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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 oral Soluble ST2 (sST2) plasmid and observe its immune response to inflammatory colonic mucosa in mice. Methods The total RNA of spleen of mice was extracted and the sST2 gene was amplified and cloned into pcDNA3.1 / myc-His A vector to construct pcDNA3.1-sST2-myc-His A plasmid, which was transfected into COS-7 cells SST2-myc-His A fusion protein. Oral sST2 plasmids were prepared by lipofectamineTM2000 encapsulation, and C57BL / 6 mice induced by dextran sulfate sodium (DSS) were given by gavage. The colon was observed by histopathology Mucosal tissue morphological changes. The expression of sST2 plasmid fusion protein was detected by Real-time PCR and the expression of cytokine (IL-4, IL-5 and IL-13) in the supernatant of intestinal lamina propria of mice was detected by ELISA. The level of secretion. Results The correct expression of sST2 gene was detected by Real-time PCR. The double-digestion and sequencing proved that plasmid pcDNA3.1-sST2-myc-His A was successfully constructed. Western blotting assay showed that sST2 was detected in transfected COS-7 cells -myc-His A fusion protein, the relative molecular mass was in line with the expectation; the morphological changes of colonic mucosa were observed; Real-time PCR results showed that the expression of sST2 in colonic mucosa of sST2 plasmid group was significantly higher The levels of IL-4, IL-5 and IL-13 in the supernatant of colon lamina propria of sST2 plasmid group were higher than that of pcDNA3.1 group (P <0.01) And saline group (P <0.05). Conclusion The sST2 plasmid was successfully prepared in this experiment. This plasmid can inhibit the Th2 immune response in inflammatory colonic mucosa of mice.