黃體生成激素與動情素共同作用下對於內皮細胞遷移的影響

Effect of combined estrogen and progesterone treatment on vascular endothelial cell migration

林正哲(Jhen-Jhe Lin)

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

有關女性荷爾蒙對血管增生的影響，過去已有相關的文獻報導，並且從女性的月經週期的荷爾蒙分泌情形我們推測雌性素(E2)及黃體素(P4)會彼此交互作用而去影響到血管增生的現象。在本實驗室先前的細胞培養研究中指出單獨處以E2 (3-300 nM)或是黃體素P4 (5-500 nM)有明顯地抑制臍靜脈內皮細胞(HUVEC)的遷移能力，且此抑制作用與投予的荷爾蒙濃度呈正相關。然而這樣的研究結果與過去文獻報導中所指出E2及P4在體內對於血管增生的影響並不一致。因此本研究的目的是想利用細胞培養方法來釐清P4和E2他們單獨對於血管內皮細胞的遷移作用是否有與其在活體內有其他促進或是抑制血管增生因子存在下有相同的影響，以及他們相互間是否會彼此影響其對內皮細胞的遷移作用，並利用細胞分子生物學的技術來探討這些相互影響的作用分子機轉。利用wound healing assay和Transwells assay的實驗結果得知， E2 在1 nM的濃度下對於HUVEC的遷移能力並無明顯的影響，然而在單獨投予50 nM 的P4或同時投於E2 (1 nM)與P4 (50 nM)則對HUVEC的遷移能力有顯著地抑制作用。給予這些濃度下的E2或P4都不會影響HUVEC的存活率、對細胞外間質的貼附能力及MMPs的活性。利用Western blot的技術來分析時，我們發現在單獨給予P4或同時給予E2和P4會促進cSrc的磷酸化，並使RhoA的表現量減少。然而事先給予PP2抑制劑(400 nM)去阻斷cSrc之後，則P4及E2+P4對於HUVEC的遷移抑制作用則消失，顯示P4可能是透過活化cSrc而造成HUVEC遷移的抑制作用。我們利用在細胞培養液中加入血管增生促進因子VEGF來模擬在子宮內膜的in vivo情況，而發現在給予VEGF (5 ng/mL)下，HUVEC移動能力會增加，然而同時給予VEGF及P4時，VEGF所引起的內皮細胞遷移的促進作用則消失。意外的，我們發現在同時投予VEGF、E2及P4時，則P4對於VEGF的影響又不見了，顯示E2、P4及VEGF三者間對於血管增生有著某種相互的影響。

關鍵字

黃體素；動情激素；遷移

並列摘要

The effect of female sex hormones on angiogenesis has been previously reported. According to the concentration profile of female sex hormones during menstrual cycle, it is reasonable to speculate that estrogen and progesterone might interact and regulate the angiogenesis process. Previously, we demonstrated that treatment with E2 (3-300 nM) or P4 (5-500 nM) alone could significantly suppress HUVEC migration ability in a concentration-dependent manner. However, these findings differ from the previous reports from in vivo studies. Accordingly, the aim of this thesis research was to clarify whether E2 and P4 alone has the same effect in vitro and in vivo on the endothelial cell migration, whether E2 and P4 will interact each other to influence the endothelial cell migration, and the molecular mechanisms underlying of the interaction. Using the wound healing assay and Transwells assay, our results showed that E2 at a concentration of 1 nM did not significantly affect the migration of HUVEC. In contrast, treatment with P4 (50 nM) alone or a combination of E2 (1 nM) and P4 (50 nM) together significantly reduced the migration ability of HUVEC. The cell survival, cell adhesion to the ECM, and the MMPs activity were not significantly affected by the same treatment conditions. Using Western blot analysis, we found that treatment with P4 (50 nM) alone or a combination of E2 (1 nM) and P4 (50 nM) increased the levels of phosphorylated cSrc and decrease the levels of RhoA protein. However, pretreatment with a cSrc inhibitor, PP2 (400 nM), which blocked cSrc, abolished the P4- or E2+P4-induced migration inhibition of HUVEC, suggesting that these inhibitions were mediated through activating the cSrc molecule. To mimic the environment of uterus endometrium in vivo, VEGF at a concentration of 5 ng/mL was added to the culture medium. In the presence of VEGF (5 ng/mL), the HUVEC migration ability was significantly increased. Combined treatment with VEGF (5 ng/mL) and P4 (50 nM) abolished the stimulatory effect of VEGF on endothelial cell migration. Surprisingly, we found that combined treatment with VEGF, E2 and P4 together did not significantly change the migration activity as compared with treatment with VEGF alone, suggesting that there might be an interaction among E2，P4 and VEGF in regulating angiogenesis processes.

並列關鍵字

Progesterone ； Estrogen ； migration

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