转化生长因子-β1对人支气管上皮细胞间充质转化及Six1基因表达影响的研究

发布时间：2018-06-13 09:56

本文选题：TGF-β1 + 支气管上皮细胞　；参考：《青岛大学》2017年硕士论文

【摘要】：目的:探讨TGF-β1对人支气管上皮细胞间充质转化及Six1基因表达的影响。方法:体外培养人支气管上皮细胞(16HBE),构建上皮-间充质转化模型。采用倒置显微镜观察各组细胞形态的变化;采用Real-time PCR检测E-钙黏蛋白、波形蛋白、α-肌动蛋白、纤维黏连蛋白及Six1基因m RNA表达的情况;采用Westernblotting检测E-钙黏素、波形蛋白、α-肌动蛋白、纤维黏连蛋白及Six1蛋白的表达变化。结果:1.倒置显微镜结果示:对照组细胞呈单层,细胞间连接较为紧密;处理组A细胞形态变化不明显;处理组B部分细胞出现肥大、拉长,细胞间连接紧密性下降;处理组C细胞形态变化较为明显,呈梭行,同时细胞间隙增大,丧失典型的铺路石样特征。2.RT-PCR检测结果示:对照组细胞可正常表达E-钙黏素基因、波形蛋白基因、纤维黏连蛋白基因、α-肌动蛋白基因及Six1基因m RNA;与对照组相比较,处理组A其E-钙粘素基因、α-肌动蛋白基因m RNA表达量降低(P值分别为0.862、0.566,均0.05),Six1基因m RNA表达量增高(P值为0.1020.05),差异均无统计学意义,波形蛋白基因、纤维黏连蛋白基因m RNA表达量显著升高(P值分别为0.003、0.0060.01),差异有统计学意义。处理组B其E-钙粘素基因m RNA表达量明显减少(P=0.042),波形蛋白基因、纤维黏连蛋白基因、α-肌动蛋白基因及Six1基因m RNA表达量均显著增加(P=0.000、0.001、0.031、0.023,均小于0.05),与对照组相比差异均有统计学意义。处理组C其E-钙粘素基因m RNA表达量显著减少(P=0.0040.01),波形蛋白基因、纤维黏连蛋白基因、α-肌动蛋白基因及Six1基因m RNA表达量均明显增加(P=0.004、0.003、0.023、0.004,均小于0.05),与对照组相比差异均有统计学意义。3.Westernblot检测结果示:对照组细胞能够正常表达E-钙粘素、α-肌动蛋白、纤维黏连蛋白、波形蛋白及Six1蛋白。与对照组相比,处理组A细胞中E-钙粘素表达减少(P=1.0230.05),α-肌动蛋白、纤维黏连蛋白及波形蛋白的表达量增高(P值分别为0.789、0.542、0.624,均0.05),但差异均无统计学意义,Six1蛋白表达量增加(P=0.0280.05),差异有统计学意义;处理组B细胞中E-钙粘素表达量明显减少(P=0.0110.05),α-肌动蛋白、纤维黏连蛋白、波形蛋白及Six1蛋白的表达量均显著增加(P值分别为0.002、0.001、0.004、0.009均0.01),与对照组相比差异均有统计学意义;处理组C细胞中E-钙粘素表达量显著减少(P=0.0090.01),α-肌动蛋白、波形蛋白及Six1蛋白的表达量显著增加(P值分别为0.008、0.003、0.007,均0.01),纤维黏连蛋白表达量明显增加(P值为0.0170.05),与对照组相比差异均有统计学意义。且随TGF-β1作用时间越长,Six1蛋白表达越高,各组间有显著性差异(F=33.50)。结论:TGF-β1可能通过上调Six1的表达诱导气道上皮细胞间充质转化,从而参与哮喘气道重塑。
[Abstract]:Aim: to investigate the effect of TGF- 尾 1 on mesenchymal transformation and expression of Six1 gene in human bronchial epithelial cells. Methods: human bronchial epithelial cells were cultured in vitro to construct epithelial-mesenchymal transformation model. The morphological changes of each group were observed by inverted microscope, the expressions of Ecadherin, vimentin, 伪 -actin, fibronectin and Six1 mRNA were detected by Real-time PCR, and Ecadherin was detected by Western blotting. The expression of vimentin, 伪-actin, fibronectin and Six1 protein. The result is 1: 1. The results of inverted microscope showed that the cells of the control group were monolayer and the intercellular junctions were close; the morphological changes of the A cells in the treatment group were not obvious; the cells in part B of the treatment group were hypertrophic, elongated, and the tight intercellular junctions decreased. The morphological changes of C cells in the treatment group were obvious, and the cell gap was enlarged, and the typical paving stone features were lost. 2. The results of RT-PCR showed that the normal expression of Ecadherin gene and vimentin gene were observed in the control group. Fibronectin gene, 伪 -actin gene and Six1 gene mRNAs were compared with control group. In the treatment group A, the mRNA expression of E-cadherin gene and 伪 -actin gene decreased by 0.862 ~ 0.566.The mRNA expression of each group was increased (P = 0.1020.05, P = 0.1020.05, respectively), vimentin gene. The mRNA expression of fibronectin gene was significantly increased (P = 0.003, P = 0.0060.01), and the difference was statistically significant. In treatment group B, the expression of Ecadherin gene mRNA was significantly reduced by P0.042, vimentin gene. The mRNA expression of fibronectin gene, 伪 -actin gene and Six1 gene were all increased significantly (P < 0.05). In treatment group C, the expression of Ecadherin gene mRNA was significantly reduced by P0. 0040.01, vimentin gene. The mRNA expression of fibronectin gene, 伪 -actin gene and Six1 gene were all increased significantly (P < 0.05). Compared with the control group, the expression of E-cadherin and 伪 -actin in the control group was significantly higher than that in the control group. The results of Western blot showed that the normal expression of E-cadherin and 伪 -actin could be observed in the control group, and the expression of E-cadherin and 伪 -actin in the control group was significantly higher than that in the control group. Fibronectin, vimentin and Six1 protein. Compared with the control group, the expression of E-cadherin in A cells of the treatment group was decreased by P1.0230.05, 伪 -actin. The expression of fibronectin and vimentin were increased (P = 0.789) 0.542n 0.624, respectively (0.05%), but there was no significant difference in the expression of Six1 protein (P < 0.05), the difference was statistically significant, and the expression of E-cadherin in B cells decreased significantly (P 0.0110.05, 伪 -actin, 伪 -actin, P < 0.05), the expression of E-cadherin and vimentin in B cells was significantly lower than that in control group (P < 0.05), but there was no significant difference in the expression of Six1 protein (P < 0.05). The expression of fibronectin, vimentin and Six1 protein increased significantly (P = 0.002, 0.001, 0.004, 0.009, respectively), which were significantly different from those of the control group, and the expression of Ecadherin in C cells of treatment group decreased significantly, and the expression of Ecadherin and 伪 -actin in C cells of treatment group were significantly lower than that of control group, and the expression of Ecadherin in C cells of treatment group was significantly lower than that of control group (P < 0.05). The expression of vimentin and Six1 protein increased significantly (P = 0.008, P = 0.003, P = 0.01g, P = 0.0170.05, P = 0.0170.05, respectively). The higher the expression of TGF- 尾 1 protein was, the higher the expression of TGF- 尾 1 protein was. Conclusion: TGF- 尾 1 may be involved in airway remodeling by up-regulating the expression of Six1 and inducing airway epithelial mesenchymal transformation.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R725.6

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