口服sST2质粒DNA在炎症性肠病小鼠中的免疫水平

Effect of orally delivered plasmid DNA expressing sST2 on inflammatory Th2 cells in intestine of mice with DSS-induced colitis

目的制备口服可溶性ST2(Soluble ST2,sST2)质粒,观察其对于小鼠炎症性结肠黏膜免疫应答水平。方法提取小鼠脾脏总RNA,扩增sST2基因,并克隆至pcDNA3.1/myc-His A载体,构建pcDNA3.1-sST2-myc-His A质粒,借此转染COS-7细胞,使之表达sST2-myc-His A融合蛋白。利用脂质体LipofectamineTM2000包裹,制备口服sST2质粒,经灌胃给予用葡聚糖硫酸钠(Dextran sulfate sodium,DSS)诱生溃疡性结肠炎的C57BL/6实验小鼠,并用组织病理学方法观察结肠黏膜组织形态变化。用Real-time PCR检测sST2基因表达,用免疫印迹检测sST2质粒融合蛋白的表达;用ELISA检测小鼠肠固有层淋巴细胞培养上清中细胞因子(IL-4、IL-5和IL-13)的分泌水平。结果Real-time PCR检测到sST2基因的正确表达,经双酶切及测序鉴定,证明质粒pcDNA3.1-sST2-myc-His A构建成功,免疫印迹试验在转染的COS-7细胞检测到sST2-myc-His A融合蛋白的表达,其相对分子质量与预期的相符;观察到结肠黏膜组织形态发生的变化;Real-time PCR结果显示,sST2质粒组在结肠黏膜组织内sST2的表达水平明显高于pcDNA3.1组和生理盐水组(P<0.01);ELISA结果显示sST2质粒组结肠固有层淋巴细胞培养上清中IL-4、IL-5和IL-13的分泌水平高于pcDNA3.1组和生理盐水组(P<0.05)。结论实验成功制备口服sST2质粒;该质粒可抑制小鼠炎症性结肠黏膜组织内Th2型免疫应答。

Objective To prepare an oral sST2 DNA plasmid and observe its effect on the immuno-regulation of intestinal mucosa of mice with DSS induced colitis. Methods The gene coding sST2 was amplified from total RNA of mice spleen and then inserted into the vector pcDNA3. 1/myc-HisA to construct a recombinant expression plasmid pcDNA3. 1-sST2-myc-HisA, which was transfected to COS-7 cells and made up of a capsule wrapped by LipofectamineTM2000. The capsule was orally dilivered to C57BL/6 mouse with DSS induced colitis, and histopathology examination was carried out in detec-tion of changes in mouse intestinal mucosa. The expression of sST-2 in intestinal tissue was analyzed by Real-time PCR and the expression of sST2-myc-HisA fusion protein was determined by Western blot. The secretion level of cytokines（ IL-4,IL-5 and IL-13）in culture supernatant of LPL （Lamina propria lymphocyte） was measured by ELISA. Results The correct expression of sST2 gene was identified by Real-time PCR. The recombinant plasmid pcDNA3. 1-sST2-myc-HisA was cor-rectly constructed as proved by restriction analysis and sequencing. The expression of sST2-myc-HisA protein was proved in transfected COS-7 cells by western blot, and with a correct relative molecular quality. The morphological changes in tested mouse intestinal mucosa were observed. The expression of sST2 in colontissue was significantly higher in sST2 plasmid group＆nbsp;than those in pcDNA3. 1 and physiological saline groups（P＜0. 01）. And secretion levels of IL-4, IL-5 and IL-13 in cul-ture supernatant of LPL in sST2 plasmid group were lower than those in pcDNA3. 1 and physiological saline groups（P＜0. 05 ） . Conclusion The oral sST2 DNA plasmid was successfully prepared. This plasmid can reduce intestinal Th2 type re-sponse in DSS induced mouse colitis.