脫氫表雄酮對支氣管上皮細(xì)胞上皮間質(zhì)轉(zhuǎn)化的作用及機(jī)制
發(fā)布時(shí)間：2018-11-05 18:05
【摘要】：目的：采用轉(zhuǎn)化生長因子-β1(transforming growth factor-β1, TGF-β1)誘導(dǎo)人支氣管上皮細(xì)胞株16HBE-14。構(gòu)建上皮-間質(zhì)轉(zhuǎn)化(Epithelial-Mesenchymal Transition, EMT)模型,觀察脫氫表雄酮(Dehydroepiandrosterone,DHEA)對TGF-β1誘導(dǎo)的EMT的作用,并探討其具體機(jī)制。 方法：1.將16HBE-14。用含有5%胎牛血清的MEM培養(yǎng)基培養(yǎng),5ng/ml TGF-β1作用16HBE-14o72h,觀察細(xì)胞的形態(tài)變化,采用Western Blot(WB)檢測E一鈣粘素(E.cadherin)、α-平滑肌肌動(dòng)蛋白(α-smooth muscle actin,α-SMA)表達(dá)。2.給予不同濃度(1μM、10μM、100μM)DHEA預(yù)處理細(xì)胞24h,后加入TGF-β1干預(yù)72h,檢測方法同1。3.設(shè)DHEA100(DHEA終濃度為100μM)+TGF-β1組和TGF-β1組,在不同時(shí)間點(diǎn)(0、0.5h、1h、2h)采用WB檢測兩組細(xì)胞p-AKT及AKT的變化過程。4.分別予雄激素受體(androgen receptor,AR)拮抗劑氟他胺、PI3K抑制劑LY294002(LY)或PI3K激動(dòng)劑IGF-1預(yù)處理細(xì)胞,設(shè)八組,分別為(1)無處理組；(2)TGF-β1組；(3)DHEA100+TGF-β1組；(4)DHEA100+TGF-β1+氟他胺組；(5)TGF-β1+LY組；(6)TGF-β1+IGF-1組；(7)TGF-β1+DHEA100+LY組；(8)TGF-β1+DHEA100+IGF-1組,TGF-β1共同干預(yù)1h后立即收集細(xì)胞,WB檢測各組細(xì)胞p-AKT及AKT的變化。各組細(xì)胞加入TGF-β1培養(yǎng)72h后立即收集細(xì)胞,WB檢測各組細(xì)胞E-cadherin.α-SMA表達(dá)。 結(jié)果：1. TGF-β1作用72h后,細(xì)胞鵝卵石外觀變?yōu)樗笮?細(xì)胞間連接疏松。E-cadherin表達(dá)下降；α-SMA表達(dá)增強(qiáng)。2.不同濃度DHEA對TGF-β1誘導(dǎo)EMT均有抑制作用,且呈濃度依賴性,100μMDHEA抑制作用最強(qiáng)。3. TGF-β1組、DHEA100+TGF-β1組均在TGF-β1處理后,p-AKT蛋白表達(dá)增加,1h達(dá)到高峰。DHEA抑制p-AKT蛋白表達(dá),AKT蛋白表達(dá)無明顯差異(P0.05)。4.氟他胺可減輕DHEA對p-AKT蛋白表達(dá)及EMT過程的抑制作用。5.LY可進(jìn)一步降低p-AKT蛋白表達(dá)及EMT過程；IGF-1促進(jìn)p-AKT蛋白表達(dá)及EMT發(fā)生。 結(jié)論：1. TGF-β1能誘導(dǎo)16HBE-14o發(fā)生EMT。2. DHEA對TGF-β1誘導(dǎo)的EMT有抑制作用,且呈濃度依賴性。3. DHEA通過AR而發(fā)揮對TGF-β1誘導(dǎo)的EMT的抑制作用。4. DHEA對EMT的抑制作用與PI3K/AKT信號途徑有關(guān)。
[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 偽 -smooth muscle actin (偽 -smooth muscle actin,) were detected by Western Blot (WB). 偽-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 偽-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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363
本文編號：2312898
[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 偽 -smooth muscle actin (偽 -smooth muscle actin,) were detected by Western Blot (WB). 偽-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 偽-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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363
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