本文關鍵詞： 肝泡型包蟲病 miRNAs表達譜 miR-133a-3p 肝纖維化　出處：《新疆醫(yī)科大學》2014年碩士論文　論文類型：學位論文

【摘要】：目的：研究多房棘球絳蟲(Echinococcus Multilocularis,Em.)感染早期所致宿主肝臟miRNAs表達譜的變化,以初步闡明miRNAs在泡球蚴感染所致宿主肝臟纖維化中的重要作用。方法：應用高通量測序方法檢測出泡球蚴感染一個月小鼠正常肝臟組織和泡球蚴病灶近旁組織miRNAs表達譜,并篩選明顯差異表達的miRNAs:應用TargetScan和miRDB兩個網(wǎng)站預測相應miRNAs的靶基因,并篩選出與炎癥反應損傷和纖維化相關的靶基因；實時熒光定量PCR(qRT-PCR)驗證測序結果；MTT法檢測Em.蛋白能否有效刺激大鼠肝星狀細胞(T6細胞)發(fā)生增殖和分化；qRT-PCR檢測Em.蛋白所致纖維化T6細胞miRNAs表達變化,并檢測纖維化相關基因α-SMA、CollA1、Col3A1、TGF-β、TGF-β受體Ⅰ和TGF-β受體Ⅱ的表達變化;Western Blot檢測Em.蛋白所致纖維化T6細胞纖維化相關蛋白α-SMA、CollA1、Col3A1、TGF-β、TGF-β受體Ⅱ和Smad-4的表達變化；T6細胞系轉染miR-133a-3p-mimic檢測miR-133a-3p及纖維化相關因子基因的表達。結果：高通量測序檢測泡球蚴感染早期小鼠肝臟差異表達miRNAs共篩選出了20個表達差異的miRNAs,13個明顯表達上調和7個明顯表達下調miRNAs;通過TargetScan和niRDB兩個網(wǎng)站共篩選出8個miRNAs參與調控參與調控組織壞死、炎癥反應和纖維化的發(fā)生發(fā)展；MTT檢測發(fā)現(xiàn)30%Em.蛋白能夠有效刺激大鼠肝星狀細胞的增殖和分化；30%Em.蛋白刺激T6細胞48h后miR-133a-3p的表達明顯下調,且肝纖維化相關TGF-β/Smad信號通路中α-SMA、Col-lA1、TGF-β和TGF-β R Ⅱ mRNA的表達明顯上調；Co1-1A1、α-SMA、Smad-4和TGF-β蛋白的表達也明顯增加。轉染miR-133a-3p-mimic后T6細胞系miR-133a-3p表達明顯增高,且纖維化相關因子表達降低。結論：成功建立了泡球蚴感染早期小鼠肝臟組織miRNAs的差異表達譜,明顯差異表的miR-133a-3p在泡球蚴感染所致肝臟組織纖維化的發(fā)生和發(fā)展過程起到關鍵作用,其發(fā)揮作用的方式可能是通過對TGF-β/Smad信號通路的調控。
[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，

本文編號：1512147