Villin1启动子调控miR-7-Sponge真核表达载体的构建及其对小鼠炎症性肠炎模型的影响  

The construction of an eukaryotic expression vector of Villin1-promoter-operating miR-7 Sponge expression and its effect on the pathology of murine IBD model

作　　者：王亚 赵娟娟 曾文焕 刘依婷 梁杰 贺钰 蒲廷莉 陈世鹏 李冬玫 徐林 Wang Ya;Zhao Juanjuan;Zeng Wenhuan;Liu Yiting;Liang Jie;He Yu;Pu Tingli;Chen Shipeng;Li Dongmei;Xu Lin(Special Key Laboratory of Gene Detection&Therapy of Guizhou Province,Zunyi Medical University,Zunyi Guizhou 563099,China;Guizhou Provincial College-based Key Lab for Tumor Prevention and Treatment with Distinctive Medicines,Zunyi Guizhou 563099,China;Department of Immunology,Zunyi Medical University,Zunyi Guizhou 563099,China)

机构地区：[1]贵州省基因检测与治疗特色重点实验室暨贵州省生物治疗人才基地,贵州遵义563099 [2]贵州省普通高等学校特色药物肿瘤防治特色重点实验室,贵州遵义563099 [3]遵义医科大学免疫学教研室,贵州遵义563099

出　　处：《遵义医科大学学报》2021年第3期269-276,共8页Journal of Zunyi Medical University

基　　金：国家自然科学基金资助项目(NO:31760258);贵州省科学技术基金重点资助项目(NO:QKH-JC-[2018]1428);遵义医科大学大学生创新创业训练计划项目(NO:ZYDC2019013)。

摘　　要：目的构建绒毛蛋白(Villin1)启动子调控的miR-7海绵体序列(简称miR-7 Sponge)的真核表达载体,观察其对小鼠炎症性肠病(IBD)模型病理损伤的影响。方法荧光原位杂交(FISH)检测葡聚糖硫酸钠盐(Dextran sulfate sodium salt,DSS)作用后小鼠结肠组织中miR-7的水平及定位情况;抽提小鼠结肠组织基因组DNA,PCR扩增Villin1启动子序列,纯化该产物并经Kpn I/Mlu I双酶切,克隆入pGL3-Basic载体,构建pGL3-Basic-Villin1启动子真核表达载体(p-V);随后将p-V和miR-7-Sponge(化学合成)分别经Mlu I/Xho I双酶切、连接,构建pGL3-Villin1-miR-7-Sponge真核表达载体(命名为p-V-miR-7sp),通过酶切和测序鉴定载体。通过2%DSS喂养野生型(WT)小鼠7 d,3 d水恢复,建立IBD小鼠模型,分别在建模的第2,4,6天通过尾部静脉注射p-V和p-V-miR-7sp载体进行治疗;Real-time PCR检测心、肝、肾、淋巴结及结肠组织中miR-7的表达,观察小鼠结肠形态和长度的变化,H&E染色分析小鼠结肠组织病理结构及炎症损伤变化。结果(1)酶切和测序结果显示成功构建p-V-miR-7sp真核表达载体;(2)Villin1启动子调控的miR-7-Sponge真核表达载体可特异降低结肠组织中miR-7的表达水平(P<0.05);(3)与对照组比,p-V-miR-7sp治疗组IBD模型小鼠结肠长度明显增加;H&E染色显示结肠组织中淋巴细胞浸润明显减少,隐窝结构轻微破坏,其损伤明显减轻。结论Villin1启动子调控的miR-7-Sponge真核表达载体不仅特异降低结肠组织中miR-7水平且明显减轻IBD模型小鼠的病理损伤。Objective To construct a miR-7-Sponge eukaryotic expression vector operated by the Villin1 promoter and to observe its effect on the development of murine IBD model.Methods FISH was used to detect the level and localization of miR-7 in colon tissue of IBD.DNA extraction from the colon tissue of WT mice,Villin1 promoter was amplified by PCR.Purification products were digested with restricted enzyme Kpn I/Mul I respectively and then sequently subcloneded into pGL3-Basic vector to construct the eukaryotic expression vector of pGL3-Basic-Villin1(p-V for short).pGL3-Basic-Villin1 and miR-7-Sponge(preliminary chemical synthesis in laboratory)were digested with restricted enzyme Mlu I/Xho I respectively and then were bonded.The pGL3-Villin1-miR-7-Sponge vector(namely p-V-miR-7sp)was identified by enzyme digestion and sequencing.The IBD model were established by feeding WT mice with 2%DSS for 7 days and recovering water for 3 days.p-V and p-V-miR-7sp vectors were injected by the tail vein on the second,fourth,and sixth days in the IBD model.The relative expression of miR-7 was determined by Real-time PCR.Then observe the changes of the size and the pathological damage of the colon.Results(1)The results of enzyme digestion and sequencing showed that the eukaryotic expression vector of p-v-miR-7sp was successfully constructed.(2)The miR-7-Sponge eukaryotic expression vector operated by the Villin1 promoter significantly downregulate the expression of miR-7 in colon tissue(P<0.05).(3)Compared with control group,in the murine IBD model,the colon length of the p-V-miR-7sp treatment group was significantly increased;the result of H&E staining showed that the infiltrating lymphocytes in the colon tissue were significantly reduced,and the crypt structure was slightly damaged.Conclusion The miR-7-Sponge eukaryotic expression vector operated by the Villin1 promoter not only specifically downregulates the expression of miR-7 in colon tissue,but also significantly alleviates the pathological damage of murine IBD model.

关 键 词：miR-7 Sponge Villin1 载体构建 miR-7表达 炎症性肠病 

分 类 号：R392.3[医药卫生—免疫学]