脱氢表雄酮对支气管上皮细胞上皮间质转化的作用及机制
发布时间：2018-11-05 18:05
【摘要】：目的：采用转化生长因子-β1(transforming growth factor-β1, TGF-β1)诱导人支气管上皮细胞株16HBE-14。构建上皮-间质转化(Epithelial-Mesenchymal Transition, EMT)模型,观察脱氢表雄酮(Dehydroepiandrosterone,DHEA)对TGF-β1诱导的EMT的作用,并探讨其具体机制。 方法：1.将16HBE-14。用含有5%胎牛血清的MEM培养基培养,5ng/ml TGF-β1作用16HBE-14o72h,观察细胞的形态变化,采用Western Blot(WB)检测E一钙粘素(E.cadherin)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)表达。2.给予不同浓度(1μM、10μM、100μM)DHEA预处理细胞24h,后加入TGF-β1干预72h,检测方法同1。3.设DHEA100(DHEA终浓度为100μM)+TGF-β1组和TGF-β1组,在不同时间点(0、0.5h、1h、2h)采用WB检测两组细胞p-AKT及AKT的变化过程。4.分别予雄激素受体(androgen receptor,AR)拮抗剂氟他胺、PI3K抑制剂LY294002(LY)或PI3K激动剂IGF-1预处理细胞,设八组,分别为(1)无处理组；(2)TGF-β1组；(3)DHEA100+TGF-β1组；(4)DHEA100+TGF-β1+氟他胺组；(5)TGF-β1+LY组；(6)TGF-β1+IGF-1组；(7)TGF-β1+DHEA100+LY组；(8)TGF-β1+DHEA100+IGF-1组,TGF-β1共同干预1h后立即收集细胞,WB检测各组细胞p-AKT及AKT的变化。各组细胞加入TGF-β1培养72h后立即收集细胞,WB检测各组细胞E-cadherin.α-SMA表达。 结果：1. TGF-β1作用72h后,细胞鹅卵石外观变为梭形,细胞间连接疏松。E-cadherin表达下降；α-SMA表达增强。2.不同浓度DHEA对TGF-β1诱导EMT均有抑制作用,且呈浓度依赖性,100μMDHEA抑制作用最强。3. TGF-β1组、DHEA100+TGF-β1组均在TGF-β1处理后,p-AKT蛋白表达增加,1h达到高峰。DHEA抑制p-AKT蛋白表达,AKT蛋白表达无明显差异(P0.05)。4.氟他胺可减轻DHEA对p-AKT蛋白表达及EMT过程的抑制作用。5.LY可进一步降低p-AKT蛋白表达及EMT过程；IGF-1促进p-AKT蛋白表达及EMT发生。 结论：1. TGF-β1能诱导16HBE-14o发生EMT。2. DHEA对TGF-β1诱导的EMT有抑制作用,且呈浓度依赖性。3. DHEA通过AR而发挥对TGF-β1诱导的EMT的抑制作用。4. DHEA对EMT的抑制作用与PI3K/AKT信号途径有关。
[Abstract]:Objective: to induce human bronchial epithelial cell line 16HBE-14 by transforming growth factor- 尾 1 (TGF- 尾 1). An epithelial-mesenchymal transformation (Epithelial-Mesenchymal Transition, EMT) model was established to investigate the effect of dehydroepiandrosterone (Dehydroepiandrosterone,DHEA) on EMT induced by TGF- 尾 1 and its mechanism. Method 1: 1. 16HBE-14. Cultured on MEM medium containing 5% fetal bovine serum, 5ng/ml TGF- 尾 1 was used for 16HBE-14o72 h. Morphological changes of cells were observed. E-cadherin (E.cadherin) and 伪 -smooth muscle actin (伪 -smooth muscle actin,) were detected by Western Blot (WB). 伪-SMA). The cells were pretreated with different concentration (1 渭 m ~ (10) 渭 M ~ (-1) 100 渭 M) DHEA for 24 h and then treated with TGF- 尾 _ (1) for 72 h. The detection method was the same as 1.3. DHEA100 (100 渭 M DHEA final concentration) TGF- 尾 1 group and TGF- 尾 1 group were used to detect the changes of p-AKT and AKT in the two groups by WB at different time points (0 ~ 0.5 h ~ 1 h ~ 2 h). The cells were pretreated with androgen receptor (androgen receptor,AR) antagonist flutamide, PI3K inhibitor LY294002 (LY) or PI3K agonist IGF-1 respectively. The cells were divided into eight groups: (1) untreated group; (2) TGF- 尾 1 group; (3) DHEA100 TGF- 尾 1 group; (4) DHEA100 TGF- 尾 1 flutamide group; (5) TGF- 尾 1 LY group; (6) TGF- 尾 1 IGF-1 group; (7) TGF- 尾 1 DHEA100 LY group; (8) in TGF- 尾 1 DHEA100 IGF-1 group, the cells were collected immediately after TGF- 尾 1 intervention for 1 h, and the changes of p-AKT and AKT were detected by WB. The cells of each group were cultured with TGF- 尾 1 for 72 h and the expression of E-cadherin. A-SMA was detected by WB. Results: 1. After treated with TGF- 尾 1 for 72 h, the appearance of cobblestone became fusiform, the intercellular junction was loose, the expression of E-cadherin decreased, and the expression of 伪-SMA increased. Different concentrations of DHEA inhibited EMT induced by TGF- 尾 1 in a concentration-dependent manner, and 100 渭 MDHEA had the strongest inhibitory effect. In TGF- 尾 1 group and DHEA100 TGF- 尾 1 group, the expression of p-AKT protein increased and reached the peak at 1 h after TGF- 尾 1 treatment. DHEA inhibited the expression of p-AKT protein, but there was no significant difference in AKT protein expression (P0.05). Flutamide could attenuate the inhibitory effect of DHEA on the expression of p-AKT protein and the process of EMT, 5.LY could further decrease the expression of p-AKT protein and EMT process, and IGF-1 could promote the expression of p-AKT protein and the development of EMT. Conclusion 1. TGF- 尾 1 induces EMT.2. in 16HBE-14o DHEA inhibited EMT induced by TGF- 尾 1 in a concentration dependent manner. DHEA inhibited EMT induced by TGF- 尾 1 by AR. 4. 4. The inhibitory effect of DHEA on EMT is related to PI3K/AKT signaling pathway.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
本文编号：2312899
[Abstract]:Objective: to induce human bronchial epithelial cell line 16HBE-14 by transforming growth factor- 尾 1 (TGF- 尾 1). An epithelial-mesenchymal transformation (Epithelial-Mesenchymal Transition, EMT) model was established to investigate the effect of dehydroepiandrosterone (Dehydroepiandrosterone,DHEA) on EMT induced by TGF- 尾 1 and its mechanism. Method 1: 1. 16HBE-14. Cultured on MEM medium containing 5% fetal bovine serum, 5ng/ml TGF- 尾 1 was used for 16HBE-14o72 h. Morphological changes of cells were observed. E-cadherin (E.cadherin) and 伪 -smooth muscle actin (伪 -smooth muscle actin,) were detected by Western Blot (WB). 伪-SMA). The cells were pretreated with different concentration (1 渭 m ~ (10) 渭 M ~ (-1) 100 渭 M) DHEA for 24 h and then treated with TGF- 尾 _ (1) for 72 h. The detection method was the same as 1.3. DHEA100 (100 渭 M DHEA final concentration) TGF- 尾 1 group and TGF- 尾 1 group were used to detect the changes of p-AKT and AKT in the two groups by WB at different time points (0 ~ 0.5 h ~ 1 h ~ 2 h). The cells were pretreated with androgen receptor (androgen receptor,AR) antagonist flutamide, PI3K inhibitor LY294002 (LY) or PI3K agonist IGF-1 respectively. The cells were divided into eight groups: (1) untreated group; (2) TGF- 尾 1 group; (3) DHEA100 TGF- 尾 1 group; (4) DHEA100 TGF- 尾 1 flutamide group; (5) TGF- 尾 1 LY group; (6) TGF- 尾 1 IGF-1 group; (7) TGF- 尾 1 DHEA100 LY group; (8) in TGF- 尾 1 DHEA100 IGF-1 group, the cells were collected immediately after TGF- 尾 1 intervention for 1 h, and the changes of p-AKT and AKT were detected by WB. The cells of each group were cultured with TGF- 尾 1 for 72 h and the expression of E-cadherin. A-SMA was detected by WB. Results: 1. After treated with TGF- 尾 1 for 72 h, the appearance of cobblestone became fusiform, the intercellular junction was loose, the expression of E-cadherin decreased, and the expression of 伪-SMA increased. Different concentrations of DHEA inhibited EMT induced by TGF- 尾 1 in a concentration-dependent manner, and 100 渭 MDHEA had the strongest inhibitory effect. In TGF- 尾 1 group and DHEA100 TGF- 尾 1 group, the expression of p-AKT protein increased and reached the peak at 1 h after TGF- 尾 1 treatment. DHEA inhibited the expression of p-AKT protein, but there was no significant difference in AKT protein expression (P0.05). Flutamide could attenuate the inhibitory effect of DHEA on the expression of p-AKT protein and the process of EMT, 5.LY could further decrease the expression of p-AKT protein and EMT process, and IGF-1 could promote the expression of p-AKT protein and the development of EMT. Conclusion 1. TGF- 尾 1 induces EMT.2. in 16HBE-14o DHEA inhibited EMT induced by TGF- 尾 1 in a concentration dependent manner. DHEA inhibited EMT induced by TGF- 尾 1 by AR. 4. 4. The inhibitory effect of DHEA on EMT is related to PI3K/AKT signaling pathway.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
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