经血干细胞以旁分泌途径抑制受损子宫内膜纤维化并促进修复的作用机制研究

发布时间：2018-01-18 10:14

本文关键词：经血干细胞以旁分泌途径抑制受损子宫内膜纤维化并促进修复的作用机制研究　出处：《浙江大学》2016年博士论文　论文类型：学位论文

【摘要】：第1章经血干细胞对受损子宫内膜的修复作用研究背景及目的：子宫内膜作为胚胎种植和发育的“土壤”,其在助孕中有举足轻重的作用。因子宫内膜损伤,粘连和缺如等导致的子宫内膜过薄可阻碍受精卵在子宫内膜着床和发育。子宫内膜间充质干细胞是子宫内膜具有高度再生能力的原因,已有大量研究证实从女性月经血中提取的经血干细胞具有间充质干细胞增殖及多向分化特性,可修复受损肝脏,改善肝脏功能。然而,经血干细胞是否具有修复受损子宫内膜能力尚未见报道。因此,本研究旨在明确经血干细胞是否能修复受损子宫内膜间质细胞的增殖及迁移能力,减轻受损子宫内膜间质细胞的凋亡。方法：本研究用米非司酮药物性损伤子宫内膜间质细胞,并利用transwell共培养体系,与经血干细胞共培养。CCK8试验用来检测子宫内膜间质细胞的增殖力改变,AV/PI双染流式用来检测子宫内膜间质细胞的凋亡变化,Western blot法用来检测子宫内膜间质细胞中VEGF的蛋白表达变化。结果：米非司酮对子宫内膜间质细胞的抑制作用呈时间及浓度依赖性,在浓度25mg/1作用48小时后,米非司酮达到最佳抑制效果。共培养经血干细胞,受损子宫内膜间质细胞的增殖能力显著增加,凋亡率显著下降。同时,共培养经血干细胞后,受损子宫内膜间质细胞的VEGF蛋白表达水平显著增加。结论：本实验在米非司酮损伤子宫内膜间质细胞的模型上,利用经血干细胞共培养体系,证实经血干细胞可通过旁分泌途径阻止或者逆转子宫内膜间质细胞的损伤。第2章经血干细胞抑制子宫内膜纤维化的作用研究研究背景及目的：宫腔粘连患者的子宫内膜基底层被大量破坏,子宫内膜再生障碍,取而代之为大量的致密纤维组织,导致子宫壁粘连,引起月经量减少、闭经及不孕等临床症状。尽管随着宫腔内药物治疗支架及抗生素的应用,宫腔粘连治疗后复发的几率在逐渐减少,然而,对于中重度粘连,宫腔镜下粘连分解术后粘连再发率仍居高不下,也是宫腔粘连治疗的一大难题。干细胞再生医学已取得可观临床试验结果,也确实有研究应用骨髓干细胞治疗宫腔粘连后,使患者月经改善及获得妊娠。经血干细胞来源于脱落的子宫内膜组织,与骨髓间充质干细胞类似,具有多向分化潜能,已被证实可修复受损心肌细胞及减轻心肌梗死后心肌细胞的纤维化形成。然而,对于经血干细胞在宫腔粘连中的治疗作用及相关机制尚未被报道。本研究通过体外实验旨在明确经血干细胞是否能抑制宫腔粘连患者子宫内膜纤维化过程,而hippo/TAZ信号通路是否参与了这个过程。方法：本研究利用transwell共培养体系,共培养经血干细胞与子宫内膜间质细胞。同时收集经血干细胞条件培养基,培养子宫内膜间质细胞。CCK8试验用来检测子宫内膜间质细胞的增殖力改变；划痕实验检测子宫内膜间质细胞迁移能力的变化；Real-time PCR及western blot法用来检测子宫内膜间质细胞中纤维化相关因子aSMA、collagen Ⅰ的mRNA与蛋白表达变化；Western blot法检测子宫内膜间质细胞中hippo信号通路相关因子phos-LATS1(1059), phos-MOB1及phos-TAZ的蛋白表达变化；免疫荧光法检测子宫内膜间质细胞中TAZ的定位变化。结果：共培养经血干细胞或经血干细胞条件培养基均显著下调aSMA与collagen Ⅰ的mRNA及蛋白表达水平,促进子宫内膜间质细胞迁移,上调hippo信号通路中的phos-LATS1(1059), phos-MOB1及phos-TAZ的表达水平。经血干细胞条件培养基显著促进子宫内膜间质细胞增殖,同时使T舵从子宫内膜间质细胞核内移出,滞留于胞浆。结论：研究中采用transwell共培养体系或干细胞条件培养基,表明经血干细胞可能是通过旁分泌途径抑制子宫内膜间质细胞向肌成纤维细胞转化,并促进间质细胞增殖及迁移,促进创伤愈合,Hippo/TAZ信号通路是被经血干细胞激活的信号通路之一。第3章经血干细胞通过抑制TGFβ作用阻止子宫内膜纤维化并促进子宫内膜修复研究背景及目的：宫腔粘连已成为女性继发不孕的第二大病因,据报道13%的不孕症患者存在宫腔粘连,43%的宫腔粘连患者合并不孕病史。目前研究认为宫腔粘连患者中残存子宫内膜组织中的干细胞在数量及功能上可能存在异常。干细胞再生医学在宫腔粘连治疗越来越受重视。我们体外研究已经发现经血干细胞可抑制子宫内膜间质细胞向肌成纤维细胞转变,降低α-SMA及collagen Ⅰ水平,表明经血干细胞可能具有减轻或阻止宫腔粘连作用。但是经血干细胞的具体作用机制仍需进一步研究。TGFβ家族是一条多功能信号通路,参与多种病理生理过程,已有研究证实在宫腔粘连患者子宫内膜组织中存在TGFβ高度表达,且随着粘连程度的增加表达增高。本研究利用体外实验验证TGFβ在促进子宫内膜间质细胞肌成纤维细胞转变中的作用,明确经血干细胞是否通过逆转TGFβ的促纤维化作用进而减轻子宫内膜纤维化,并促进子宫内膜修复。方法：本研究评估TGFβ对子宫内膜间质细胞增殖力、迁移力及纤维化相关因子αSMA、collagen Ⅰ、CTGF及fibronectin表达的影响。利用transwell共培养体系,共培养经血干细胞与子宫内膜间质细胞,同时收集经血干细胞条件培养基,培养子宫内膜间质细胞,评估经血干细胞能否逆转TGFβ的上述作用。CCK8试验用来检测子宫内膜间质细胞的增殖力改变；划痕实验检测子宫内膜间质细胞的迁移能力变化；real-time PCR法用来检测子宫内膜间质细胞中纤维化相关因子αSMA、 collagen Ⅰ、CTGF及fibronectin的]mRNA表达变化。Western blot及免疫荧光法检测子宫内膜间质细胞中phos-TAZ的蛋白表达变化及TAZ的定位变化。结果：TGFβ显著上调子宫内膜间质细胞中纤维化相关因子αSMA、collagen Ⅰ、CTGF及fibronectin的mRNA表达水平,抑制子宫内膜间质细胞的迁移,但对子宫内膜间质细胞的增殖力无影响。TGFβ可使phos-TAZ的蛋白表达水平下调,并使TAZ滞留于细胞核内。共培养经血干细胞或经血干细胞条件培养基可显著逆转TGFβ的作用,使TGFβ诱导的αSMA、collagen Ⅰ、 CTGF及fibronectin表达下调,恢复子宫内膜间质细胞的迁移力,促进创伤修复。结论：TGFβ信号通路的激活可能介导了子宫内膜创伤后的纤维化,促使宫腔粘连,经血干细胞以旁分泌途径激活hippo信号通路,抑制TGFβ的促纤维化作用。
[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.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R711.6

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