本文關(guān)鍵詞：經(jīng)血干細胞以旁分泌途徑抑制受損子宮內(nèi)膜纖維化并促進修復(fù)的作用機制研究　出處：《浙江大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 經(jīng)血干細胞 子宮內(nèi)膜間質(zhì)細胞 子宮內(nèi)膜修復(fù) 米非司酮 經(jīng)血干細胞 子宮內(nèi)膜纖維化 Hippo信號通路 旁分泌 經(jīng)血干細胞 TGFβ 子宮內(nèi)膜纖維化 旁分泌

【摘要】：第1章 經(jīng)血干細胞對受損子宮內(nèi)膜的修復(fù)作用研究背景及目的：子宮內(nèi)膜作為胚胎種植和發(fā)育的“土壤”,其在助孕中有舉足輕重的作用。因子宮內(nèi)膜損傷,粘連和缺如等導(dǎo)致的子宮內(nèi)膜過薄可阻礙受精卵在子宮內(nèi)膜著床和發(fā)育。子宮內(nèi)膜間充質(zhì)干細胞是子宮內(nèi)膜具有高度再生能力的原因,已有大量研究證實從女性月經(jīng)血中提取的經(jīng)血干細胞具有間充質(zhì)干細胞增殖及多向分化特性,可修復(fù)受損肝臟,改善肝臟功能。然而,經(jīng)血干細胞是否具有修復(fù)受損子宮內(nèi)膜能力尚未見報道。因此,本研究旨在明確經(jīng)血干細胞是否能修復(fù)受損子宮內(nèi)膜間質(zhì)細胞的增殖及遷移能力,減輕受損子宮內(nèi)膜間質(zhì)細胞的凋亡。方法：本研究用米非司酮藥物性損傷子宮內(nèi)膜間質(zhì)細胞,并利用transwell共培養(yǎng)體系,與經(jīng)血干細胞共培養(yǎng)。CCK8試驗用來檢測子宮內(nèi)膜間質(zhì)細胞的增殖力改變,AV/PI雙染流式用來檢測子宮內(nèi)膜間質(zhì)細胞的凋亡變化,Western blot法用來檢測子宮內(nèi)膜間質(zhì)細胞中VEGF的蛋白表達變化。結(jié)果：米非司酮對子宮內(nèi)膜間質(zhì)細胞的抑制作用呈時間及濃度依賴性,在濃度25mg/1作用48小時后,米非司酮達到最佳抑制效果。共培養(yǎng)經(jīng)血干細胞,受損子宮內(nèi)膜間質(zhì)細胞的增殖能力顯著增加,凋亡率顯著下降。同時,共培養(yǎng)經(jīng)血干細胞后,受損子宮內(nèi)膜間質(zhì)細胞的VEGF蛋白表達水平顯著增加。結(jié)論：本實驗在米非司酮損傷子宮內(nèi)膜間質(zhì)細胞的模型上,利用經(jīng)血干細胞共培養(yǎng)體系,證實經(jīng)血干細胞可通過旁分泌途徑阻止或者逆轉(zhuǎn)子宮內(nèi)膜間質(zhì)細胞的損傷。第2章經(jīng)血干細胞抑制子宮內(nèi)膜纖維化的作用研究研究背景及目的：宮腔粘連患者的子宮內(nèi)膜基底層被大量破壞,子宮內(nèi)膜再生障礙,取而代之為大量的致密纖維組織,導(dǎo)致子宮壁粘連,引起月經(jīng)量減少、閉經(jīng)及不孕等臨床癥狀。盡管隨著宮腔內(nèi)藥物治療支架及抗生素的應(yīng)用,宮腔粘連治療后復(fù)發(fā)的幾率在逐漸減少,然而,對于中重度粘連,宮腔鏡下粘連分解術(shù)后粘連再發(fā)率仍居高不下,也是宮腔粘連治療的一大難題。干細胞再生醫(yī)學(xué)已取得可觀臨床試驗結(jié)果,也確實有研究應(yīng)用骨髓干細胞治療宮腔粘連后,使患者月經(jīng)改善及獲得妊娠。經(jīng)血干細胞來源于脫落的子宮內(nèi)膜組織,與骨髓間充質(zhì)干細胞類似,具有多向分化潛能,已被證實可修復(fù)受損心肌細胞及減輕心肌梗死后心肌細胞的纖維化形成。然而,對于經(jīng)血干細胞在宮腔粘連中的治療作用及相關(guān)機制尚未被報道。本研究通過體外實驗旨在明確經(jīng)血干細胞是否能抑制宮腔粘連患者子宮內(nèi)膜纖維化過程,而hippo/TAZ信號通路是否參與了這個過程。方法：本研究利用transwell共培養(yǎng)體系,共培養(yǎng)經(jīng)血干細胞與子宮內(nèi)膜間質(zhì)細胞。同時收集經(jīng)血干細胞條件培養(yǎng)基,培養(yǎng)子宮內(nèi)膜間質(zhì)細胞。CCK8試驗用來檢測子宮內(nèi)膜間質(zhì)細胞的增殖力改變；劃痕實驗檢測子宮內(nèi)膜間質(zhì)細胞遷移能力的變化；Real-time PCR及western blot法用來檢測子宮內(nèi)膜間質(zhì)細胞中纖維化相關(guān)因子aSMA、collagen Ⅰ的mRNA與蛋白表達變化；Western blot法檢測子宮內(nèi)膜間質(zhì)細胞中hippo信號通路相關(guān)因子phos-LATS1(1059), phos-MOB1及phos-TAZ的蛋白表達變化；免疫熒光法檢測子宮內(nèi)膜間質(zhì)細胞中TAZ的定位變化。結(jié)果：共培養(yǎng)經(jīng)血干細胞或經(jīng)血干細胞條件培養(yǎng)基均顯著下調(diào)aSMA與collagen Ⅰ的mRNA及蛋白表達水平,促進子宮內(nèi)膜間質(zhì)細胞遷移,上調(diào)hippo信號通路中的phos-LATS1(1059), phos-MOB1及phos-TAZ的表達水平。經(jīng)血干細胞條件培養(yǎng)基顯著促進子宮內(nèi)膜間質(zhì)細胞增殖,同時使T舵從子宮內(nèi)膜間質(zhì)細胞核內(nèi)移出,滯留于胞漿。結(jié)論：研究中采用transwell共培養(yǎng)體系或干細胞條件培養(yǎng)基,表明經(jīng)血干細胞可能是通過旁分泌途徑抑制子宮內(nèi)膜間質(zhì)細胞向肌成纖維細胞轉(zhuǎn)化,并促進間質(zhì)細胞增殖及遷移,促進創(chuàng)傷愈合,Hippo/TAZ信號通路是被經(jīng)血干細胞激活的信號通路之一。第3章 經(jīng)血干細胞通過抑制TGFβ作用阻止子宮內(nèi)膜纖維化并促進子宮內(nèi)膜修復(fù)研究背景及目的：宮腔粘連已成為女性繼發(fā)不孕的第二大病因,據(jù)報道13%的不孕癥患者存在宮腔粘連,43%的宮腔粘連患者合并不孕病史。目前研究認(rèn)為宮腔粘連患者中殘存子宮內(nèi)膜組織中的干細胞在數(shù)量及功能上可能存在異常。干細胞再生醫(yī)學(xué)在宮腔粘連治療越來越受重視。我們體外研究已經(jīng)發(fā)現(xiàn)經(jīng)血干細胞可抑制子宮內(nèi)膜間質(zhì)細胞向肌成纖維細胞轉(zhuǎn)變,降低α-SMA及collagen Ⅰ水平,表明經(jīng)血干細胞可能具有減輕或阻止宮腔粘連作用。但是經(jīng)血干細胞的具體作用機制仍需進一步研究。TGFβ家族是一條多功能信號通路,參與多種病理生理過程,已有研究證實在宮腔粘連患者子宮內(nèi)膜組織中存在TGFβ高度表達,且隨著粘連程度的增加表達增高。本研究利用體外實驗驗證TGFβ在促進子宮內(nèi)膜間質(zhì)細胞肌成纖維細胞轉(zhuǎn)變中的作用,明確經(jīng)血干細胞是否通過逆轉(zhuǎn)TGFβ的促纖維化作用進而減輕子宮內(nèi)膜纖維化,并促進子宮內(nèi)膜修復(fù)。方法：本研究評估TGFβ對子宮內(nèi)膜間質(zhì)細胞增殖力、遷移力及纖維化相關(guān)因子αSMA、collagen Ⅰ、CTGF及fibronectin表達的影響。利用transwell共培養(yǎng)體系,共培養(yǎng)經(jīng)血干細胞與子宮內(nèi)膜間質(zhì)細胞,同時收集經(jīng)血干細胞條件培養(yǎng)基,培養(yǎng)子宮內(nèi)膜間質(zhì)細胞,評估經(jīng)血干細胞能否逆轉(zhuǎn)TGFβ的上述作用。CCK8試驗用來檢測子宮內(nèi)膜間質(zhì)細胞的增殖力改變；劃痕實驗檢測子宮內(nèi)膜間質(zhì)細胞的遷移能力變化；real-time PCR法用來檢測子宮內(nèi)膜間質(zhì)細胞中纖維化相關(guān)因子αSMA、 collagen Ⅰ、CTGF及fibronectin的]mRNA表達變化。Western blot及免疫熒光法檢測子宮內(nèi)膜間質(zhì)細胞中phos-TAZ的蛋白表達變化及TAZ的定位變化。結(jié)果：TGFβ顯著上調(diào)子宮內(nèi)膜間質(zhì)細胞中纖維化相關(guān)因子αSMA、collagen Ⅰ、CTGF及fibronectin的mRNA表達水平,抑制子宮內(nèi)膜間質(zhì)細胞的遷移,但對子宮內(nèi)膜間質(zhì)細胞的增殖力無影響。TGFβ可使phos-TAZ的蛋白表達水平下調(diào),并使TAZ滯留于細胞核內(nèi)。共培養(yǎng)經(jīng)血干細胞或經(jīng)血干細胞條件培養(yǎng)基可顯著逆轉(zhuǎn)TGFβ的作用,使TGFβ誘導(dǎo)的αSMA、collagen Ⅰ、 CTGF及fibronectin表達下調(diào),恢復(fù)子宮內(nèi)膜間質(zhì)細胞的遷移力,促進創(chuàng)傷修復(fù)。結(jié)論：TGFβ信號通路的激活可能介導(dǎo)了子宮內(nèi)膜創(chuàng)傷后的纖維化,促使宮腔粘連,經(jīng)血干細胞以旁分泌途徑激活hippo信號通路,抑制TGFβ的促纖維化作用。
[Abstract]:The first chapter of blood stem cells on the damaged endometrial repair effect of background and objective: endometrial implantation and development as the "soil", which plays an important role in helping pregnancy. Because of endometrial injury, adhesion and absence as a result of thin endometrium Kezu because the fertilized eggs in the implantation and development of endometrium endometrial mesenchymal stem cells are highly endometrial regeneration, a large number of studies have confirmed that the extraction from the female menstrual blood in the blood stem cells have mesenchymal stem cells proliferation and differentiation characteristics, can repair the damaged liver and improve liver function. However, blood stem cells repair damaged uterus endometrial ability has not been reported. Therefore, the purpose of this study is to clarify whether blood stem cells can repair damaged endometrial stromal cell proliferation and migration ability, reduce the damage to the uterus Membrane interstitial cell apoptosis. Methods: This study used mifepristone induced endometrial stromal cells, and the use of Transwell co culture system, co cultured with blood stem cells.CCK8 test used to detect endometrial stromal cell proliferation, AV/PI double staining flow cytometry to detect endometrial stromal cells apoptosis Western, blot method was used to detect endometrial stromal cells in VEGF expression. Results: Mifepristone on endometrial stromal cells showed the inhibition effect between time and concentration, the concentration at 48 hours after 25mg/1, to achieve the best effect. Mifepristone blood stem cells were cultured, the proliferation ability of endometrial stromal cells damage increased significantly, the apoptosis rate was significantly decreased. At the same time, Co cultured blood stem cells, damaged endometrial stromal cells VEGF protein expression level increased significantly. Conclusion The experimental injury of endometrial stromal cells in model of mifepristone on blood stem cells using co culture system, menstrual blood stem cells can prevent or reverse the secretory pathway in endometrial stromal cell damage through the side. In the second chapter, blood stem cells inhibit endometrial fibrosis effect of research background and objective: Endometrial basal layer of intrauterine adhesions in patients with broken, endometrial regeneration disorder, replaced by dense large amount of fibrous tissue, leading to uterine adhesions caused by oligomenorrhea, amenorrhea and infertility and other clinical symptoms. Although with the application of intrauterine drug treated stents and antibiotics, intrauterine adhesions after treatment, the rate of recurrence in the gradually reduced however, for moderate and severe adhesions, hysteroscopic adhesiolysis adhesion after recurrence rate remains high, it is a difficult problem for treatment of intrauterine adhesions. Stem cells Medical students have achieved considerable results of clinical trials, there are research and application of bone marrow stem cells for the treatment of intrauterine adhesions, menstrual improvement and make patients got pregnancy. Blood stem cells derived from endometrial tissue loss, and bone marrow mesenchymal stem cells, have the potential of multi-directional differentiation, has been shown to repair damaged myocardial cells and reduce the myocardial infarction fibrosis. However, for the blood stem cells in the treatment of intrauterine adhesions and the related mechanism has not yet been reported. This study by in vitro experiments to clear blood stem cells is not inhibited the process of intrauterine adhesions in patients with endometrial fibrosis, and hippo/TAZ signaling pathway is involved in this process methods: This study co culture system by Transwell, blood stem cells and endometrial stromal cell co culture medium. At the same time to collect blood stem cell conditions, Cultured endometrial stromal cells.CCK8 test is used to detect changes in endometrial stromal cell proliferation; interstitial cell migration changes of endometrial scratch assay. Real-time PCR and Western; blot method was used to detect endometrial stromal cells in fibrosis related factors between aSMA and protein expression changes of mRNA collagen 1; endomentrium the Western blot Hippo pathway between cell associated factor phos-LATS1 (1059), the expression changes of phos-MOB1 and phos-TAZ protein in TAZ cells; location change detection of endometrial immunofluorescence. Results: blood stem cell culture medium and mRNA protein or blood stem cell conditions were significantly lower aSMA and collagen I expression the level of co culture, promote endometrial stromal cell migration, upregulation of Hippo signaling pathway in phos-LATS1 (1059), phos-MOB1 and phos-TAZ of the table The level of blood. Stem cell conditioned medium significantly promote endometrial stromal cell proliferation, while the T rudder from endometrial stromal cell nucleus removed, retained in the cytoplasm. Conclusion: the study used Transwell coculture system or stem cell conditioned medium showed that blood stem cells may be through paracrine pathway inhibition of uterus endometrial stromal cells into myofibroblast transformation and promote stromal cell proliferation and migration, promote wound healing, Hippo/TAZ signaling pathway is one of the signal transduction pathway is blood stem cell activation. The third chapter of blood stem cells through inhibition of TGF beta to prevent endometrial fibrosis and promote endometrial repair background and objective: uterine cavity the adhesion has become the second largest cause of female infertility, it is reported that 13% of infertility patients have intrauterine adhesion, intrauterine adhesions in patients with history of infertility. The present study 43% Think of intrauterine adhesions in patients with residual endometrial tissue stem cells in number and function may be abnormal. Stem cells for regenerative medicine in the treatment of intrauterine adhesions is paid more and more attention. Our in vitro studies have found that blood stem cells can inhibit endometrial stromal cells into myofibroblast transformation, reduce the alpha -SMA and collagen I show that the level of blood stem cells could significantly reduce or prevent intrauterine adhesions. But the specific mechanism of blood stem cells still need further study of.TGF beta family is a multifunctional signaling pathway involved in various pathophysiological processes, studies have confirmed the presence of TGF beta is highly expressed in intrauterine adhesions in patients with endometrial tissues. With the increase of the degree of adhesion and increased expression. This study by in vitro experiments of TGF beta in promoting endometrial stromal cells myofibroblast transformation in the role of Ming Dynasty That blood stem cells by promoting fibrosis reversal of TGF beta and reduce endometrial fibrosis, and promote endometrial repair. Methods: This study evaluated the quality of the TGF beta cell proliferation in endometrial, migration and fibrosis related factor alpha SMA, collagen 1, the expressions of CTGF and fibronectin. The co culture system Transwell, blood stem cells and endometrial stromal cells were cultured, while collecting medium blood stem cells, cultured endometrial stromal cells, evaluation of blood stem cells can reverse TGF beta to test the effect of.CCK8 on endometrial stromal cell proliferation; endometrial stromal cell scratch assay. The migration of real-time; PCR method was used to detect endometrial stromal cells fibrosis related factors between alpha SMA, collagen I, the expression of CTGF and fibronectin]mRNA Location changes detection of endometrial.Western blot and immunofluorescence method between phos-TAZ cells in protein expression and TAZ. Results: TGF significantly upregulated beta endometrial stromal cells fibrosis related factors between alpha SMA, collagen 1, CTGF and fibronectin mRNA expression, inhibit endometrial stromal cell migration, but the endometrial stromal cell proliferation without affecting.TGF beta can make phos-TAZ protein expression, and TAZ retention in the nucleus. The co culture medium could significantly reverse the blood stem cells or blood stem cells TGF beta condition, alpha SMA, TGF induced collagen and fibronectin expression of CTGF. Cut, recovery of endometrial stromal cell migration between stress, promote wound healing. Conclusion: TGF beta signaling pathway may mediate the endometrial fibrosis after trauma, promote intrauterine adhesions, menstrual blood The stem cells activate the Hippo signaling pathway by paracrine pathway and inhibit the fibrotic effect of TGF beta.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R711.6

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