miR146a在肺成纤维细胞表型转化中的作用和机制

发布时间：2018-04-29 09:07

本文选题：miR146a + 肺纤维化　；参考：《桂林医学院》2017年硕士论文

【摘要】：目的:1、本研究通过小鼠肺纤维化造模,体外培养小鼠肺成纤维细胞,TGF-β1(transforming growth factor-β1)刺激肺成纤维细胞发生表型转化后,蛋白质印迹法(Western-blotting,WB)、实时荧光定量聚合酶链式反应(Quantitative real-time polymerase chain reaction,qRT-PCR)检测在不同转化程度中肿瘤坏死因子受体相关因子6(TNF receptor associated factor 6,TRAF6)蛋白、microRNA-146a(miR146a)的动态表达变化,以探讨miR146a在肺成纤维细胞表型转化中的的作用机制。方法:1、将40只小鼠随机分为两组:对照组、博来霉素肺纤维化组(模型组);对照组予以气管内灌注生理盐水,模型组则气管内滴注博来霉素溶液(2.5mg/kg),于造模后第4周处死小鼠。2、采用肺组织苏木精一伊红染色法(hematoxylin-eosin staining,HE染色)的方法来验证纤维化模型是否构建成功;3、肺成纤维细胞的培养和鉴定,用细胞贴壁法结合胰酶法获得肺成纤维细胞,在倒置显微镜观察细胞形态和数量的变化,并拍照记录以及免疫细胞组织化学染色测定成纤维细胞表面标记物两种方法鉴定肺成纤维细胞;3.将细胞分成四个组,标记为A(空白对照组)、B、C、D组,分别用浓度为0、5、10、15ug/l的TGF-β1刺激肺成纤维细胞发生表型转化,用WB检测α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)TRAF6蛋白的表达,qRT-PCR法检测细胞中miR146a的相对表达量。结果:成功构建博来霉素诱导的小鼠肺纤维化小鼠模型,成功培养出肺成纤维细胞,并用TGF-β1进行诱导24小时。1、随着TGF-β1浓度的提高,α-SMA蛋白、TRAF6蛋白的表达均呈浓度依赖性增高,四组细胞的α-SMA蛋白、TRAF6蛋白表达有统计学意义:((TRAF6)F=41.25;(a-SMA)F=150.04,P0.01,P0.05);2、四组间miR146a基因的表达有统计学意义(F=335.00,P0.01,P0.05),无TGF-β1刺激时,miR146a呈低表达,B组TGF-β1浓度为5ug/l刺激下的表达最高,然后呈浓度依赖性下降趋势;3、在TGF-β1诱导下的肺成纤维细胞表型转化率越高(α-SMA表达越高),miR146a的表达越低。结论:(1)TGF-β1在一定浓度范围内能促进肺成纤维细胞转化成肌成纤维细胞;(2)miR146a能负向调节肺成纤维细胞向肌成纤维细胞的转化
[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 伪 -smooth muscle actin (伪 -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 伪 -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 伪 -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 (伪 -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.
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563;R593.2

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