Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, China.
Dig Dis Sci. 2010 Oct;55(10):2929-39. doi: 10.1007/s10620-010-1227-0. Epub 2010 May 26.
To study the expression of human intestinal trefoil factor (hITF) mRNA in Crohn's disease and to screen the cellular proteins that can interact with the hITF protein by a yeast two-hybrid system in order to explore the mechanism of hITF in protecting intestinal mucosa from injury.
Seventy-eight patients underwent double-balloon enteroscopy (DBE). Expression of hITF mRNA was detected by quantitative real-time polymerase chain reaction analysis (qRT-PCR). The hITF gene was amplified by PCR and cloned into vector pDEST32. The yeast cells cotransformed with pDEST32-hITF and the human jejunal cDNA library were plated in a selective SC-Leu-Trp-His-Ura medium. The subsequent screen was performed with χ-gal detection, and true-positive clones were sequenced and analyzed with bioinformatics. Co-immunoprecipitation (Co-IP) was performed to confirm the binding of putative proteins to the hITF protein.
Thirty-nine patients were diagnosed with Crohn's disease. We found that the expression of hITF mRNA is significantly increased in Crohn's disease compared to normal controls. A total of ten colonies were selected and sequenced. Among these, six colonies were Homo sapiens zinc finger protein 193 (ZNF193), three colonies were Homo sapiens Aldo-keto reductase family 1C 1 (AKR1C1), and one colony was of an unknown gene. A reverse two-hybrid experiment and Co-IP indicated that ZNF193 and AKR1C1 might interact with hITF.
The expression of hITF mRNA is increased in Crohn's disease. ZNF193 and AKR1C1 are proteins that can interact with the hITF protein by a yeast two-hybrid system and Co-IP, hITF may contribute to the mucosal repair through this interaction.
研究人类三叶因子（hITF）mRNA 在克罗恩病中的表达，并通过酵母双杂交系统筛选与 hITF 蛋白相互作用的细胞蛋白，以探讨 hITF 保护肠黏膜免受损伤的机制。
对 78 例患者行双气囊小肠镜检查（DBE）。采用实时定量聚合酶链反应分析（qRT-PCR）检测 hITF mRNA 的表达。采用 PCR 扩增 hITF 基因并克隆到载体 pDEST32 中。将 pDEST32-hITF 与人类空肠 cDNA 文库共转化的酵母细胞接种在选择性 SC-Leu-Trp-His-Ura 培养基中。随后用 χ-gal 检测进行筛选，对阳性克隆进行测序和生物信息学分析。采用免疫共沉淀（Co-IP）验证假定蛋白与 hITF 蛋白的结合。
39 例患者被诊断为克罗恩病。我们发现 hITF mRNA 的表达在克罗恩病中明显高于正常对照。共选择了 10 个菌落进行测序。其中 6 个为 Homo sapiens zinc finger protein 193（ZNF193），3 个为 Homo sapiens Aldo-keto reductase family 1C 1（AKR1C1），1 个为未知基因。反向双杂交实验和 Co-IP 表明 ZNF193 和 AKR1C1 可能与 hITF 相互作用。
hITF mRNA 在克罗恩病中表达增加。ZNF193 和 AKR1C1 是通过酵母双杂交系统和 Co-IP 与 hITF 蛋白相互作用的蛋白质，hITF 可能通过这种相互作用促进黏膜修复。
