【作者】 王绍成；

【导师】 赵雨杰；

【作者基本信息】 中国医科大学 ， 细胞生物学， 2005， 硕士

【摘要】 随着人类基因组测序的逐步完成,功能基因组的研究成为今后的主要研究方向之一。我们采用消减杂交技术（Subtractive Hybridization）筛选与肺癌发生、发展相关的功能基因,通过临床病人肺癌组织与其癌旁正常组织基因表达谱的变化,构建差异表达的cDNA文库,经测序和RT-PCR验证,筛选出与肺癌有重要贡献的一组基因。我们从中选择TIG2（Tazaroteneinduced gene 2）基因深入研究其对肺癌发生、发展的作用。 经RT-PCR,我们发现TIG2 mRNA在肺癌细胞中低表达或沉默,而在正常肺细胞呈程序性中表达。从正常肺细胞WI-38中克隆TIG2 cDNA,通过体外构建哺乳动物表达重组质粒（pFLAG-CMV/TIG2;pCI-neo/TIG2）,然后分别转染肺癌细胞（A2,LH7）,免疫荧光揭示TIG2编码蛋白主要定位于细胞质内,且分泌出去。培养的肺癌细胞（A2,LH7,BE1）经维甲酸不同浓度处理,发现TIG2基因仍表达沉默,因此,肺癌细胞（A2,LH7,BE1）是维甲酸无应答细胞;然后经去甲基化试剂（5-Aza-CdR）处理后,发现TIG2基因获得重新表达,因此,推断TIG2基因在肺癌细胞中表达沉默与DNA甲基化有关,经CpG岛软件预测,发现TIG2基因调控区存在CpG岛,文献报道,在肺癌细胞中,维甲酸受体β（RARβ）常出现甲基化,抑制其表达,所以,TIG2基因的表达沉默可能与RARβ和/或TIG2甲基化有关。对稳定转染TIG2 cDNA的肺癌细胞株（LH7-TIG2）功能变化作深入研究,流式细胞仪分析其细胞周期,与稳定转染相应空载质粒肺癌细胞株（LH7-mock）比较,发现G₁期细胞数明显增加（25%→49%）,G₂/M期明显下降（18.18%→8.00%）;LH7-TIG2细胞增殖和侵袭能力明显减弱等。同时,TIG2基因的表达使ERK2（p42）活性降低或消失。综合以上实验结果初步更多还原

【Abstract】 The studies on functional genome become one of directions with the complement of human genome sequence. Our focus on screening related functional genes to lung carcinogenesis progression using subtractive hybridization method. We have constructed differentially expressed cDNA libraries by gene expression profile changes in clinical lung tumor tissue compared with its parental normal lung tissue. We have screened a group of genes proofed by DNA sequencing and RT - PCR, which is the most important contributes to lung tu-morigenesis. We selected TIG2 ( Tazarotene - induced gene 2 ) from screened genes to further insight into the effect on lung tumorigenesis.We found that TIG2 mRNA is downregulated or silenced in lung cancer cells ,but constitutively expressed in normal lung cells by RT - PCR. TIG2 cDNA was cloned from normal lung cell(WI -38) , and mammalian recombinant expression plasmids containing TIG2, named pFLAG - CMV/HG2, pCI - neo/ HG2, respectively, then lung cancer cells ( A2,LH7 ) were transfected with these recombinant plasmids. The encoded product of TIG2 was located cytoplasm and secreted by immunofluorescence assay. TIG2 gene was still silenced in lung cancer cells during treatment with variant concentration of retinoic acid, so the cultured lung cancer cells is refractory cells. Interestingly, TIG2 gene was re -expressed after treatment with demethylating reagent (5 - Aza - CdR) , so our inference is that the silence of TIG2 gene in lung cancer was associated with DNA methylation. The promoter region of TIG2 gene existed CpG islands predicted with MethPrimer software, combined with retinoic acid receptor β( RARβ ) usually being methylation and silence in lung cancer,so the silence of TIG2 gene in lung cancer cells was related to the DNA methylation of TIG2 and/or retionic更多还原