cx43、Cx40蛋白表达及定位,Western blot法检测细胞中Cx43、Cx40蛋白表达,染料示踪分子传递法(划痕标记染料传输法)检测细胞的缝隙连接功能。结果:①与对照组相比,Hcy组MTT法测得A值及细胞周期测得S值增高(P0.05),18α-GA组降低(P0.05);与Hcy组比,TGFβ-1+18α-GA组A值及S值均降低(P0.05)。②免疫荧光技术检测细胞中Cx43、Cx40蛋白表达呈阳性,两者共定位于胞浆。③与对照组相比,Hcy组Cx43、Cx40蛋白表达增强(P0.05),18α-GA组Cx43、Cx40表达均减弱(P0.05);与Hcy组比,Hcy+18α-GA组Cx43、Cx40表达均减弱(P0.05)。④与对照组相比,Hcy组缝隙连接功能明显增强(P0.05),18α-GA组缝隙连接功能明显减弱(P0.05),与Hcy组比,Hcy+18α-GA组缝隙连接功能显著降低(P0.05)。结论:Hcy通过上调SHR VSMCs Cx43和Cx40的蛋白表达,引起缝隙连接通讯功能的增强,促进了SHR VSMCs增殖。     

Connexin31 显性听力下降相关突变体研究

Cx31 突变可以导致常染色体显性听力下降、常染色体隐性听力下降、周围神经疾病伴听力丧失,以及变性红皮肤病角化病,其导致不同疾病的机理一直是研究的重点 . 利用定点突变技术 (site-directed mutagenesis, SDM) 构建 connexin31 显性听力下降相关突变体 Cx31R180X , Cx31E183K 并插入到真核表达载体 pEGFP-N1 ,转染 HeLa 细胞,转染 Cx31R180X-pEGFP-N1 和 Cx31E183K-pEGFP-N1 Cx31 突变体质粒的 HeLa 细胞质膜上没有出现斑块状染色和聚集现象,分别用内质网染料 conA 和高尔基体染料 WGA 进行免疫荧光染色,结果显示 Cx31 显性听力下降相关突变体蛋白主要分布在细胞质内,且大部分定位在内质网和高尔基体上 . 同时分别用抗 Cx31 和抗 GFP 抗体进行蛋白质印迹检测,证实 Cx31R180X , Cx31E183K 在转染的 HeLa 细胞中都有表达 . 研究发现 Connexin31 显性耳聋相关突变体 Cx31R180X , Cx31E183K 不能正常地形成间隙连接通道,这与 connexin31 EKV ( 变性红皮肤病角化病 ) 相关突变体能够运输到细胞膜上形成间隙连接通道的报道不相同,提示 connexin31 不同部位突变导致不同疾病的致病机理可能不一样 , 从而为解释“ one connexin two diseases ”提供分子水平的依据 .     

Cx26基因重表达抑制人鼻咽癌细胞系HNE1的恶性增殖

间隙连接蛋白 (Cx)基因在胚胎发育、细胞生长、分化以及细胞内环境的稳定过程中起重要调节作用 .肿瘤发生与Cx基因的表达及功能异常密切相关 ,肿瘤细胞常存在Cx基因表达下调或缺失 .将人Cx2 6基因编码区cDNA序列 ,亚克隆于真核表达载体pcDNA3 1(+) ,采用脂质体转染 ,将重组表达载体pcDNA3 1(+) Cx2 6转入鼻咽癌细胞系HNE1,使Cx2 6基因在HNE1中重表达 ,探讨Cx2 6基因对鼻咽癌细胞系HNE1的生物学功能的影响 .研究结果表明 :Cx2 6基因的重表达 ,抑制HNE1细胞生长 ,细胞周期阻滞于G0 G1期 ,HNE1细胞的克隆形成能力下降 ,裸鼠致瘤能力减弱 .     

Ubiquitin B在HSP90抑制剂17-AAG诱导人宫颈癌HeLa细胞周期阻滞中的作用

目的 研究Ubiquitin B（Ubb）在热休克蛋白90（HSP90）抑制剂17-AAG诱导人宫颈癌HeLa细胞周期阻滞中的作用及机制.方法 不同浓度17-AAG处理HeLa细胞后,流式细胞术检测细胞周期分布,荧光分光光度法检测细胞蛋白酶体活性变化;Ubb siRNA 转染HeLa细胞后,Real Time PCR法检测Ubb干扰效应,Western 印迹检测细胞周期相关蛋白的表达改变.结果 17-AAG可以诱导HeLa细胞阻滞于G2/M期,同时显著增强细胞内糜蛋白酶样蛋白酶体活性,并且两者的变化均呈现剂量依赖性;干扰HeLa细胞内Ubb后,可以逆转17-AAG引起的G2/M期阻滞;17-AAG可明显下调HeLa细胞周期相关蛋白Cdk1和Hec1的表达,并且这一变化也是Ubb依赖的.结论 Ubb在17-AAG诱导的HeLa细胞周期阻滞中发挥重要作用,Ubb和HSP90抑制剂17-AAG在功能上相互关联,可能成为宫颈癌治疗的新靶点.     

十字孢碱对宫颈癌细胞中Btk表达的影响

目的探讨十字孢碱对宫颈癌细胞生长及酪氨酸激酶Btk（tyrosine kinase）基因和蛋白表达的影响。方法用不同浓度的十字孢碱处理宫颈癌HeLa细胞24h,采用MTT比色法分析IC50。用不同浓度十字孢碱处理HeLa细胞24h,Real—time PCR检测酪氨酸激酶BtkmRNA表达的变化,Western印迹检测酪氨酸激酶Btk蛋白表达的变化。结果随着十字孢碱浓度及处理时间的增加,宫颈癌HeLa细胞生长抑制率升高;随着十字孢碱剂量的增加,酪氨酸激酶BtkmRNA及蛋白的表达水平逐渐减少。结论十字孢碱可呈剂量依赖性抑制HeLa细胞的生长,呈浓度依赖性减少酪氨酸激酶BtkmRNA及蛋白的表达。     

Transport and Function of Cx26 Mutants Involved in Skin and Deafness Disorders

We examined the subcellular localization and function of several Cx26 mutants that exhibit both sensorineural deafness and various skin disease phenotypes. To facilitate these aims, all Cx26 mutants were tagged at the carboxyl-terminal with green fluorescent protein (GFP), which has previously been shown not to affect Cx26 transport, assembly or function. In this article we focus on two point mutations (R75W and ΔE42) that occur in the first extracellular loop region of Cx26, a region hypothesized to be critical for correct hemichannel docking between contacting cells. In gap junctional intercellular communication (GJIC)-deficient HeLa cells, both R75W-GFP and ΔE42-GFP were transported to the cell surface and assembled into gap junction-like structures. Neither R75W-GFP nor ΔE42-GFP formed gap junctions that were permeable to Lucifer Yellow suggesting they are loss-of-function mutations. We also examined the phenotype of these two mutations in a rat epidermal keratinocyte (REK) cell line that is capable of undergoing differentiation. Using antibodies against several members of the connexin family reportedly expressed by epidermal keratinocytes, we found these cells endogenously expressed Cx43 and Cx26 but not Cx30, Cx32, or Cx37. When expressed in REK cells, similar to in HeLa cells, R75W-GFP and ΔE42-GFP were assembled at the cell surface into structures that resembled gap junctions. Future experiments will examine the effect of the Cx26 mutants on the function and differentiation of these epidermal keratinocytes.     

Identification of cells expressing Cx43, Cx30, Cx26, Cx32 and Cx36 in gap junctions of rat brain and spinal cord

We have identified cells expressing Cx26, Cx30, Cx32, Cx36 and Cx43 in gap junctions of rat central nervous system (CNS) using confocal light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling (FRIL). Confocal microscopy was used to assess general distributions of connexins, whereas the 100-fold higher resolution of FRIL allowed co-localization of several different connexins within individual ultrastructurally-defined gap junction plaques in ultrastructurally and immunologically identified cell types. In >4000 labeled gap junctions found in >370 FRIL replicas of gray matter in adult rats, Cx26, Cx30 and Cx43 were found only in astrocyte gap junctions; Cx32 was only in oligodendrocytes, and Cx36 was only in neurons. Moreover, Cx26, Cx30 and Cx43 were co-localized in most astrocyte gap junctions. Oligodendrocytes shared intercellular gap junctions only with astrocytes, and these heterologous junctions had Cx32 on the oligodendrocyte side and Cx26, Cx30 and Cx43 on the astrocyte side. In 4 and 18 day postnatal rat spinal cord, neuronal gap junctions contained Cx36, whereas Cx26 was present in leptomenigeal gap junctions. Thus, in adult rat CNS, neurons and glia express different connexins, with "permissive" connexin pairing combinations apparently defining separate pathways for neuronal vs. glial gap junctional communication.     

Nature of Cx30-containing channels in the adult mouse mammary gland

Oligonucleotide microarray analysis uniquely shows that several members of the connexin family of gap junction proteins are expressed by the epithelium during mouse mammary gland development. Connexin 26 (Cx26) is present throughout pregnancy and lactation, is then undetectable shortly after weaning, but reappears during involution. Additionally, Cx30 is abundant in late-pregnant and early lactating gland epithelium. From mid-pregnancy into early lactation, Cx26 and Cx30 co-localize in junctional plaques between epithelial cells, forming hemichannels of mixed connexin content. Microarray analysis also shows Cx32 is developmentally restricted to parturition, suggesting that specific modification of gap junction channel composition and/or intercellular communication pathways occurs at parturition. Specifically, heteromeric channels of all pairwise combinations are formed when these connexins are expressed within the same cells. Of these hemichannels, Cx26/Cx32 pores are increasingly sensitive to closure by taurine (an osmolyte implicated in milk protein synthesis) with increasing Cx26 content. In contrast, physiological taurine concentrations have no effect on Cx26/Cx30 and Cx30/Cx32 channel activity. Such changes in connexin expression and channel composition and their chemical modulation are discussed in relation to the various stages of mammary gland development in the adult mouse. This work was supported by grants GM36044 and GM61406 from the NIH to A.L. Harris and by generous funding from Breakthrough Breast Cancer Research to B. Gusterson.     

Identification of Cells Expressing Cx43, Cx30, Cx26, Cx32 and Cx36 in Gap Junctions of Rat Brain and Spinal Cord

We have identified cells expressing Cx26, Cx30, Cx32, Cx36 and Cx43 in gap junctions of rat central nervous system (CNS) using confocal light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling (FRIL). Confocal microscopy was used to assess general distributions of connexins, whereas the 100-fold higher resolution of FRIL allowed co-localization of several different connexins within individual ultrastructurally-defined gap junction plaques in ultrastructurally and immunologically identified cell types. In >4000 labeled gap junctions found in >370 FRIL replicas of gray matter in adult rats, Cx26, Cx30 and Cx43 were found only in astrocyte gap junctions; Cx32 was only in oligodendrocytes, and Cx36 was only in neurons. Moreover, Cx26, Cx30 and Cx43 were co-localized in most astrocyte gap junctions. Oligodendrocytes shared intercellular gap junctions only with astrocytes, and these heterologous junctions had Cx32 on the oligodendrocyte side and Cx26, Cx30 and Cx43 on the astrocyte side. In 4 and 18 day postnatal rat spinal cord, neuronal gap junctions contained Cx36, whereas Cx26 was present in leptomenigeal gap junctions. Thus, in adult rat CNS, neurons and glia express different connexins, with “permissive” connexin pairing combinations apparently defining separate pathways for neuronal vs. glial gap junctional communication.     

Connexin 26 expression prevents down-regulation of barrier and fence functions of tight junctions by Na+/K+-ATPase inhibitor ouabain in human airway epithelial cell line Calu-3

Gap junctions are considered to play a crucial role in differentiation of epithelial cells and to be associated with tight junction proteins. In this study, to investigate the role of gap junctions in regulation of the barrier function and fence function on the tight junctions, we introduced the Cx26 gene into human airway epithelial cell line Clau-3 and used a disruption model of tight junctions employing the Na(+)/K(+)-ATPase inhibitor ouabain. In parental Calu-3 cells, gap junction proteins Cx32 and Cx43, but not Cx26, and tight junction proteins occludin, JAM-1, ZO-1, claudin-1, -2, -3, -4, -5, -6, -7, -8, -9, and -14 were detected by RT-PCR. The barrier function and fence function of tight junctions were well maintained, whereas the GJIC was low level. Treatment with ouabain caused disruption of the barrier function and fence function of tight junctions together with down-regulation of occludin, JAM-1, claudin-2, and -4 and up-regulation of ZO-1 and claudin-14. In Cx26 transfectants, Cx26 protein was detected by Western blotting and immunocytochemistry, and many gap junction plaques were observed with well-developed tight junction strands. Expression of claudin-14 was significantly increased in Cx26 transfectants compared to parental cells, and in some cells, Cx26 was co-localized with claudin-14. Interestingly, transfection with Cx26 prevented disruption of both functions of tight junctions by treatment with ouabain without changes in the tight junction proteins. Pretreatment with the GJIC blockers 18beta-glycyrrhetinic acid and oleamide did not affect the changes induced by Cx26 transfection. These results suggest that Cx26 expression, but not the mediated intercellular communication, may regulate tight junction barrier and fence functions in human airway epithelial cell line Calu-3.     

Growth-suppressing activity of the transfected Cx26 on BICR-M1Rk breast cancer cell line

There are accumulating evidences suggesting that connexin (Cx), a gap junction channel-forming protein, acts as a growth suppressor in various cancer cells, and this effect is attributed to the gap junction-mediated intercellular communication (GJIC). In order to characterize the relationship between the growth-arresting activity of Cx26 and its cytoplasmic localizations after expression, we linked a nuclear export signal (NES) sequence to Cx26 cDNA before transfecting into a rat breast cancer cell line. A confocal fluorescent microscopic observation revealed that the insertion of NES minimized the nuclear expression of Cx26, and increased its cytoplasmic expression, including plasma membrane junctions. Total cell counting and BrdUrd-labeling experiments showed that the growth of the breast cancer cells was inhibited by 74% upon transfection of Cx26-NES, whereas only 9% inhibition was observed with only Cx26 cDNA.     

Connexins Induce and Maintain Tight Junctions in Epithelial Cells

Connexins (Cx) are considered to play a crucial role in the differentiation of epithelial cells and to be associated with adherens and tight junctions. This review describes how connexins contribute to the induction and maintenance of tight junctions in epithelial cells, hepatic cells and airway epithelial cells. Endogenous Cx32 expression and mediated intercellular communication are associated with the expression of tight junction proteins of primary cultured rat hepatocytes. We introduced the human Cx32 gene into immortalized mouse hepatic cells derived from Cx32-deficient mice. Exogenous Cx32 expression and the mediated intercellular communication by transfection could induce the expression and function of tight junctions. Transfection also induced expression of MAGI-1, which localized at adherens and tight junction areas in a gap junctional intercellular communication (GJIC)–independent manner. Furthermore, expression of Cx32 was related to the formation of single epithelial cell polarity of the hepatic cells. On the other hand, Cx26 expression, but not mediated intercellular communication, contributed to the expression and function of tight junctions in human airway epithelial cells. We introduced the human Cx26 gene into the human airway epithelial cell line Calu-3 and used a model of tight junction disruption by the Na⁺/K⁺-ATPase inhibitor ouabain. Transfection with Cx26 prevented disruption of both tight junction functions, the fence and barrier, and the changes of tight junction proteins by treatment with ouabain in a GJIC–independent manner. These results suggest that connexins can induce and maintain tight junctions in both GJIC-dependent and –independent manners in epithelial cells.     

Some Oculodentodigital Dysplasia-Associated Cx43 Mutations Cause Increased Hemichannel Activity in Addition to Deficient Gap Junction Channels

Oculodentodigital dysplasia (ODDD) is a dominantly inherited human disorder associated with different symptoms like craniofacial anomalies, syndactyly and heart dysfunction. ODDD is caused by mutations in the GJA1 gene encoding the gap junction protein connexin43 (Cx43). Here, we have characterized four Cx43 mutations (I31M, G138R, G143S and H194P) after stable expression in HeLa cells. In patients, the I31M and G138R mutations showed all phenotypic characteristics of ODDD, whereas G143S did not result in facial abnormalities and H194P mutated patients exhibited no syndactylies. In transfected HeLa cells, these mutations led to lack of the P2 phosphorylation state of the Cx43 protein, complete inhibition of gap junctional coupling measured by neurobiotin transfer and increased hemichannel activity. In addition, altered trafficking and delayed degradation were found in these mutants by immunofluorescence and pulse-chase analyses. In G138R and G143S mutants, the increased hemichannel activity correlated with an increased half-time of the Cx43 protein. However, the I31M mutated protein showed no extended half-time. Thus, the increased hemichannel activity may be directly caused by an altered conformation of the mutated channel forming protein. We hypothesize that increased hemichannel activity may aggravate the phenotypic abnormalities in ODDD patients who are deficient in Cx43 gap junction channels. Radoslaw Dobrowolski and Annette Sommershof contributed equally to this work.     

Suppression of the transformed phenotype of breast cancer by tropomyosin-1

Gap junctional intercellular communication (GJIC) is thought to play a crucial role in cell differentiation. Small gap junction plaques are frequently associated with tight junction strands in hepatocytes, suggesting that gap junctions may be closely related to the role of tight junctions in the establishment of cell polarity. To examine the exact role of gap junctions in regulating tight junctions, we transfected connexin 32 (Cx32), Cx26, or Cx43 cDNAs into immortalized mouse hepatocytes derived from Cx32-deficient mice and examined the expression and function of the endogenous tight junction molecules. In transient wild-type Cx32 transfectants, immunocytochemistry revealed that endogenous occludin was in part localized at cell borders, where it was colocalized with Cx32, whereas neither was detected in parental cells. In Cx32 null hepatocytes transfected with Cx32 truncated at position 220 (R220stop), wild-type Cx26, or wild-type Cx43 cDNAs, occludin was not detected at cell borders. In stable wild-type Cx32 transfectants, occludin, claudin-1, and ZO-1 mRNAs and proteins were significantly increased compared to parental cells and all of the proteins were colocalized with Cx32 at cell borders. Treatment with a GJIC blocker, 18 beta-glycyrrhetinic acid, resulted in decreases of occludin and claudin-1 at cell borders in the stable transfectants. The induction of tight junction proteins in the stable transfectants was accompanied by an increase in both fence and barrier functions of tight junctions. Furthermore, in the stable transfectants, circumferencial actin filaments were also increased without a change of actin protein. These results indicate that Cx32 formation and/or Cx32-mediated intercellular communication may participate in the formation of functional tight junctions and actin organization.