泡球蚴感染早期小鼠肝脏microRNAs表达谱的变化及miR-133a-3p初步功能研究

发布时间：2018-02-15 01:55

  本文关键词： 肝泡型包虫病 miRNAs表达谱 miR-133a-3p 肝纤维化　出处：《新疆医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：研究多房棘球绦虫(Echinococcus Multilocularis,Em.)感染早期所致宿主肝脏miRNAs表达谱的变化,以初步阐明miRNAs在泡球蚴感染所致宿主肝脏纤维化中的重要作用。方法：应用高通量测序方法检测出泡球蚴感染一个月小鼠正常肝脏组织和泡球蚴病灶近旁组织miRNAs表达谱,并筛选明显差异表达的miRNAs:应用TargetScan和miRDB两个网站预测相应miRNAs的靶基因,并筛选出与炎症反应损伤和纤维化相关的靶基因；实时荧光定量PCR(qRT-PCR)验证测序结果；MTT法检测Em.蛋白能否有效刺激大鼠肝星状细胞(T6细胞)发生增殖和分化；qRT-PCR检测Em.蛋白所致纤维化T6细胞miRNAs表达变化,并检测纤维化相关基因α-SMA、CollA1、Col3A1、TGF-β、TGF-β受体Ⅰ和TGF-β受体Ⅱ的表达变化;Western Blot检测Em.蛋白所致纤维化T6细胞纤维化相关蛋白α-SMA、CollA1、Col3A1、TGF-β、TGF-β受体Ⅱ和Smad-4的表达变化；T6细胞系转染miR-133a-3p-mimic检测miR-133a-3p及纤维化相关因子基因的表达。结果：高通量测序检测泡球蚴感染早期小鼠肝脏差异表达miRNAs共筛选出了20个表达差异的miRNAs,13个明显表达上调和7个明显表达下调miRNAs;通过TargetScan和niRDB两个网站共筛选出8个miRNAs参与调控参与调控组织坏死、炎症反应和纤维化的发生发展；MTT检测发现30%Em.蛋白能够有效刺激大鼠肝星状细胞的增殖和分化；30%Em.蛋白刺激T6细胞48h后miR-133a-3p的表达明显下调,且肝纤维化相关TGF-β/Smad信号通路中α-SMA、Col-lA1、TGF-β和TGF-β R Ⅱ mRNA的表达明显上调；Co1-1A1、α-SMA、Smad-4和TGF-β蛋白的表达也明显增加。转染miR-133a-3p-mimic后T6细胞系miR-133a-3p表达明显增高,且纤维化相关因子表达降低。结论：成功建立了泡球蚴感染早期小鼠肝脏组织miRNAs的差异表达谱,明显差异表的miR-133a-3p在泡球蚴感染所致肝脏组织纤维化的发生和发展过程起到关键作用,其发挥作用的方式可能是通过对TGF-β/Smad信号通路的调控。
[Abstract]:Objective: to study the changes of miRNAs expression profile of host liver caused by Echinococcus multilocularis Em.in the early stage of Echinococcus multilocularis infection. To elucidate the important role of miRNAs in hepatic fibrosis induced by alveolar hydatid infection, high throughput sequencing method was used to detect the miRNAs expression profile in normal liver and adjacent tissues of alveolar hydatid lesion in mice infected with alveolar hydatid for one month. TargetScan and miRDB were used to predict the target genes of corresponding miRNAs, and the target genes related to inflammatory reaction injury and fibrosis were screened. The results of real-time fluorescence quantitative PCRQRT-PCR were used to detect the expression of miRNAs in rat hepatic stellate cells (T6 cells) induced by Em. protein, and whether the protein could effectively stimulate the proliferation and differentiation of T6 cells. The expression changes of TGF- 尾 receptor 鈪，

本文编号：1512147