【作者】 李静；

【导师】 耿美玉；

【作者基本信息】 中国海洋大学 ， 水产品加工与贮藏， 2008， 博士

【摘要】 间隙连接通讯(gapjunction intercellular communication，GJIC)是以细胞间的间隙连接(gapjunction，GJ)为途径进行的细胞间直接的信息交流的一种方式，可允许小于1-2kDa的小分子物质如金属离子、第二信使及其它小分子代谢产物通过，使细胞间可以不通过细胞外空间而达到直接通讯的目的。GJIC与肿瘤密切相关，多数肿瘤细胞表现为GJIC能力降低或丧失，这是肿瘤细胞内间隙连接异常，导致间隙连接功能障碍所致。间隙连接异常主要表现为连接蛋白(Cx)的异常，其分子机制可能有以下4个方面：(1)Cx基因突变；(2)Cx mRNA表达降低或异常表达；(3)Cx蛋白分布异常(分布在细胞质内而不是细胞膜上)；(4)其它蛋白对Cx功能的异常调节。由于间隙连接减少，GJIC水平降低，肿瘤细胞内生长刺激信号超过阀值或生长抑制信号达不到阀值，因此肿瘤细胞表现为过度增生和恶性表型。细胞膜表面的糖链有三种存在形式，即支链中性糖苷、酸性直链蛋白聚糖及糖脂，这些糖缀合物通过糖基化影响蛋白质功能，在细胞表面形成分支状的糖链宛如“天线”，传递着细胞间信息，参与细胞通讯和信号传递。细胞表面糖链结构的改变与疾病的发生、发展紧密相关。肿瘤细胞表面往往表现糖基异常化，导致糖链异常增多，如出现β(1，6)N-乙酰氨基葡萄糖过度表达，N-乙酰乳糖胺重复序列、唾液酸化和岩藻糖化升高等。这些变化可放大细胞内增殖信号，影响细胞间质的粘附、迁移和侵袭特性，导致肿瘤恶性增殖与转移。一方面肿瘤细胞间的间隙连接功能异常，一方面肿瘤细胞表面表达多种异常糖链，间隙连接与糖链在肿瘤中的异常增殖及转移中都起着重要作用，但两者的联系及糖链对肿瘤间隙连接功能形成的影响研究的较少。肿瘤细胞的一个重要特点之一是细胞膜表面唾液酸糖链含量增加。文献报道Birgit Rose小组采用衣霉素、苦马豆碱、粟精胺可以促进细胞间隙连接形成，促进细胞间隙功能增强。衣霉素、苦马豆碱、粟精胺作用可使细胞表面产生含有不同单糖组成的糖链，进而影响唾液酸在糖链的末端的形成，考虑到唾液酸在肿瘤中异常高表达是一种普遍现象，上述研究结果提示我们可能是肿瘤中细胞表面的唾液酸残基影响了肿瘤细胞的间隙连接功能，从而导致细胞间隙连接通讯降低，发生了肿瘤细胞的恶性增殖及转移。基于上述假设，本论文旨在利用多种手段，如分子生物学手段、激光共聚焦显微镜成像技术、流式细胞术，借助于化学调节剂观察唾液酸改变对细胞间隙连接功能的影响，在此基础上观察唾液酸糖链改变前后连接蛋白的表达、磷酸化水平及细胞上分布及动态变化，分析与连接蛋白相互作用的其它蛋白，探讨唾液酸对细胞间隙连接功能影响的机制。该论文的研究将从一个侧面阐明肿瘤异常糖链对肿瘤发生发展的影响，为糖生物学前沿探索性研究提供实验依据。第一部分：α—(2，3)／(2，6)唾液酸对肿瘤细胞Cx43间隙连接功能的影响第一章构建Hela-Cx43、Hela-Cx43-EGFP稳定细胞株采用逆转录病毒载体pBABE，成功构建了稳定高表达的Hela-Cx43、Hela-Cx43-EGFP细胞株，为后续的研究提供实验材料。第二章α—(2，3)／(2,6)唾液酸对肿瘤细胞Cx43间隙连接功能的影响1．衣霉素抑制Hela细胞膜表面N-糖链的生成，引起间隙连接功能显著增强。2．瞬时干扰Hela细胞糖基转移酶GnTV，降低细胞膜表面β1，6分支，促进间隙连接功能。3．唾液酸酶降低Hela细胞膜表面唾液酸，间隙连接功能显著增强。4．唾液酸酶抑制剂增加NIH-3T3细胞表面唾液酸，间隙连接功能显著降低。5、NIH-3T3-STX高表达α2,8 sialic acid，间隙连接功能无明显变化。上述实验结果表明，肿瘤细胞中唾液酸的高表达，参与介导了细胞间隙连接功能的降低，并且这种降低主要与α2,3 sialic acid与α2,6 sialic acid有关，而与α2,8 sialic acid无关。上述结果也说明了肿瘤由于唾液酸的增加促进肿瘤发生发展与细胞间隙连接功能降低有关。第二部分：α—(2，3)／(2，6)唾液酸对肿瘤细胞CX43间隙连接功能影响的机制研究1、Westernblotting结果表明降低细胞膜表面唾液酸并不改变Cx43的表达及其磷酸化水平：但Cx43连接斑形成增多；2、Westernblotting结果表明唾液酸酶作用后细胞ZO—1表达没有改变，IP及免疫荧光共定位结果表明唾液酸酶作用后促进了Cx43与ZO-1的结合；3、Westernblotting结果表明唾液酸酶作用后细胞N-cadherin表达没有改变，IP及免疫荧光共定位结果表明唾液酸酶作用后促进了Cx43与N-Cadherin复合物的形成；4、唾液酸酶作用后细胞内ERK1／2磷酸化水平明显增加，细胞间同质粘附增加，以及免疫荧光表明唾液酸降低后可促进N-cadherin的膜成簇。β-catenin可通过α-catenin介导和细胞骨架系统(如微管蛋白)相连，IP及免疫荧光共定位结果发现唾液酸酶作用后促进了细胞β—catenin与N-cadherin的结合，IP实验结果表明唾液酸酶作用后Cx43与β-catenin结合增多；5、免疫荧光实验表明唾液酸酶作用后CX43与微管末端共定位增加；6、FRAP实验结果表明降低细胞膜表面唾液酸能加快Cx43连接斑形成；7、Pull down实验结果表明唾液酸酶作用后N-cadherin分子上唾液酸修饰明显减少。上述结果表明，N-cadherin分子的唾液酸减少后，能够导致细胞上N-cadherin在细胞间聚集成簇，引起ERK1／2磷酸化水平增加，促进N-cadherin通过β-catenin与细胞骨架的结合，进而导致Cx43从胞内转运至细胞膜上，并与Z0—1结合增多，促进膜上连接斑的形成，从而促进CX43细胞间隙连接功能。更多还原

【Abstract】 Gap junction intercellular communication (GJIC),is a pathway of the direct cellto cell communication through the gap junctions(GJs) of the neighboring cells.It cantransfer the small molecules less than 1-2KDa via this pathway,for example metalions,second messenger and other small molecule metabolites and so on.GJIC plays a key role in carcinogenesis and frequently decreases or is absent inthe most cancer cells,which are also regarded as aberrant gap-junction functioninduced by abnomal connexins including four molecular mechanisms:(1)the mutantof Cx;(2) mRNA decrease or abnomal of Cx;(3)the abnomal distribute of Cx (in thecytoplasma other than membrance);(4) the abnomal regulation by other protein.The GJIC decreasing with the gap junction reduction leads the growthstimulation signals to overrun the threshold or leads the inhibition signals not to reachto the threshold,favoring the cancer cells abnomal proliferation and phenotypictransformationThere exist three types of sugar chain on the cell membrance,namely branchedneutral-a-glucoside,linear acidic proteoglycans and glycosphingolipids,which like aantenna on the surface of cell membrance transfering the interce-llular messages,and taking part in the cell communication.The alteration of structureof sugar chain is considered to be related to many diseases.Changes in the expression and structure of carbohydrates can be considered as anuniversal feature of malignant transformation.Changes in glycosylation include boththe under-and overexpression of naturally-occurring glycans,as well as new structureof glycans accompanying by increasedβ1,6GlcNAc-branching of N-glycans,N-Acetyl-Glucosamine,N-Acetyl-lactosamine,sialyl production and polyfucosylation,etc.These alteration may enlarge the proliferation signaling,therefore influence theadhesion,migration and invasion,and lead to cancer growth and metastasis at last. Aberrant gap-junction function and abnomal structure of carbohydrates are allplay important roles in the cancer cell proliferation and metastasis,however,therelationship of them was still rarely studied.Sialic acid on tumor cell surface is obviously expressed higher than normal cells.It is reported that the Birgit Rose group found the tunicamycin,swainsonine orcastanospermine could suppress glycosylation,and therefore promote the gapjunctions assembly,and increase the gap-junction function.In fact,tunicamycin,swainsonine or castanospermine may induce variable sugar chain structure withdifferent monosaccharides,and at last effect the form of the terminal of sialic acid.Thinking about the increase of sialic acid is very popular phenominona,the resultsabove suggest us that the aberrant sialic acid on cancer cell suface may effect the gap-junction function,decrease of GJIC,and lead cancer cell to progressively proliferationand metastasis.Base on the above hypothesis,the paper will integrate different technologies,including molecular biology technologies,confocal optical imaging technologies,flow cytometry,and chemistry reagents to study the effects of sialic acid on thegap-junction functions,furthermore,to research the its mechanisms by observatingthe alteration of connexins expression,dynamic distribution and colocation with itsrelated molecules.The paper results will be helpful to illustrate the influence of theaberrant glycosylation on cancer evolutionary development from one side view andlay a foundation for the forefront exploration of glycobiology.PartⅠThe effects ofα-(2,3)/(2,6) sialic acid on the Cx43 gap-junctionfunctions1.Construction of pBABE-Cx43,pBABE-Cx43-EGFP plasmid and the Cx43 andCx43-EGFP highly expressing cell lineUsing retrovirus vector pBABE-puro,we constructed successfully a stable highlyexpressing cell lines as experiment materials for further study.2.The effects ofα-(2,3)/(2,6)sialic acid on the Cx43 gap-junction functions.(1) Tunicamycin inhibitted the N-glycan expression on the surface of Hela cells,and increased notably gap-junction functions.(2) Employing instant RNA interference technology to reduce the glycotransferaseGnTV expression induced to decreaseβ1,6GlcNAc-branching of N-glycan,thereforepromoted gap-junction functions.(3) Sialidase effectively decreased sialic acid on the surface of the Hela cells,inducedincrease of gap-junction functions.(4) Sialidase inhibitor enchanced sialic acid of the surface of the NIH-3T3 cells,induced increase of gap-junction functions(5) NIH-3T3 cells highly expressed theα2,8 sialic acid,and no obvious change wasnoted in gap-junction functions.The results above indicate that the highly expressed sialic acid on cancer cells candecrease the gap-junction functions,which much more involves with the oα2,3 sialic acidorα2,6 sialic acid,but is none ofα2,8 sialic acid.Also,these results also show thatthe aberrant sialic acid on cancer evolutionary development is related to decrease ofthe gap-junction functions.PartⅡThe mechanism elucidation about effects ofα-(2,3)/(2,6)sialic acid on the Cx43gap-junction functions.(1) Westernblotting experiment showed that the decrease of sialic acid didn’t changethe Cx43 expression and its phospholation level.(2) Westernblotting experiment showed sialidase didn,t change the ZO-1 expression,IP and confocal experiment showed sialidase improved the interaction of Cx43 andZO-1.(3) Westernblotting experiment showed sialidase didn’t change N-cadherin expression,IP and confocal experiment showed sialidase promoted the complex formation ofCx43 and N-Cadherin.(4)Sialidase could increase the ERK1/2 phospholation level,and enchanedintercellular homotypic adhesion,Immunofluorometric assay showed sialidase couldpromote the N-cadherin cluster on the membrance.β-catenin can connect to thecytoskeleton(such as tubulin) byα-catenin,IP and confocal experiment showedsialidase could improve theβ-catenin binding to N-cadherin. (5) confocal experiment showed sialidase could increase the collocation of CX43 andthe terminal of tubulin.(6) FRAP results indicated that sialidase could increase the formation of Cx43 plague.(7)With Pull down experiment,we found that sialidase significantly reduce sialic acidmodification of N-cadherin.The results above indicate that the reduce of sialic acid modification ofN-cadherin can induce N-cacherin cluster on the cell membrance,increase theERK1/2 phospholation and promote the N-cadherin connection to cytoskeletonthroughβ-catenin,therefore lead Cx43 to migrate from cytoplasm to membrance,futhermore interate with ZO-1,increase the formation of Cx43 plague,and improvethe Cx43 gap junctions function.更多还原