【摘要】：目的:觀察17-β雌二醇(E2)對(duì)低氧誘導(dǎo)的人肺動(dòng)脈平滑肌細(xì)胞增殖(hPASMCs)的影響,并探討雌激素受體(estrogen receptor,ER)在其中的介導(dǎo)作用。方法:1細(xì)胞培養(yǎng):常氧組hPASMCs置于37℃、5%CO_2、95%氧氣的培養(yǎng)箱中進(jìn)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(jìn)行消化傳代,實(shí)驗(yàn)所用細(xì)胞為第5~8代,均處于對(duì)數(shù)生長(zhǎng)期。實(shí)驗(yàn)前,細(xì)胞均在不含F(xiàn)BS的無(wú)酚紅DMEM培養(yǎng)液中培養(yǎng)24h進(jìn)行同步化。2細(xì)胞干預(yù):應(yīng)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(shí)驗(yàn)測(cè)定不同干預(yù)條件下細(xì)胞增殖情況,篩選最適E2干預(yù)濃度。其次,將細(xì)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(shí)驗(yàn)測(cè)定各組細(xì)胞增殖情況,并采用RT-PCR法測(cè)定不同組hPASMCs中ERα、ERβ及PCNA的mRNA表達(dá)水平,采用Western blot法測(cè)定不同組hPASMCs中ERα、ERβ及PCNA的蛋白表達(dá)水平。結(jié)果:慢性低氧可顯著刺激hPASMCs增殖(P0.01),應(yīng)用E2干預(yù)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(dǎo)細(xì)胞增殖的同時(shí)伴隨ERαmRNA及蛋白表達(dá)下降(P0.01),PCNAmRNA及蛋白表達(dá)升高(P0.01),而ERβmRNA及蛋白表達(dá)無(wú)明顯變化(P0.05);應(yīng)用E2干預(yù)后,抑制了細(xì)胞的增殖并逆轉(zhuǎn)了ERα、PCNA指標(biāo)的以上變化(P0.01),但對(duì)ERβ表達(dá)無(wú)明顯影響(P0.05)。在低氧+E2基礎(chǔ)上,應(yīng)用MPP后可顯著削弱E2的以上作用(P0.01),而應(yīng)用ERβ抑制劑PHTPP干預(yù)則無(wú)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(guò)上調(diào)ERα的表達(dá)抑制hPASMCs的增殖,進(jìn)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(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R544.1

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