【作者】 胡多沙；

【导师】 曹亚；

【作者基本信息】 中南大学 ， 病理与病理生理学， 2009， 博士

【摘要】 肿瘤的发生发展过程中与免疫系统有十分密切的相互作用,肿瘤细胞可逃避、干扰以及抑制机体免疫系统,众多相关免疫蛋白和免疫分子参与其中。免疫球蛋白(Ig)是介导体液免疫反应的核心分子。经典免疫学理论认为,人类所有的细胞都含有胚系免疫球蛋白基因,但只有B淋巴细胞中,免疫球蛋白基因才具备转录和表达的可行性。新近多项研究发现,上皮性肿瘤细胞同样能够完成B淋巴细胞中独有的V(D)J重排以及类别转换等过程,进行Ig分子的转录与表达。我们前期工作从鼻咽癌中克隆了异常的人类Igκ轻链基因(Tx),具备软琼脂集落形成,裸鼠成瘤等恶性转化功能。在对Igκ基因研究中发现存在4个SNP位点。在鼻咽癌样本中分析这些SNP与鼻咽癌易感性之间的关联,结果显示SNP4-3基因型在正常人群和鼻咽癌人群中的分布具有显著性差异。其中杂合型AT-GC是鼻咽癌患病的遗传风险因子,SNP6-5则可能通过调节基因的重组和表达方式起到辅助作用;综合分析显示,4个位点均为杂合型的单倍型,在鼻咽癌患者人群中的分布频率高于其在正常人群中的分布,是鼻咽癌患病的风险因子。Ig在多种肿瘤细胞中表达,我们又证明了Igκ基因SNP群与鼻咽癌易感性相关。在此基础上,本论文第一部分研究Igκ轻链基因上的SNP位点与肺癌、乳腺癌、食管癌、胃癌、结肠癌以及宫颈癌遗传易感之间的关联。通过生物信息学预测Igκ基因恒定区SNP位点对Ig蛋白结构的影响,发现SNP3位点不同基因型可形成Igκ轻链蛋白空间结构上的差异。差异结构位于分子表面,不直接影响抗原抗体结合,可能对其功能造成一定影响。测定Igκ基因SNP位点基因型,分析基因型分布与多种上皮性肿瘤易感性的相关性,结果发现,SNP3、5位点不同基因型在胃癌与乳腺癌病例及对照中的分布存在统计学差异,提示与胃癌及乳腺癌发病相关。SNP3位点GG、GC型,SNP5位点CC、CT型,为胃癌、乳腺癌发病的风险因素。在胃癌全体病例与对照中,风险基因型SNP3-G,SNP5-C的OR值分别为1.64与1.67,代表携带这两种基因型的人群胃癌的发病风险分别是正常人的1.64倍与1.67倍;在乳腺癌全体病例与对照中,风险基因型SNP3-G,SNP5-C的OR值分别为1.94与1.56,代表携带这两种基因型的人群乳腺癌的发病风险分别是正常人的1.94倍与1.56倍。经测序新发现SNP位点nt3324,基因型为G/A,与SNP6-5完全连锁,连锁基因型为G-A-T与A-G-C。SNP具有微量效应和累积效应,SNP位点相关的发病风险可能与疾病的其他风险因素存在关联。本论文的第二部分针对每种病例中各因素对SNP位点基因型与肿瘤发病风险之间的关联进行分层分析。结果显示,在胃癌中,Igκ基因上SNP位点风险基因型SNP3-G与SNP5-C在幽门螺旋杆菌(HP)感染为阴性的人群中与胃癌发病相关,OR值为2.61(1.22—5.57)与2.57(1.20—5.49),表明SNP3、SNP5风险基因型主要在HP感染阴性的人群中发挥作用。同时,风险基因型SNP3-G与SNP5-C在＜49年龄段与胃癌发病相关,OR值分别为3.06(1.23-7.60)与2.83(1.24-6.43);在58-65年龄段,风险基因型SNP3-G与胃癌发病相关,OR值为2.89(1.28-6.53)。表明SNP3、SNP5风险基因型可能在特定范围年龄的人群中发挥作用。乳腺癌病例与对照中,风险基因型SNP3-G与SNP5-C在＞56年龄分组中与乳腺癌发病相关,OR值分别为2.53(1.31-4.88)与2.28(1.19-4.35),表明SNP3、SNP5风险基因型主要在年龄＞56人群中发挥作用,可能与激素分泌水平相关。同时,雌激素受体ER与孕激素受体PR表达为阴性的乳腺癌中,风险基因型SNP3-G与SNP5-C作用更为明显。在乳腺癌ER表达为阴性的病例中,OR值分别为2.71(1.52-4.85),2.41(1.32-4.41)。在PR表达为阴性的病例中,OR值分别为2.42(1.42-4.14),2.00(1.16-3.44)。上述研究表明Igκ基因上的SNP位点与胃癌以及乳腺癌的遗传易感性相关,是胃癌及乳腺癌的患病风险因子,并与HP感染、年龄、ER/PR表达等因素相关。肿瘤是遗传和环境因素多方面共同作用的疾病。我们的研究首次明确了Ig基因上SNP位点与肿瘤遗传易感之间的关联,为进一步理解环境及宿主因素等与肿瘤表达Ig的内在联系提供了新的角度,也为Ig在肿瘤与免疫系统相互作用中所发挥的作用提供了实验依据。本论文的第三部分以包括鼻咽癌、宫颈癌、乳腺癌、胃癌与结肠癌在内的细胞系为模型,通过检测Ig的不同存在方式,探讨肿瘤细胞表达Ig的异质性。正常Igκ轻链单体为26KD左右分子,两条轻链与两条重链结合为一个完整的Ig四聚体。我们发现肿瘤细胞中κ轻链单体呈26KD与55KD两种分子量大小,以55KD大小为主。其中26KD的κ轻链单体可与重链结合,55KDκ轻链不与重链结合。缺陷型Ig(truncatedIg)是一类在病理状态下存在的分子量与分子结构上都不同于正常Ig的分子,根据实验结果,我们推测肿瘤细胞表达缺陷型κ轻链。正常的α重链分子量为55KD,与轻链结合为IgA四聚体。研究发现肿瘤细胞表达的α重链单体呈40KD与60KD两种分子量大小。其中40KD分子具备Fc段,60KD分子不具备可识别的Fc段,均为缺陷型。这些新发现提示肿瘤细胞表达Ig具有异质性,这为理解其生物学功能提供了新的启示。同时研究发现,上皮性肿瘤细胞中存在膜上的mIg与分泌至细胞外的sIg,其中至少包含κ轻链与α重链成分,并且在不同组织来源的肿瘤细胞中其表达水平有差异。Hela细胞中具有较高的α重链分泌水平。以鼻咽癌细胞为模型,从信号传导的角度,我们还初步探讨了外源性致瘤蛋白EB病毒编码的潜伏膜蛋白LMP1对肿瘤细胞mIg与sIg的调控。发现LMP1可上调细胞的sIgκ分泌水平。我们的研究在病毒致瘤因素与肿瘤细胞表达Ig的调控机制间建立了新的联系,EB病毒编码的LMP1能够上调肿瘤细胞表达的Ig,可能在LMP1致瘤中有其特殊的功能与生物学意义。肿瘤的发病与免疫系统密切相关,涉及多个相关免疫分子。上皮性肿瘤细胞表达Ig的异质性及其相关机制的阐明,为我们从一个新角度理解缺陷型免疫分子在肿瘤发生发展中的意义提供了启示。更多还原

【Abstract】 Immunoglobulin(Ig) is the one of most important molecules mediating humoral immune response.By classic immunology theory, although all of the body cell contains germline gDNA that encodes Ig chains,only those in B lymphocytes are transcripted and expressed. Recently,accumulating evidences showed that cancer cells of epithelium origins could also carry out unique B cell processings such as V(D)J rearrangement and class switching,which led to Ig molecule transcription and expression.From a human nasopharyngeal carcinoma cell line,we have cloned an aberrant Igκgene(Tx).Aberrant Igκgene gene was shown to have transforming activity,including clone formation in soft agar and tumorigenesis in mude mice.Previous study identified 4 SNP loci on Igκgene,and their relationship with nasopharyngeal carcinoma(NPC) was analyzed.Results suggested that SNP3 and SNP5 were associated with NPC susceptibility. SNP3-CG genotype was distributed at a higher frequency in NPC patients than that in healthy individuals.Thus SNP loci on Igκgene gene is possibly a risk factor for NPC carcinogenesis.Based on that Ig expressed in cancer cells of various tissue origion and was we have confirmed its associated with NPC susceptibility,the first part of this project is focused on the relationship of SNPs on Igκgene with the susceptibility of lung,breast,esophagus,gastric,colon,and cervics cancers.Using bioinformatic methods,we predicted the changes of the 3-dimensional structures of Ig molecule caused by the different genotypes of SNP3,which located in the C region of Igκgene.Results implied that SNP3 could lead an amino acid change located on the surface of the Ig molecule,which had no direct impact on specific antigen-antibody binding,but might still have certain effects on its function,yet to be defined.Distribution for genotypes of SNPs on Igκgene was determined and analysed in all the clinical samples,which revealed significant differences between gastric,breast cancer cases and corresponding controls,implied that SNPs on Igκgene was associated with susceptibility of these two cancers.In gastric cancer cases and controls,the OR values for risk genotype SNP3-G and SNP5-G were 1.64 and 1.67 respectively, suggesting that the carriers might have 1.64 and 1.67 folds risks for developing gastric cancer compared with noncarriers.In breast cancer, the OR values were 1.94 and 1.57,suggesting that the risks for developing breast cancer was 1.94 and 1.56 folds higher in carriers than in noncarriers.By sequencing,a new SNP locus nt3324 was identified.Its genotype was G/A,completely linked with SNP6-5,the linkage genotype is G-A-T&A-G-C.It is known that SNPs have a micro-effect and cumulative effect.The risks for diseases associated with SNPs may correlate with other risk factors.In the second part of the research,stratified analyses for SNPs on Igκgene were carried out.The results revealed that in gastric cancer,the risks associated with SNP3-G and SNP5-C mainly presented in the cases with negative helicobacter pylori(HP) infection,OR value were 2.61(1.22-5.57) and 2.57(1.20-5.49) respectively,which suggested that risk genotypes of SNP3,5 played a role in gastric cancer carcinogenesis only in population without HP infection.Risk genotypes of SNP3 and SNP5 were associated with gastric cancer susceptibility in population aged below 49,with OR value being 3.06(1.23-7.60),2.83(1.24-6.43) respectively;SNP3-G also related to gastric cancer susceptibility in population aged between 58 and 65,with OR value being 2.89(1.28-6.53). The results implied that the risk genotypes of SNP3 and SNP5 may play a role in gastric cancer carcinogenesis in a population of a particular age group.In breast cancer cases and controls,the risk genotypes SNP3-G and SNP5-C were related to susceptibility in a population aged above 56,with OR value being 2.53(1.31-4.88) and 2.28(1.19-4.35),while,increased risk associated with SNP3G and SNP5 was observed in cases with negative estrogen receptor(ER) and progestogen receptor(PR) expression.The OR values for SNP3-G and SNP5-C in ER(-) cases were 2.71(1.52-4.85) and 2.41(1.32-4.41),in PR(-) cases were 2.42(1.42-4.41) and 2.00(1.16-3.44).The studies above revealed that SNP loci on Igκgene were risk factors for gastric cancer and breast cancer,associated with the diseases’ susceptibility,and correlated with other risk factors such as HP infection, age and ER/PR expression.The investigation provided new insights into the phenomenon of cancers expressing the Ig molecule.The third part of this research focused on the heterogeneity of Ig expression in different cancer cells including NPC,cervical cancer,breast cancer,gastric cancer and colon cancer.Normal Igκis a 26KD molecule combined with heavy chains to form an integral Ig tetramer.It was found that there were 2 types of Igκlight chain monomers expressed in cancer cells with molecular weight being 26KD and 55KD respectively.The 55KD chain was the major one and could not combine with heavy chains. It has been reported that truncated Ig is a class of Ig molecules with abnormal molecular weight and varied structures,exsiting under pathological conditions.We speculated that the 55KD Igκbelongs to the class of truncated Ig.Normalαheavy chain is a 55KD molecule,combined with light chains to form an integral IgA.In cancer cells,αheavy chain also had two types of Igαmonomers,molecular weight being 40KD and 60KD respectively.The 40KD chain has an Fc domain recognizable by protein A,and the 60KD chain lacks an Fc domain,they are both truncated.The epithelial cancer cells were also capable of expressing mIg in cell membrane and secreting sIg into cell culture medium.The expression and secretion level were varied in cell lines of different tissue origins.Theαheavy chain secreted by Hela cell line was moderately higher than that of other cells.Using NPC cell lines as a model,the regulation of cancer cell’s mIg and sIg by an exogenous oncoprotein latent membrane protein 1(LMP 1) encoded by EB virus was also explored.It was found that LMP1 could up-regulate the level of sIgκsecreted by NPC cancer cells.Our research established a new connection between virus oncogenic factors and the regulation mechanism of Ig expressed in cancer cells.This finding provided clues for exploring the biological significance of Ig expression in cancer cells.Tumor is a kind of immune deficiency disease;various clusters of related immuno-proteins are involved in carcinogenesis and tumor development.Our research focused on the SNP loci on Igκgene associated with cancer susceptibility;established the correlationship between risk genotypes of SNP loci on Igκgene and other environmental/host risk factors;and clarified heterogeneity of Ig expressed in cancer cells and its regulation mechanism.Our research have shed light on the significance of deficiency immuno-molecule in carcinogenesis.更多还原