核转录因子Snail调控二氧化硅诱导的人支气管上皮细胞上皮—间质转型作用机制的研究

发布时间：2017-12-31 12:20

本文关键词：核转录因子Snail调控二氧化硅诱导的人支气管上皮细胞上皮—间质转型作用机制的研究　出处：《中南大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的：研究核转录因子Snail在二氧化硅(SiO2)·诱导的人支气管上皮细胞(HBE)上皮-间质转型(EMT)中的作用机制。方法：以200μg/mlSiO2刺激的HBE细胞为模型,(1)不同时间点(0、1、6、12、18、24h)用倒置显微镜观察HBE细胞形态学改变；(2)不同时间点(0、24h)用电子显微镜观察HBE细胞超微结构改变；(3)不同时间点(0、1、6、12、18、24h)用Western-Blot检测HBE细胞Snail总蛋白表达水平的变化；(4)不同时间点(0、1、6、12、18、24h)用Western-Blot检测HBE细胞Snail核蛋白表达水平的变化；(5)不同时间点(0、1、6、12、18、24h)用免疫细胞荧光检测HBE细胞Snail的核定位情况；(6)不同时间点(0、12、18h)用EMSA检测HBE细胞Snail DNA结合活性的变化情况；(7)RNA干扰Snail的表达后,Western-Blot检测HBE细胞Snail、α-SMA、Vimentin、E-cad蛋白表达水平的变化。结果：(1)倒置显微镜观察结果显示：HBE细胞与SiO2共育后,随着时间的延长,原呈铺路石样排列的HBE细胞出现梭形、纺锤形改变,间隙略微增宽。(2)电镜结果显示：对照组的细胞大多为圆形,表面微绒毛丰富；SiO2诱导HBE细胞24h时梭形多见,细胞核多形性,细胞表面微绒毛结构减少甚至缺失,代之表现为突起,胞浆内出现大量管状结构,次级溶酶体内可见细胞吞噬的SiO2颗粒。(3)Western-blot检测总蛋白的结果显示：在SiO2作用下,Snail表达随着时间的延长逐渐上调,18h表达达到顶峰,之后逐渐下降；12h组和18h组Snail蛋白的表达水平分别为(2.88±0.63)和(4.51±1.15),与对照组的(0.57±0.04)相比,差异均有统计学意义(P＜0.05)。(4) Western-blot检测核蛋白的结果显示：在SiO2作用下,Snail表达随着时间的延长逐渐上调,12h表达达到顶峰,之后逐渐下降；12h组和18h组Snail蛋白的表达水平分别为(4.51±0.81)和(2.76±0.77),与对照组的(0.67±0.15)相比,差异均有统计学意义(P＜0.05)。(5)免疫细胞荧光结果显示：对照组可见到微弱荧光表达于胞浆,核内未见表达；实验组荧光主要表达于细胞核,随着SiO2作用时间的延长,Snail表达逐渐向核内移位。(6)EMSA结果显示：在SiO2作用下,12h组和18h组Snail DNA结合活性的表达水平分别为(0.020±0.00047)和(0.023±0.0011),与对照组的(0.0014±0.00026)相比,差异均有统计学意义(P＜0.05),表明Snail DNA结合活性明显升高。(7)siRNA结果显示：①刺激组和干扰组Snail蛋白的表达水平分别为(20.02±1.44)和(6.73±1.03),差异有统计学意义(P＜0.05),表明siRNA干扰后Snail表达下调,抑制率为66.38%；②刺激组和干扰组E-cad蛋白的表达水平分别为(0.18±0.03)和(0.84±0.01),差异有统计学意义(P＜0.05),表明siRNA干扰后E-cad表达上调；③刺激组和干扰组α-SMA和vimentin蛋白的表达水平分别为(2.35±0.45)和(0.23±0.05),(13.12±1.09)和(2.33±0.54),差异均有统计学意义(P＜0.05),表明siRNA干扰后α-SMA和vimentin表达下调,抑制率分别为90.21%和82.24%。结论：(1)SiO2可诱导人支气管上皮细胞(HBE)发生上皮-间质转型(EMT); (2) SiO2可诱导HBE细胞Snail的表达和活化；(3)Snail参与调节SiO2诱导的上皮-间质转型(EMT)。
[Abstract]:Objective: To study the mechanism of nuclear transcription factor Snail in the epithelial mesenchymal transition (EMT) of human bronchial epithelial cells (HBE) induced by silica (SiO2).
Methods: 200 g/mlSiO2 stimulated HBE cells as a model, (1) at different time points (0,1,6,12,18,24h) inverted microscope was used to observe the morphological changes of HBE; (2) at different time points (0,24h) to observe the changes of ultrastructure of HBE cells by electron microscopy; (3) at different time points (0,1,6,12,18,24h) expression in Western-Blot detection of HBE Snail cell total protein; (4) at different time points (0,1,6,12,18,24h) expression in Western-Blot cells was detected by HBE Snail nuclear protein; (5) at different time points (0,1,6,12,18,24h) by immunofluorescence detection of HBE nuclear localization of Snail cells; (6) at different time points (0,12,18h) detected by EMSA HBE cell Snail DNA binding activity changes; (7) the expression of RNA Snail interference, Western-Blot detection of HBE cell Snail, alpha -SMA, Vimentin, E-cad protein expression of.
Results: (1) the inverted microscope observation showed: HBE cells with SiO2 after co culture, with the extension of time, the original is paving stone like HBE cells arranged fusiform, spindle shaped, slightly widened the gap. (2) electron microscopy showed that the control cells were round, microvilli on the surface of the rich SiO2; HBE cells induced by 24h spindle rare, pleomorphic nuclei, cell surface microvilli reduced or missing, the performance for the generation processes, large tubular structures appeared in cytoplasm, secondary dissolution of SiO2 particles in vivo phagocytosis. Visible (3) detection of Western-blot protein results showed that in the presence of SiO2 with the extension of time, the expression of Snail gradually increased, the expression of 18h reached the peak, then decreased gradually; the expression level of Snail protein in 12h group and 18h group respectively (2.88 + 0.63) and (4.51 + 1.15), and control group (0.57 + 0.04) compared to the difference was Statistical significance (P < 0.05). (4) the nuclear protein Western-blot detection results showed that in the presence of SiO2, Snail expression gradually increased with the extension of time, the expression of 12h reached the peak, then decreased gradually; the expression level of Snail protein in 12h group and 18h group respectively (4.51 + 0.81) and (2.76 + 0.77), and control group (0.67 + 0.15) compared, the differences were statistically significant (P < 0.05). (5) immunofluorescence results showed that the control group showed weak fluorescence expression in the cytoplasm, but no expression in the nucleus of experimental group; fluorescence was mainly expressed in the nucleus, with the prolongation of SiO2 action time the expression of Snail, gradually shift to the nucleus (6). EMSA results showed that in the presence of SiO2, 12h group and 18h group Snail DNA binding activity expression level respectively (0.020 + 0.00047) and (0.023 + 0.0011), and control group (0.0014 + 0.00026) compared, the differences were statistically significant (P < 0.05), Show that the Snail binding activity of DNA was significantly increased. (7) the results showed that the expression level of siRNA stimulation group and interference group Snail protein respectively (20.02 + 1.44) and (6.73 + 1.03), the difference was statistically significant (P < 0.05), indicated that the down-regulation of Snail expression after siRNA interference, the inhibition rate was 66.38%; expression the level of stimulation group and interference group E-cad protein respectively (0.18 + 0.03) and (0.84 + 0.01), the difference was statistically significant (P < 0.05), expression of E-cad showed that after siRNA interference; the expression level of the stimulation group and interference group, alpha -SMA and vimentin protein respectively (2.35 + 0.45) and (0.23 + 0.05), (13.12 + 1.09) and (2.33 + 0.54), the differences were statistically significant (P < 0.05), showed that alpha -SMA and downregulation of vimentin expression after siRNA interference, the inhibition rate were 90.21% and 82.24%.
Conclusion: (1) SiO2 can induce epithelial mesenchymal transition (EMT) in human bronchial epithelial cells (HBE). (2) SiO2 can induce the expression and activation of Snail in HBE cells. (3) Snail is involved in regulating SiO2 induced epithelial mesenchymal transition (EMT).

【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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