女性荷爾蒙對於葉酸誘導的人類內皮細胞生長與遷移抑制之影響

Effects of female sex hormones on folic acid induced inhibitions of proliferation and migration of human endothelial cells

盧沂榛(Yi-Chen Lu)

指導教授 ： 李文森

臺北醫學大學/醫學院/醫學科學研究所/碩士(2013年)

摘要

本實驗室先前的研究已證實葉酸具有抗血管增生的功能。然而，懷孕婦女被推薦日常攝取葉酸做為膳食補充，以防止一些嚴重的新生兒缺陷，如脊柱裂（spina bifida）等。由於血管增生對於子宮內膜重組和胚胎發育是非常重要的，應該有某些機制以允許懷孕母親和胎兒脫離葉酸-誘導的抗血管生成作用。雖然真正的機轉仍是未知，但其中一個可能的機制是，在懷孕母體血液中高濃度的女性荷爾蒙也許會拮抗葉酸對血管增生的影響。為了解決這個疑問，我們初步測試女性荷爾蒙是否對於葉酸引起的抑制血管內皮細胞增生及遷移作用是否有影響。實驗結果顯示黃體素(Progesterone, P4)及雌激素(Estrogen, E2)都可中止葉酸引起的抑制血管內皮細胞增生及遷移作用。這結果顯示女性荷爾蒙可以調控葉酸對於血管增生的影響，而這些影響可以分別被黃體素受體及雌激素受體的拮抗劑所抑制。使用免疫沉澱（immunoprecipitation）技術，我們進一步發現FR，ER，PR和Src之間可形成一個複合體。我們之前的研究顯示，黃體素和葉酸抑制細胞增生的機轉都是透過活化cSrc，進而活化ERK，接著促進p21和p27的蛋白質表現量，進而抑制細胞增殖；同時它們可利用抑制RhoA及Rac-1的活性，而抑制內皮細胞的遷移現象。利用西方墨點法（Western blot analysis）分析蛋白質表現結果顯示在單獨給予葉酸、黃體素及雌激素時人類臍靜脈內皮細胞（human umbilical vein endothelial cells; HUVEC）的pSrc，pERK，p21和p27蛋白質表現量都有顯著增加，且RhoA及Rac-1的活性都有受到抑制，然而在同時加入葉酸及女性荷爾蒙之後，則這些蛋白質增加的現象就不見了。而黃體素及雌激素對於葉酸的影響可因分別投予黃體素受體及雌激素受體的拮抗劑而抑制。而我們的實驗更證實了雌激素對此的作用可被雌激素受體β型的拮抗劑所抑制。根據上述實驗結果，我們推測女性荷爾蒙可以保護防止由葉酸誘發的人類臍靜脈內皮細胞遷移與增殖的抑制。

關鍵字

細胞增生 ； 細胞遷移 ； 雌激素 ； 黃體素 ； 人類臍靜脈內皮細胞

並列摘要

Previously, we demonstrated that folic acid (FA) exerted an anti-angiogenic activity. However, pregnant women were recommended to intake dietary FA supplements to prevent some severe birth defects, such as spina bifida. Since angiogenesis is very important for endometrial reorganization and embryonic development, there should be some mechanisms to allow the pregnant mother and the fetus to escape from the FA-induced anti-angiogenic action. Although the mechanism underlying is still unknown, one of the possible mechanisms is that a high concentration of female sex hormones in maternal blood might antagonize the FA effect on angiogenesis. To address this issue, we initially examined the effects of female sex hormones on the FA-induced proliferation and migration inhibitions of endothelial cells. The data revealed that both progesterone (P4) and estrogen (E2) can antagonize the FA-induced proliferation and migration inhibitions of endothelial cells. These results might suggest that female sex hormones regulate the anti-angiogenic action of FA, which could be suppressed by the antagonists of P4 and E2 receptors, respectively. Immunoprecipitation assay further revealed that FA receptor (FR), estrogen receptor (ER), progesterone receptor (PR) and cSrc formed a complex. Our previous studies showed that both P4 and FA inhibited HUVEC proliferation through cSrc-mediated up-regulation of p21 and p27, and inhibited HUVEC migration through cSrc-mediated suppression of RhoA and Rac-1 activities. By using Western blotting analyses, our data revealed significant increases of protein levels of pSrc, pERK, p21 and p27, as well as the activities of RhoA and Rac-1 in HUVEC treated with P4, E2 or FA alone. However, the elevated protein levels of pSrc, pERK, p21 and p27, as well as the activities of RhoA and Rac-1 were diminished while cells were co-treated with FA and female sex hormones together. Moreover, treatment with PR or ER antagonists inhibited the effects of P4 or E2 on the anti-proliferation and anti-migration actions of FA. Moreover, our data also showed that the effects of E2 on the FA-induced endothelial cell behaviors were abolished by pretreatment of the cell with an ER β antagonist (PHTPP). Taken together, the findings from the present study suggest that female sex hormones could protect against the FA-induced migration and proliferative inhibitions of HUVEC.

並列關鍵字

Proliferation ； Migration ； Estrogen ； Progesterone ； HUVEC

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