【摘要】：研究背景:宮腔粘連(IUAs)是指創(chuàng)傷、感染等各種原因所致子宮內(nèi)膜基底層損傷后,宮腔部分或全部粘連。近年研究表明,子宮內(nèi)膜纖維化是IUAs的主要病理學(xué)特征,是其本質(zhì)。叉頭框F2(FoxF2)是器官發(fā)育、細(xì)胞外基質(zhì)合成、上皮-間質(zhì)細(xì)胞轉(zhuǎn)化中的重要轉(zhuǎn)錄調(diào)節(jié)因子,參與了多種器官的纖維化過程。FoxF2在IUAs的作用尚未見文獻(xiàn)報道。雌激素是存在于女性體內(nèi)的最主要性激素,在IUAs治療中常作為經(jīng)驗性治療被臨床廣泛應(yīng)用,但因療效不一、基礎(chǔ)研究缺乏,其應(yīng)用劑量仍存在廣泛爭議。本實驗擬探討FoxF2在IUAs中的表達(dá)及不同濃度雌激素對IUAs纖維化進(jìn)程和FoxF2表達(dá)的影響。第一章FoxF2在宮腔粘連細(xì)胞模型中的表達(dá)及意義目的:探討FoxF2在IUAs細(xì)胞模型中的表達(dá)。方法:1.原代培養(yǎng)人子宮內(nèi)膜基質(zhì)細(xì)胞(HESCs)。2.免疫細(xì)胞化學(xué)法進(jìn)行細(xì)胞鑒定。3.建立IUAs細(xì)胞模型:用0和10ng/ml轉(zhuǎn)化生長因子β1(TGF-β1)分別作用于HESCs 48 h作為對照組和模型組。4.實時熒光定量PCR(qPCR)和蛋白質(zhì)印跡法(WB)檢測α-平滑肌肌動蛋白(α-SMA)、膠原I(COLI)及FoxF2的mRNA和蛋白表達(dá)。結(jié)果:1.原代培養(yǎng)的HESCs形態(tài)穩(wěn)定,生長活性好。2.免疫細(xì)胞化學(xué)法檢測細(xì)胞波形蛋白表達(dá)陽性,且波形蛋白陽性細(xì)胞比率高,而角蛋白18表達(dá)陰性,證實為HESCs,且純度高。3.與對照組相比,模型組α-SMA、COLI及FoxF2的mRNA和蛋白表達(dá)均顯著性增高(P0.05)。結(jié)論:1.10ng/mlTGF-β1可誘導(dǎo)HESCs向肌成纖維細(xì)胞轉(zhuǎn)化,引起纖維化標(biāo)志物α-SMA和COLI的表達(dá)穩(wěn)定性增加,從而建立IUAs細(xì)胞模型。2.FoxF2在IUAs中高表達(dá),提示FoxF2可能參與IUAs纖維化進(jìn)程。第二章雌激素對宮腔粘連纖維化進(jìn)程及FoxF2表達(dá)的影響目的:探討不同濃度雌激素對IUAs纖維化進(jìn)程及FoxF2表達(dá)的影響。方法:1.配置10-6、10-8 10-10、10-12mol/L的雌激素工作液。2.本實驗分為5組,分別為模型組、10-6mol/L E2組、10-8mol/LE2組、10-10 mol/L E2組、10-12 mol/L E2組。先用 10 ng/ml TGF-β1作用于HESCs 48 h以建立IUAs細(xì)胞模型,再用上述不同濃度雌激素作用于該模型中48 h。3.qPCR和WB法檢測α-SMA、COLI及FoxF2的mRNA和蛋白表達(dá)。結(jié)果:1.qPCR 結(jié)果:與模型組相比,10-6、10-8、10-10mol/LE2 組 α-SMA、COLI、FoxF2mRNA 表達(dá)均下降(10-10mol/LE2組 COLI:P0.05,其余各組P0.05);10-12 mol/LE2組 α-SMA、COLImRNA 表達(dá)上升(P0.05),FoxF2mRNA 表達(dá)下降(P0.05)。2.WB 結(jié)果:與模型組相比,10-6、10-8、10-10 mol/LE2組 α-SMA、COLI、FoxF2蛋白表達(dá)均下降(P0.05);10-12 mol/LE2組α-SMA蛋白表達(dá)上升(P0.05),COLI和FoxF2蛋白表達(dá)均下降(P0.05)。結(jié)論:17β-雌二醇可在一定范圍內(nèi)下調(diào)α-SMA和COLI的表達(dá),逆轉(zhuǎn)IUAs纖維化進(jìn)程,并抑制FoxF2的表達(dá)。
[Abstract]:Background: intrauterine adhesions (IUAs) refer to partial or total adhesions of uterine cavity after endometrial basal layer injury caused by trauma and infection. Recent studies have shown that endometrial fibrosis is the main pathological feature and essence of IUAs. Forked frame F2 (FoxF2) is an important transcriptional regulator in organ development, extracellular matrix synthesis and epithelial-interstitial cell transformation. The role of FoxF2 in the process of organ fibrosis has not been reported in the literature. Estrogen is the most important sex hormone in women. It is often used as empirical therapy in IUAs. However, because of the difference of curative effect and lack of basic research, the dosage of estrogen is still controversial. The purpose of this study was to investigate the expression of FoxF2 in IUAs and the effects of different concentrations of estrogen on the progression of IUAs fibrosis and the expression of FoxF2. The expression and significance of FoxF2 in uterine Adhesion Cell Model objective: to investigate the expression of FoxF2 in IUAs cell model. Method 1: 1. Primary culture of human endometrial stromal cells (HESCs). 2. Immunocytochemistry method for cell identification. IUAs cell model was established: 0 and 10ng/ml transforming growth factor 尾 1 (TGF- 尾 1) were treated with HESCs for 48 h as control group and model group respectively. The expression of 偽 -smooth muscle actin (偽 -SMA), collagen I (COLI) and FoxF2 was detected by real-time quantitative PCR (qPCR) and Western blot (WB). The result is 1: 1. The primary culture of HESCs was stable in morphology and good in growth activity. 2. The positive expression of vimentin was detected by immunocytochemistry, and the positive rate of vimentin was high, but the expression of keratin 18 was negative, which was proved to be HESCs, with high purity. 3. Compared with the control group, the mRNA and protein expressions of 偽 -SMA-COLI and FoxF2 in the model group were significantly higher than those in the control group (P0.05). Conclusion: 1. 10 ng / ml TGF- 尾 1 can induce the transformation of HESCs to myofibroblasts and increase the stability of 偽 -SMA and COLI expression, thus establishing the IUAs cell model. 2. FoxF2 is highly expressed in IUAs, suggesting that FoxF2 may participate in the process of IUAs fibrosis. The effect of estrogen on the process of intrauterine adhesion fibrosis and the expression of FoxF2 objective: to investigate the effects of different concentrations of estrogen on the progression of IUAs fibrosis and the expression of FoxF2. Method 1: 1. Oestrogen working solution. 2 with 10-6, 10-8, 10-10, 10-12 mol / L, 10-8, 10-10, 10-12 mol / L. The experiment was divided into five groups: model group 10-6 mol / L E2 group 10-8 mol / L E _ 2 group 10-10 mol/L E _ 2 group 10-12 mol/L E _ 2 group. The IUAs cell model was established by 10 ng/ml TGF- 尾 1 treatment with HESCs for 48 h. The mRNA and protein expressions of 偽 -SMA-COLI and FoxF2 were detected by 48 h.3.qPCR and WB methods. Results 1. QPCR results: compared with the model group, the expression of 偽 -SMACOLICOLI FoxF2 mRNA in 10-6 + 10-8 10-10 mol / L 2 group decreased (P0.05) in 10-12 mol/LE2 group (P0.05). 2.WB results: compared with the model group, the expression of 偽 -SMACOLICOLI FoxF2 protein in 10-6 10-8 10-10 mol/LE2 group decreased (P0.05) in 10-12 mol/LE2 group (P0.05). 2. WB results: compared with the model group, the expression of 偽 -SMACOLICOLICOLI FoxF2 protein in 10-12 mol/LE2 group was decreased (P0.05), and the expression of 偽 -SMACOLICOLI FoxF2 protein in 10-12 mol/LE2 group was significantly decreased compared with the model group (P0.05). The expression of coli and FoxF2 decreased significantly (P0.05). Conclusion the expression of 偽 -SMA and COLI can be down-regulated by 尾 -estradiol in a certain range, which can reverse the process of IUAs fibrosis and inhibit the expression of FoxF2.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R711.74

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