RL95-2-BeWo共培养体外着床模型的建立及粘附—植入功能的多因素调节

发布时间：2018-03-14 21:38

本文选题：雌二醇　切入点：孕酮　出处：《扬州大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的建立一种理想的体外共培养胚胎着床模型,研究不同浓度的雌二醇(E2)、孕酮(P4)和肝素结合表皮生长因子(HB-EGF)的刺激对子宫内膜上皮细胞整合素β3及其配体骨桥蛋白表达的影响,以及对共培养模型粘附和铺展能力的影响,初步探讨该模型在胚胎粘附-植入方面的多因素调节。方法 1、建立RL95-2-BeWo共培养体外着床模型将人子宫内膜癌细胞株RL95-2制成单层细胞用于模拟子宫内膜上皮,通过无血清培养和低吸附培养皿将人绒毛膜癌细胞株BeWo制成球状体用于模拟胚胎,将两者共培养模拟胚胎着床过程,观察形态并计算球状体粘附率(本实验以在澳大利亚亨利王子研究所PHI培训期间所做的球状体形态和粘附率作为对照)。 2、多因素调节RL95-2-BeWo共培养体外着床模型粘附-植入功能： (1)粘附因子整合素β3及其配体OPN检测通过间接免疫荧光技术检测RL95-2单层细胞、BeWo单层细胞和BeWo球状体整合素β3及OPN的定位表达；用不同浓度的E2、P4、E2+P4及HB-EGF处理RL95-2细胞,通过实时定量PCR技术比较各组整合素β3及OPN的mRNA表达水平。 (2)粘附实验和铺展实验用不同浓度的E2、P4、E2+P4及HB-EGF处理RL95-2细胞后与BeWo球状体共培养,比较各组球状体粘附率；用高浓度E2、P4及HB-EGF分别处理RL95-2细胞后与BeWo球状体共培养,第1h和第24h时拍照并测量球状体的直径,比较各组球状体铺展倍数。结果 1、建立RL95-2-BeWo体外共培养模型,本实验组改良方法制备的BeWo球状体与PHI在形态和粘附率上无明显差异(P0.05)。 2、整合素β3和OPN在RL95-2单层细胞、BeWo单层细胞和BeWo球状体的细胞质和膜表面表达。 3、E2对RL95-2细胞的整合素β3和OPN mRNA的表达无明显影响(P0.05)；高浓度P4上调OPN mRNA的表达,低浓度P4明显抑制OPN mRNA的表达(P0.05)；雌孕激素联合应用与各自单独应用无明显差异(P0.05)；高浓度HB-EGF能够促进整合素β3(P0.05)和OPN mRNA (P0.05)的表达。 4、高浓度E2抑制BeWo球状体粘附(P0.05),高浓度P4促进球状体粘附(P0.05),高浓度HB-EGF明显增加球状体粘附(P0.05)；高浓度P4和HB-EGF明显促进球状体在RL95-2单层细胞上的铺展(P0.05),并与作用时间有关。结论 1、成功建立了RL95-2-BeWo共培养体外着床模型。 2、整合素p3和OPN表达于RL95-2细胞和BeWo球状体的粘附接触面,其在子宫内膜细胞的表达受多因素调节。 3、高浓度E2不利于胚胎粘附,高浓度P4和HB-EGF能促进胚胎粘附和植入。
[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.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.8

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