本文關(guān)鍵詞：核轉(zhuǎn)錄因子Snail調(diào)控二氧化硅誘導的人支氣管上皮細胞上皮—間質(zhì)轉(zhuǎn)型作用機制的研究　出處：《中南大學》2011年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 二氧化硅(SiO_2) Snail 人支氣管上皮細胞(HBE) 上皮-間質(zhì)轉(zhuǎn)型(EMT)

【摘要】：目的：研究核轉(zhuǎn)錄因子Snail在二氧化硅(SiO2)·誘導的人支氣管上皮細胞(HBE)上皮-間質(zhì)轉(zhuǎn)型(EMT)中的作用機制。 方法：以200μg/mlSiO2刺激的HBE細胞為模型,(1)不同時間點(0、1、6、12、18、24h)用倒置顯微鏡觀察HBE細胞形態(tài)學改變；(2)不同時間點(0、24h)用電子顯微鏡觀察HBE細胞超微結(jié)構(gòu)改變；(3)不同時間點(0、1、6、12、18、24h)用Western-Blot檢測HBE細胞Snail總蛋白表達水平的變化；(4)不同時間點(0、1、6、12、18、24h)用Western-Blot檢測HBE細胞Snail核蛋白表達水平的變化；(5)不同時間點(0、1、6、12、18、24h)用免疫細胞熒光檢測HBE細胞Snail的核定位情況；(6)不同時間點(0、12、18h)用EMSA檢測HBE細胞Snail DNA結(jié)合活性的變化情況；(7)RNA干擾Snail的表達后,Western-Blot檢測HBE細胞Snail、α-SMA、Vimentin、E-cad蛋白表達水平的變化。 結(jié)果：(1)倒置顯微鏡觀察結(jié)果顯示：HBE細胞與SiO2共育后,隨著時間的延長,原呈鋪路石樣排列的HBE細胞出現(xiàn)梭形、紡錘形改變,間隙略微增寬。(2)電鏡結(jié)果顯示：對照組的細胞大多為圓形,表面微絨毛豐富；SiO2誘導HBE細胞24h時梭形多見,細胞核多形性,細胞表面微絨毛結(jié)構(gòu)減少甚至缺失,代之表現(xiàn)為突起,胞漿內(nèi)出現(xiàn)大量管狀結(jié)構(gòu),次級溶酶體內(nèi)可見細胞吞噬的SiO2顆粒。(3)Western-blot檢測總蛋白的結(jié)果顯示：在SiO2作用下,Snail表達隨著時間的延長逐漸上調(diào),18h表達達到頂峰,之后逐漸下降；12h組和18h組Snail蛋白的表達水平分別為(2.88±0.63)和(4.51±1.15),與對照組的(0.57±0.04)相比,差異均有統(tǒng)計學意義(P＜0.05)。(4) Western-blot檢測核蛋白的結(jié)果顯示：在SiO2作用下,Snail表達隨著時間的延長逐漸上調(diào),12h表達達到頂峰,之后逐漸下降；12h組和18h組Snail蛋白的表達水平分別為(4.51±0.81)和(2.76±0.77),與對照組的(0.67±0.15)相比,差異均有統(tǒng)計學意義(P＜0.05)。(5)免疫細胞熒光結(jié)果顯示：對照組可見到微弱熒光表達于胞漿,核內(nèi)未見表達；實驗組熒光主要表達于細胞核,隨著SiO2作用時間的延長,Snail表達逐漸向核內(nèi)移位。(6)EMSA結(jié)果顯示：在SiO2作用下,12h組和18h組Snail DNA結(jié)合活性的表達水平分別為(0.020±0.00047)和(0.023±0.0011),與對照組的(0.0014±0.00026)相比,差異均有統(tǒng)計學意義(P＜0.05),表明Snail DNA結(jié)合活性明顯升高。(7)siRNA結(jié)果顯示：①刺激組和干擾組Snail蛋白的表達水平分別為(20.02±1.44)和(6.73±1.03),差異有統(tǒng)計學意義(P＜0.05),表明siRNA干擾后Snail表達下調(diào),抑制率為66.38%；②刺激組和干擾組E-cad蛋白的表達水平分別為(0.18±0.03)和(0.84±0.01),差異有統(tǒng)計學意義(P＜0.05),表明siRNA干擾后E-cad表達上調(diào)；③刺激組和干擾組α-SMA和vimentin蛋白的表達水平分別為(2.35±0.45)和(0.23±0.05),(13.12±1.09)和(2.33±0.54),差異均有統(tǒng)計學意義(P＜0.05),表明siRNA干擾后α-SMA和vimentin表達下調(diào),抑制率分別為90.21%和82.24%。 結(jié)論：(1)SiO2可誘導人支氣管上皮細胞(HBE)發(fā)生上皮-間質(zhì)轉(zhuǎn)型(EMT); (2) SiO2可誘導HBE細胞Snail的表達和活化；(3)Snail參與調(diào)節(jié)SiO2誘導的上皮-間質(zhì)轉(zhuǎn)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).

【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

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