本文選題：miR146a + 肺纖維化　； 參考：《桂林醫(yī)學院》2017年碩士論文

【摘要】：目的:1、本研究通過小鼠肺纖維化造模,體外培養(yǎng)小鼠肺成纖維細胞,TGF-β1(transforming growth factor-β1)刺激肺成纖維細胞發(fā)生表型轉(zhuǎn)化后,蛋白質(zhì)印跡法(Western-blotting,WB)、實時熒光定量聚合酶鏈式反應(Quantitative real-time polymerase chain reaction,qRT-PCR)檢測在不同轉(zhuǎn)化程度中腫瘤壞死因子受體相關因子6(TNF receptor associated factor 6,TRAF6)蛋白、microRNA-146a(miR146a)的動態(tài)表達變化,以探討miR146a在肺成纖維細胞表型轉(zhuǎn)化中的的作用機制。方法:1、將40只小鼠隨機分為兩組:對照組、博來霉素肺纖維化組(模型組);對照組予以氣管內(nèi)灌注生理鹽水,模型組則氣管內(nèi)滴注博來霉素溶液(2.5mg/kg),于造模后第4周處死小鼠。2、采用肺組織蘇木精一伊紅染色法(hematoxylin-eosin staining,HE染色)的方法來驗證纖維化模型是否構建成功;3、肺成纖維細胞的培養(yǎng)和鑒定,用細胞貼壁法結合胰酶法獲得肺成纖維細胞,在倒置顯微鏡觀察細胞形態(tài)和數(shù)量的變化,并拍照記錄以及免疫細胞組織化學染色測定成纖維細胞表面標記物兩種方法鑒定肺成纖維細胞;3.將細胞分成四個組,標記為A(空白對照組)、B、C、D組,分別用濃度為0、5、10、15ug/l的TGF-β1刺激肺成纖維細胞發(fā)生表型轉(zhuǎn)化,用WB檢測α-平滑肌肌動蛋白(α-smooth muscle actin,α-SMA)TRAF6蛋白的表達,qRT-PCR法檢測細胞中miR146a的相對表達量。結果:成功構建博來霉素誘導的小鼠肺纖維化小鼠模型,成功培養(yǎng)出肺成纖維細胞,并用TGF-β1進行誘導24小時。1、隨著TGF-β1濃度的提高,α-SMA蛋白、TRAF6蛋白的表達均呈濃度依賴性增高,四組細胞的α-SMA蛋白、TRAF6蛋白表達有統(tǒng)計學意義:((TRAF6)F=41.25;(a-SMA)F=150.04,P0.01,P0.05);2、四組間miR146a基因的表達有統(tǒng)計學意義(F=335.00,P0.01,P0.05),無TGF-β1刺激時,miR146a呈低表達,B組TGF-β1濃度為5ug/l刺激下的表達最高,然后呈濃度依賴性下降趨勢;3、在TGF-β1誘導下的肺成纖維細胞表型轉(zhuǎn)化率越高(α-SMA表達越高),miR146a的表達越低。結論:(1)TGF-β1在一定濃度范圍內(nèi)能促進肺成纖維細胞轉(zhuǎn)化成肌成纖維細胞;(2)miR146a能負向調(diào)節(jié)肺成纖維細胞向肌成纖維細胞的轉(zhuǎn)化
[Abstract]:Objective to investigate the effects of TGF- 尾 1(transforming growth factor- 尾 1 (TGF- 尾 1(transforming growth factor- 尾 1) on the phenotype transformation of lung fibroblasts induced by pulmonary fibrosis in mice. Western-blotting method was used to detect the dynamic expression of tumor necrosis factor receptor related factor (6(TNF receptor associated factor 6 / TRAF6) protein microRNA-146a miR146a in different degree of transformation by real-time quantitative real-time polymerase chain reactions-qRT-PCR. To explore the mechanism of miR146a in phenotypic transformation of pulmonary fibroblasts. Methods: one, 40 mice were randomly divided into two groups: control group, bleomycin pulmonary fibrosis group (model group), and control group were given intratracheal instillation of normal saline. The model group was given intratracheal instillation of bleomycin solution 2.5 mg / kg of bleomycin. Mice were killed at the 4th week after the model. Hematoxylin-eosin staininginginginginginginghe staining was used to verify whether the fibrosis model was successfully constructed and the lung fibroblasts were fine. Cell culture and identification, Lung fibroblasts were obtained by cell adhesion method and trypsin method. The morphological and quantitative changes of lung fibroblasts were observed under inverted microscope. The lung fibroblasts were identified by two methods: photo recording and immunocytochemical staining. The cells were divided into four groups, labeled as A (blank control group) and treated with TGF- 尾 1 at a concentration of 0 ~ 5 ~ 10U / 1 to induce phenotypic transformation of lung fibroblasts. The expression of 偽 -smooth muscle actin (偽 -SMA-TRAF6) was detected by Western blot. The relative expression of miR146a was detected by qRT-PCR. Results: the mouse model of pulmonary fibrosis induced by bleomycin was successfully constructed, and lung fibroblasts were successfully cultured and induced by TGF- 尾 1 for 24 hours. The expression of 偽 -SMA protein TRAF6 protein increased in a concentration-dependent manner with the increase of TGF- 尾 1 concentration. There was significant difference in the expression of 偽 -SMA protein and TRAF6 protein in the four groups. The expression of miR146a gene in the four groups was significantly higher than that in the 5ug/l stimulated group. The expression of TGF- 尾 1 in the B group was the highest in the 5ug/l stimulated group, and the expression of TGF- 尾 1 in the B group was the highest in the low expression of TGF- 尾 1 group, and the expression of TGF- 尾 1 in the B group was the highest under the stimulation of the 5ug/l, and the expression of TGF- 尾 1 in the B group was the highest in the low expression of TGF- 尾 1 group, and the expression of TGF- 尾 1 in the B group was the highest under the stimulation of 5ug/l. The phenotypic transformation rate of lung fibroblasts induced by TGF- 尾 1 was higher (偽 -SMA expression was higher) and the expression of miR146a was lower. Conclusion TGF- 尾 1 can promote the transformation of lung fibroblasts into myofibroblasts in a certain concentration range. TGF- 尾 1 can negatively regulate the transformation of lung fibroblasts into myofibroblasts.
【學位授予單位】：桂林醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R563;R593.2

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