本文關(guān)鍵詞： 肝泡型包蟲(chóng)病 miRNAs表達(dá)譜 miR-133a-3p 肝纖維化　出處：《新疆醫(yī)科大學(xué)》2014年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的：研究多房棘球絳蟲(chóng)(Echinococcus Multilocularis,Em.)感染早期所致宿主肝臟miRNAs表達(dá)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(yīng)用高通量測(cè)序方法檢測(cè)出泡球蚴感染一個(gè)月小鼠正常肝臟組織和泡球蚴病灶近旁組織miRNAs表達(dá)譜,并篩選明顯差異表達(dá)的miRNAs:應(yīng)用TargetScan和miRDB兩個(gè)網(wǎng)站預(yù)測(cè)相應(yīng)miRNAs的靶基因,并篩選出與炎癥反應(yīng)損傷和纖維化相關(guān)的靶基因；實(shí)時(shí)熒光定量PCR(qRT-PCR)驗(yàn)證測(cè)序結(jié)果；MTT法檢測(cè)Em.蛋白能否有效刺激大鼠肝星狀細(xì)胞(T6細(xì)胞)發(fā)生增殖和分化；qRT-PCR檢測(cè)Em.蛋白所致纖維化T6細(xì)胞miRNAs表達(dá)變化,并檢測(cè)纖維化相關(guān)基因α-SMA、CollA1、Col3A1、TGF-β、TGF-β受體Ⅰ和TGF-β受體Ⅱ的表達(dá)變化;Western Blot檢測(cè)Em.蛋白所致纖維化T6細(xì)胞纖維化相關(guān)蛋白α-SMA、CollA1、Col3A1、TGF-β、TGF-β受體Ⅱ和Smad-4的表達(dá)變化；T6細(xì)胞系轉(zhuǎn)染miR-133a-3p-mimic檢測(cè)miR-133a-3p及纖維化相關(guān)因子基因的表達(dá)。結(jié)果：高通量測(cè)序檢測(cè)泡球蚴感染早期小鼠肝臟差異表達(dá)miRNAs共篩選出了20個(gè)表達(dá)差異的miRNAs,13個(gè)明顯表達(dá)上調(diào)和7個(gè)明顯表達(dá)下調(diào)miRNAs;通過(guò)TargetScan和niRDB兩個(gè)網(wǎng)站共篩選出8個(gè)miRNAs參與調(diào)控參與調(diào)控組織壞死、炎癥反應(yīng)和纖維化的發(fā)生發(fā)展；MTT檢測(cè)發(fā)現(xiàn)30%Em.蛋白能夠有效刺激大鼠肝星狀細(xì)胞的增殖和分化；30%Em.蛋白刺激T6細(xì)胞48h后miR-133a-3p的表達(dá)明顯下調(diào),且肝纖維化相關(guān)TGF-β/Smad信號(hào)通路中α-SMA、Col-lA1、TGF-β和TGF-β R Ⅱ mRNA的表達(dá)明顯上調(diào)；Co1-1A1、α-SMA、Smad-4和TGF-β蛋白的表達(dá)也明顯增加。轉(zhuǎn)染miR-133a-3p-mimic后T6細(xì)胞系miR-133a-3p表達(dá)明顯增高,且纖維化相關(guān)因子表達(dá)降低。結(jié)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(dá)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(guò)程起到關(guān)鍵作用,其發(fā)揮作用的方式可能是通過(guò)對(duì)TGF-β/Smad信號(hào)通路的調(diào)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(hào)：1512147