17-β雌二醇通過雌激素受體α抑制肺動脈平滑肌細胞的增殖
發(fā)布時間：2018-08-16 18:16
【摘要】：目的:觀察17-β雌二醇(E2)對低氧誘導的人肺動脈平滑肌細胞增殖(hPASMCs)的影響,并探討雌激素受體(estrogen receptor,ER)在其中的介導作用。方法:1細胞培養(yǎng):常氧組hPASMCs置于37℃、5%CO_2、95%氧氣的培養(yǎng)箱中進行培養(yǎng),低氧組hPASMCs置于37℃、5%CO_2、92%N2的三氣培養(yǎng)箱中培養(yǎng),每2d換液1次,并以0.25%胰蛋白酶進行消化傳代,實驗所用細胞為第5~8代,均處于對數(shù)生長期。實驗前,細胞均在不含F(xiàn)BS的無酚紅DMEM培養(yǎng)液中培養(yǎng)24h進行同步化。2細胞干預:應用第5~8代hPASMCs,分為常氧組、低氧組、低氧+E2(10-6mol/L)組、低氧+E2(10-7mol/L)組、低氧+E2(10-8mol/L)組、低氧+E2(10-9mol/L)組,行MTT實驗測定不同干預條件下細胞增殖情況,篩選最適E2干預濃度。其次,將細胞分為常氧組、低氧組、低氧+E2(10-6mol/L)、低氧+E2(10-6mol/L)+MPP組、低氧+E2(10-6mol/L)+PHTPP組,行MTT實驗測定各組細胞增殖情況,并采用RT-PCR法測定不同組hPASMCs中ERα、ERβ及PCNA的mRNA表達水平,采用Western blot法測定不同組hPASMCs中ERα、ERβ及PCNA的蛋白表達水平。結(jié)果:慢性低氧可顯著刺激hPASMCs增殖(P0.01),應用E2干預后hPASMCs增殖減少(P0.01),且最佳抑制濃度為10-6mol/l。另外,慢性低氧誘導細胞增殖的同時伴隨ERαmRNA及蛋白表達下降(P0.01),PCNAmRNA及蛋白表達升高(P0.01),而ERβmRNA及蛋白表達無明顯變化(P0.05);應用E2干預后,抑制了細胞的增殖并逆轉(zhuǎn)了ERα、PCNA指標的以上變化(P0.01),但對ERβ表達無明顯影響(P0.05)。在低氧+E2基礎(chǔ)上,應用MPP后可顯著削弱E2的以上作用(P0.01),而應用ERβ抑制劑PHTPP干預則無明顯影響(P0.05)。結(jié)論:1 E2能有效抑制hPASMCs的增殖。2 E2可能通過上調(diào)ERα的表達抑制hPASMCs的增殖,進而發(fā)揮其拮抗HPH的作用。
[Abstract]:AIM: To observe the effect of 17-beta estradiol (E2) on hypoxia-induced proliferation of human pulmonary artery smooth muscle cells (hPASMCs) and explore the mediating effect of estrogen receptor (ER). METHODS: 1 Cell culture: hPASMCs in normoxia group were cultured at 37 C, 5% CO_2, 95% oxygen, and hPASMCs in hypoxia group were cultured at 37 C and 5% CO_2. The cells were cultured in a three-gas incubator containing 92% N 2 and were subcultured with 0.25% trypsin every 2 days. The cells of the 5th to 8th passages were all in logarithmic growth phase. The cells were divided into normal oxygen group, hypoxia + E2 (10-6 mol/L), hypoxia + E2 (10-7 mol/L), hypoxia + E2 (10-8 mol/L), hypoxia + E2 (10-8 mol/L), hypoxia + E2 (10-8 mol/L), hypoxia + E2 (10-9 mol/L) group, and hypoxia + E2 (10-9 mol/L) group. MTT experiment was used to determine the cell proliferation under different intervention conditions, and the optimal concentration of E2 intervention was screen. Second, the cells were divided into normoxygen group, hypooxygen group, hypoxia + E2 (10-6 mol/L), hypoxia + E2 (10-6 mol/L), E2 (10-6 mol/+PHTPP group, MTT assay was used to determine the proliferation of hPASMCs, and RT-PCR was used to determine the mRNA expression levels of ERa, ERbeta and PCNA in hPASMCs of different groups. Western blot was used to determine the protein expression levels of ERa, ERbeta and PCNA in hPASMCs of different groups. The optimal inhibitory concentration was 10-6 mol/l. In addition, chronic hypoxia-induced cell proliferation was accompanied by decreased expression of ER-alpha mRNA and protein (P 0.01), increased expression of PCNA mRNA and protein (P 0.01), but no significant change of ER-beta mRNA and protein expression (P 0.05). On the basis of hypoxia + E2, MPP could significantly weaken the above effect of E2 (P 0.01), but PHTPP intervention had no significant effect (P 0.05). Conclusion: 1 E2 can effectively inhibit the proliferation of hPASMCs. 2 E2 may inhibit the proliferation of hPASMCs by up-regulating the expression of ERa, and then play its antagonism. Anti HPH effect.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R544.1
本文編號：2186784
[Abstract]:AIM: To observe the effect of 17-beta estradiol (E2) on hypoxia-induced proliferation of human pulmonary artery smooth muscle cells (hPASMCs) and explore the mediating effect of estrogen receptor (ER). METHODS: 1 Cell culture: hPASMCs in normoxia group were cultured at 37 C, 5% CO_2, 95% oxygen, and hPASMCs in hypoxia group were cultured at 37 C and 5% CO_2. The cells were cultured in a three-gas incubator containing 92% N 2 and were subcultured with 0.25% trypsin every 2 days. The cells of the 5th to 8th passages were all in logarithmic growth phase. The cells were divided into normal oxygen group, hypoxia + E2 (10-6 mol/L), hypoxia + E2 (10-7 mol/L), hypoxia + E2 (10-8 mol/L), hypoxia + E2 (10-8 mol/L), hypoxia + E2 (10-8 mol/L), hypoxia + E2 (10-9 mol/L) group, and hypoxia + E2 (10-9 mol/L) group. MTT experiment was used to determine the cell proliferation under different intervention conditions, and the optimal concentration of E2 intervention was screen. Second, the cells were divided into normoxygen group, hypooxygen group, hypoxia + E2 (10-6 mol/L), hypoxia + E2 (10-6 mol/L), E2 (10-6 mol/+PHTPP group, MTT assay was used to determine the proliferation of hPASMCs, and RT-PCR was used to determine the mRNA expression levels of ERa, ERbeta and PCNA in hPASMCs of different groups. Western blot was used to determine the protein expression levels of ERa, ERbeta and PCNA in hPASMCs of different groups. The optimal inhibitory concentration was 10-6 mol/l. In addition, chronic hypoxia-induced cell proliferation was accompanied by decreased expression of ER-alpha mRNA and protein (P 0.01), increased expression of PCNA mRNA and protein (P 0.01), but no significant change of ER-beta mRNA and protein expression (P 0.05). On the basis of hypoxia + E2, MPP could significantly weaken the above effect of E2 (P 0.01), but PHTPP intervention had no significant effect (P 0.05). Conclusion: 1 E2 can effectively inhibit the proliferation of hPASMCs. 2 E2 may inhibit the proliferation of hPASMCs by up-regulating the expression of ERa, and then play its antagonism. Anti HPH effect.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R544.1
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相關(guān)期刊論文 前2條
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2 許麗輝;何強;;Myocardin在大鼠血管平滑肌細胞表型轉(zhuǎn)化中的作用機制[J];中國老年學雜志;2010年17期
，本文編號：2186784
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