【作者】 郑慧；

【导师】 曹亚；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2006， 博士

【摘要】 我们的前期工作从中国人鼻咽癌细胞株CNE2的基因组DNA文库中克隆了一个具有细胞恶性转化功能的基因Tx。通过序列分析发现Tx基因是一个缺乏可变区(variable region，V区)的Ig kappa轻链基因。进一步的EST数据库分析提示，上皮性肿瘤细胞可能表达Ig kappa轻链。我们通过一系列分子生物学实验证实了上皮性肿瘤细胞表达Ig kappa轻链的转录本和蛋白质。 在上皮性肿瘤细胞表达Ig kappa轻链这一原创性发现的基础上，我们发现肿瘤组织中也存在Ig alpha重链蛋白的阳性信号。这使我们继Ig kappa轻链的研究之后，进一步对Ig alpha重链在上皮性肿瘤细胞中表达的现象和功能等进行深入而系统的研究，旨在为认识上皮性肿瘤细胞表达Ig这一异常现象及其潜在的生物学意义提供重要的理论依据。 1．利用生物信息学分析和预测Ig alpha重链在多种肿瘤组织中的表达 EST(expressed sequence tag)数据库常被用于鉴定某一基因在哪些组织或细胞中表达。因此，我们首先将Ig alpha重链恒定区(constant region，C区)基因进行了EST的搜索分析，发现大部分EST来源于B淋巴细胞，但有部分EST来源于肺癌、胃癌、结肠癌、膀胱癌、肾癌、宫颈癌等上皮性肿瘤组织或细胞。 进一步，我们通过Virtual Northern Blot分析了Ig alpha重链的表达谱，结果表明Ig alpha重链表达于B淋巴细胞、肺癌、肾癌、前列腺癌、宫颈癌、结肠癌、软骨肉瘤、卵巢癌、鳞状细胞癌等一系列上皮性肿瘤组织或细胞中。这为我们发现癌细胞表达Ig alpha重链这一现象提供了有力的佐证。 2．确定上皮性肿瘤细胞系和组织表达Ig alpha重链 生物信息学的分析提供了初步的证据。进一步，我们通过一系列实验方法来证实上皮性肿瘤细胞能够表达Ig alpha重链。考虑到Ig V(variable gene)，D(diversity gene)，J(joining gene)区基因片段众多，重组后的VDJ基因序列变异性较大，因此，我们首先分析了Ig保守的恒定区C区(constant gene)的表达。利用SW480(人结肠腺癌细胞系)、MCF-7(人乳腺腺癌细胞系)、HeLa(人宫颈低分化鳞状上皮癌)、MGC(人胃癌细胞系)、CNE1(人鼻咽癌细胞系)五种上皮性肿瘤细胞系，通过RT-PCR扩增Ig alpha重链恒定区(Cα区)，并通过测序，结果证实了多种上皮性肿瘤细胞系中存在Ig alpha重链Cα区mRNA基因的表达。 进一步，我们通过Northern Blot检测上述五种上皮性肿瘤细胞系中Ig alpha重链mRNA的表达。结果显示存在2.2Kb和1.5Kb左右的两个转录本，这更多还原

【Abstract】 We previously found a transforming gene Tx from CNE2 genomic DNA library, which has the function of cell malignant transformation. By Tx sequence Blast, we found that in fact Tx is a Ig kappa light chain lacking V region. Using dbEST analyse, we found that some kinds of cancer cell lines can express EST of Ig kappa. So we further confirmed that human cancer cells express Ig kappa mRNA and protein by a series of molecular biology experiments.We found Ig alpha heavy chain in some tumor tissues unexpected. It raised the issue whether human cancer cells could express Ig alpha heavy chain. So we will focus on the expression and function of Ig alpha heavy chain in human epithelial cancer cells. We hope that by studying the Ig alpha chain in cancer cells, people can further understand the significance of Ig expression in cancer cells.1. The prediction of EST of Ig alpha chain in cancers by bioinformation.EST(expressed sequence tag) database is often used to identify in which tissues or cells a given gene can express. By EST search of Ig alpha C region(constant region), we found that the majority of EST come from B lymphocytes, but some ESTs come from lung, stomach, colon, bladder, kidney and cervical cancers. It provides strong proofs of the expression of Ig alpha chain in cancer cells.Furthermore, we analyzed the expression profile of Ig alpha heavy chain in cancer cells, using Virtual Northern Blot. The results showed that Ig alpha chain was expressed in a series of cancer cells including lung, colon, prostate, kidney, ovary and cervical cancers.2. The expression of Ig alpha heavy chain in human cancer cells and tissues.Next, we tried to confirm that human epithelial cancers can express Ig alpha heavy chain. Because of variable V (variable gene), D (diversity gene), J (joining gene) region of Ig, we first analyzed the Ig Ca mRNA. We found Ig Ca mRNA sequences in Hela, MCF-7, SW480, MGC, CNE1 cell lines by RT-PCR and sequencing.Further, we used Northern Blot to determine the mRNA of Ig alpha in Hela, MCF-7, SW480, MGC, CNE1 cell lines. The results showed two transcriptions of Ig更多还原