小鼠小肠上皮内淋巴细胞与脾T淋巴细胞间基因表达差异分析

Gene expression differences between murine intestine intraepithelial lymphocytes and splenic T lymphocytes

目的 :比较小鼠小肠上皮内淋巴细胞 (intestine intraepithelial lymphocyte,i IEL )与脾脏 T淋巴细胞的基因表达差异 ,建立两者的 c DNA差异减除文库 ,分析 i IEL优势表达的基因。方法 :采用 Percoll密度梯度法分离 i IEL ,尼龙毛柱法分离脾脏 T淋巴细胞。应用改良后的差减杂交法 ,连接特异的衔接子 ,通过减除杂交从 i IEL中去除与脾 T淋巴细胞相同的表达基因后 ,经抑制性 PCR扩增 ,并与质粒载体连接 ,得到二者的 c DNA减除文库。用 Northern印迹分析法从中筛选 i IEL优势表达的 EST。 结果 :从 i IEL特异的 c DNA减除文库中筛选出 5 0条有明显差异的 EST,进行测序 ,并取得登录号。对所得 EST用基本局域联配搜索工具 (basic local alignmentsearch tool,BL AST)进行比对 ,分析得出 14个 i IEL优势表达的基因。这些基因分别与病原体的模式识别、细胞因子相关信号转导以及蛋白酶抑制剂有关。 结论 :i IEL是肠道天然免疫的重要组成部分 ,能识别细菌的保守抗原。细胞因子是影响其行为的重要方式 。

Objective: To compare the differences in gene expression between murine intestine intraepithelial lymphocytes (iIELs) and splenic T lymphocytes, establishing the cDNA subtractive library of iIELs, analyzing the iIELs special genes. Methods: The iIELs and splenic T lymphocytes were isolated by density gradient in Percoll and nylon column respectively. cDNA subtractive library of iIELs was established by improved subtractive hybridization. The cDNA was ligated with a different adaptor. After 2 rounds of hybridization and suppression PCR amplification, the PCR products were directly inserted into the plasmid vectors. The ESTs special for iIELs were screened by reverse Northern blot. Results: Fifty ESTs with obvious difference in the cDNA subtractive library of iIELs were screened. These ESTs were squenced and submitted to Genebank. The ESTs were analyzed through BLAST, and 14 genes special for iIELs were obtained. These genes were associated with the pattern recognition of pathogen, cytokine signal transduction and proteinase inhibitors. Conclusion: iIELs are important components of intestine natural immunity, it can recognize the conservative antigens of bacteria and can be affected by cytokines and proteinase inhibitors, leading to unique cellular behavior--more resting and less activation.