本文選題：雌二醇　切入點：孕酮　出處：《揚州大學》2014年碩士論文　論文類型：學位論文

【摘要】：目的建立一種理想的體外共培養(yǎng)胚胎著床模型,研究不同濃度的雌二醇(E2)、孕酮(P4)和肝素結合表皮生長因子(HB-EGF)的刺激對子宮內(nèi)膜上皮細胞整合素β3及其配體骨橋蛋白表達的影響,以及對共培養(yǎng)模型粘附和鋪展能力的影響,初步探討該模型在胚胎粘附-植入方面的多因素調(diào)節(jié)。 方法 1、建立RL95-2-BeWo共培養(yǎng)體外著床模型 將人子宮內(nèi)膜癌細胞株RL95-2制成單層細胞用于模擬子宮內(nèi)膜上皮,通過無血清培養(yǎng)和低吸附培養(yǎng)皿將人絨毛膜癌細胞株BeWo制成球狀體用于模擬胚胎,將兩者共培養(yǎng)模擬胚胎著床過程,觀察形態(tài)并計算球狀體粘附率(本實驗以在澳大利亞亨利王子研究所PHI培訓期間所做的球狀體形態(tài)和粘附率作為對照)。 2、多因素調(diào)節(jié)RL95-2-BeWo共培養(yǎng)體外著床模型粘附-植入功能： (1)粘附因子整合素β3及其配體OPN檢測 通過間接免疫熒光技術檢測RL95-2單層細胞、BeWo單層細胞和BeWo球狀體整合素β3及OPN的定位表達；用不同濃度的E2、P4、E2+P4及HB-EGF處理RL95-2細胞,通過實時定量PCR技術比較各組整合素β3及OPN的mRNA表達水平。 (2)粘附實驗和鋪展實驗 用不同濃度的E2、P4、E2+P4及HB-EGF處理RL95-2細胞后與BeWo球狀體共培養(yǎng),比較各組球狀體粘附率；用高濃度E2、P4及HB-EGF分別處理RL95-2細胞后與BeWo球狀體共培養(yǎng),第1h和第24h時拍照并測量球狀體的直徑,比較各組球狀體鋪展倍數(shù)。 結果 1、建立RL95-2-BeWo體外共培養(yǎng)模型,本實驗組改良方法制備的BeWo球狀體與PHI在形態(tài)和粘附率上無明顯差異(P0.05)。 2、整合素β3和OPN在RL95-2單層細胞、BeWo單層細胞和BeWo球狀體的細胞質(zhì)和膜表面表達。 3、E2對RL95-2細胞的整合素β3和OPN mRNA的表達無明顯影響(P0.05)；高濃度P4上調(diào)OPN mRNA的表達,低濃度P4明顯抑制OPN mRNA的表達(P0.05)；雌孕激素聯(lián)合應用與各自單獨應用無明顯差異(P0.05)；高濃度HB-EGF能夠促進整合素β3(P0.05)和OPN mRNA (P0.05)的表達。 4、高濃度E2抑制BeWo球狀體粘附(P0.05),高濃度P4促進球狀體粘附(P0.05),高濃度HB-EGF明顯增加球狀體粘附(P0.05)；高濃度P4和HB-EGF明顯促進球狀體在RL95-2單層細胞上的鋪展(P0.05),并與作用時間有關。 結論 1、成功建立了RL95-2-BeWo共培養(yǎng)體外著床模型。 2、整合素p3和OPN表達于RL95-2細胞和BeWo球狀體的粘附接觸面,其在子宮內(nèi)膜細胞的表達受多因素調(diào)節(jié)。 3、高濃度E2不利于胚胎粘附,高濃度P4和HB-EGF能促進胚胎粘附和植入。
[Abstract]:Objective to establish an ideal co-cultured embryo implantation model in vitro to study the effects of different concentrations of estradiol E2P, progesterone P4) and heparin-bound epidermal growth factor (HB-EGF) on the expression of integrin 尾 3 and its ligand osteopontin in endometrial epithelial cells. And the effect on the adhesion and spreading ability of co-culture model was discussed, and the multifactor regulation of the model in embryo adhesion and implantation was discussed. Method. 1. To establish the model of RL95-2-BeWo co-culture implantation in vitro. Human endometrial carcinoma cell line RL95-2 was made into monolayer cells to mimic endometrial epithelium, and human choriocarcinoma cell line BeWo was made into spherical body to simulate embryo by serum-free culture and low adsorption culture dish. The embryo implantation process was simulated by co-culture, morphology was observed and the adhesion rate of globular bodies was calculated. (in this experiment, the morphology and adhesion rate of globular bodies made during PHI training at the Prince Henry Institute of Australia were used as the control. 2, multivariate regulation of adhesion-implant function in RL95-2-BeWo co-culture model in vitro:. Detection of integrin 尾 3 and its ligand OPN. The localization expression of integrin 尾 3 and OPN in RL95-2 monolayer cells and BeWo spheroid bodies were detected by indirect immunofluorescence technique, and RL95-2 cells were treated with different concentrations of E2P4P4E2P4 and HB-EGF. The mRNA expression levels of integrin 尾 3 and OPN were compared by real time quantitative PCR. (2) Adhesion experiment and spreading experiment. RL95-2 cells were treated with different concentrations of E2P4P4E2P4 and HB-EGF and cocultured with BeWo globules, and the adhesion rates of BeWo spheroid cells were compared, and RL95-2 cells treated with high concentrations of E2P4 and HB-EGF were co-cultured with BeWo spheroid cells. The diameters of globular bodies were measured at 1 h and 24 h, and the spheroidal spreading times of each group were compared. Results. 1. The model of co-culture of RL95-2-BeWo in vitro was established. There was no significant difference in morphology and adhesion rate between BeWo spheroid and PHI prepared by the modified method in this experimental group. 2. Integrin 尾 3 and OPN were expressed on the cytoplasm and membrane of RL95-2 monolayer cells and BeWo globules. 3The expression of integrin 尾 _ 3 and OPN mRNA in RL95-2 cells was not affected by E _ 2, and the expression of OPN mRNA was up-regulated by high concentration of P4. The low concentration of P4 significantly inhibited the expression of OPN mRNA (P0.05), the combination of estrogen and progesterone had no significant difference from that of each other (P0.05), and the high concentration of HB-EGF could promote the expression of integrin 尾 _ 3 (P _ (0.05)) and OPN mRNA (P _ (0.05)). 4. High concentration of E2 inhibited BeWo spheroid adhesion (P0.05), high concentration of P4 promoted spheroid adhesion (P0.05), high concentration of HB-EGF significantly increased spheroid adhesion (P0.05), and high concentration of P4 and HB-EGF significantly promoted spheroids spreading on RL95-2 monolayers, which was related to the time of action. Conclusion. 1. RL95-2-BeWo co-culture in vitro implantation model was successfully established. 2. Integrin p3 and OPN were expressed on the adhesion surface of RL95-2 cells and BeWo globules, and their expression in endometrial cells was regulated by many factors. 3. High concentration of E 2 was not conducive to embryo adhesion, and high concentration of P 4 and HB-EGF could promote embryo adhesion and implantation.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R714.8

【參考文獻】

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本文編號：1613079