间充质干细胞诱导调节性T细胞治疗卵巢早衰的实验研究

发布时间：2018-02-15 05:58

本文关键词： 卵巢早衰自身免疫性卵巢疾病间充质干细胞调节性T细胞　出处：《南方医科大学》2017年博士论文　论文类型：学位论文

【摘要】：目的自身免疫性卵巢疾病是卵巢早衰(POF)的重要病因之一,调节性T细胞(Treg)及相关细胞因子的异常可能参与其发病。在动物模型上不同来源间充质干细胞(MSCs)均能修复受损的卵巢功能,但免疫调控机制是否参与其中,未曾报道。本研究首次从免疫调控的角度研究MSCs对免疫损伤的卵巢功能的修复作用,验证MSCs是否通过调控Treg细胞及相关细胞因子修复受损的卵巢功能,为阐明MSCs治疗POF的作用机制、指导其临床应用提供依据。方法第一章:检测不同卵巢功能女性外周血雌二醇(E2)、卵泡刺激素(FSH)水平、CD4+Foxp3+Treg细胞及干扰素-γ(IFN-γ)、转化生长因子-β1(TGF-β1)和白介素-10(IL-10)水平。第二章:以透明带3多肽免疫小鼠构建POF模型,通过阴道脱落细胞涂片、血清性激素、卵巢组织形态、抗透明带3抗体(AZP3Ab)及CD45鉴定建模成功,检测脾脏CD4+CD25+Foxp3+Treg细胞、卵巢Foxp3及外周血细胞因子。第三章:在细胞接触或Transwell条件下,小鼠BMSCs与抗CD3/CD28激活的POF小鼠脾细胞增殖模型共培养96h,加入TGF-β1中和抗体、前列腺素E2(PGE2)抑制剂,检测CD4+T细胞增殖、Treg细胞及上清细胞因子;分选POF小鼠脾脏CD4+CD25-T细胞,加入BMSCs共培养96h,检测CD4+CD25+T细胞及上清细胞因子。第四章:POF小鼠经尾静脉或卵巢移植BMSCs,体内示踪,检测卵巢内分泌功能(血清性激素、卵巢组织形态、抗苗勒管激素(AMH)蛋白);免疫功能(脾脏Treg细胞、卵巢Foxp3及细胞因子);生育能力(移植后4w合笼,孕18.5天观察胎鼠)。结果第一章:与健康育龄女性相比,POF患者血清E2下降、FSH上升,外周血CD4+Foxp3+Treg细胞减少,TGF-β1和IL-10表达下调,IFN-γ则上调。第二章:与对照组比较,模型组小鼠动情周期紊乱比例增加,血清E2下降、FSH上升,各级卵泡减少,卵巢AZP3Ab和CD45表达上调,Foxp3下调,脾脏CD4+CD25+Foxp3+Treg细胞减少,外周血TGF-β1和IL-10表达下调,IFN-y上调。第三章:在细胞接触条件下,高剂量BMSCs可抑制POF小鼠的CD4+T细胞增殖、诱导Treg细胞生成,促进TGF-β1和IL-10产生以及抑制IFN-γ分泌;在Transwell条件下,BMSCs与脾细胞增殖模型共培养所产生的免疫抑制效应比细胞接触条件更明显,且TGF-β1中和抗体和PGE2抑制剂可以阻断该效应。高剂量BMSCs可以诱导CD4+CD25-T细胞分化为CD4+CD25+T细胞。第四章:BMSCs主要迁移至POF小鼠卵巢间质,卵泡部位未见其分布;使POF小鼠的血清E2上升,FSH下降;增加各级卵泡及黄体数量,上调卵巢AMH的表达;改善小鼠的妊娠率和平均产鼠数;增加小鼠脾脏Treg细胞比例以及卵巢Foxp3、TGF-β1和IL-10的表达,抑制卵巢内IFN-y的产生。结论BMSCs可以通过诱导POF小鼠体内Treg细胞、TGF-β1和IL-10的生成以及抑制IFN-y分泌而发挥其免疫调控机制,恢复机体免疫耐受,从而改善免疫损伤的卵巢功能。
[Abstract]:Objective autoimmune ovarian disease is one of the important causes of premature ovarian failure (POF). The abnormalities of regulatory T cell Tregand related cytokines may be involved in the pathogenesis. In animal models, MSCs from different sources can repair the damaged ovarian function, but whether the immune regulation mechanism is involved, In this study, we first studied the effect of MSCs on the repair of immune damaged ovary function from the perspective of immune regulation, and verified whether MSCs can repair the damaged ovarian function by regulating Treg cells and related cytokines. In order to elucidate the mechanism of MSCs in the treatment of POF, Methods: in the first chapter, the levels of CD4 Foxp3 Treg cells and IFN- 纬, TGF- 尾 1 and IL-10 in peripheral blood of women with different ovarian functions were detected. The levels of estradiol E _ 2 and follicle stimulating hormone (FSH) in peripheral blood of female patients with different ovarian functions were determined, and the levels of IFN- 纬 -IFN- 纬, TGF- 尾 _ (1) and IL-10) were determined. Chapter: POF model was established by immunizing mice with pellucida 3 peptide. Vaginal exfoliative cell smear, serum sex hormone, ovarian tissue morphology, anti-pellucida 3 antibody AZP3Aband CD45 were used to identify the spleen CD4 CD25 Foxp3 Treg cells. Ovarian Foxp3 and peripheral blood cytokines. Chapter 3: in the presence of cell contact or Transwell, mouse BMSCs was co-cultured for 96 h with the spleen cell proliferation model of POF mice activated by anti-#en3#. TGF- 尾 1 neutralizing antibody, prostaglandin E2PGE2) inhibitor was added. The proliferation of CD4 T cells and their supernatant cytokines were detected, the spleen CD4 CD25-T cells of POF mice were isolated and co-cultured with BMSCs for 96 h, and the CD4 CD25 T cells and supernatant cytokines were detected. Ovarian endocrine function (serum sex hormone, ovarian histopathology, anti-mullerian hormone Treg) protein, immune function (spleen Treg cell, ovarian Foxp3 and cytokine), fertility (4 weeks after transplantation) were detected. Results: compared with healthy women of childbearing age, serum E2 decreased and FSH increased, while the expression of TGF- 尾 1 and IL-10 decreased in peripheral blood CD4 Foxp3 Treg cells. Chapter 2: compared with the control group, the expression of TGF- 尾 1 and IL-10 was down-regulated. In the model group, the proportion of estrous cycle disorder increased, serum E2 decreased and FSH increased, follicle decreased, AZP3Ab and CD45 expression in ovary increased and Foxp3 down-regulated, spleen CD4 CD25 Foxp3 Treg cells decreased. The expression of TGF- 尾 1 and IL-10 in peripheral blood was down-regulated and IFN-y was up-regulated. Chapter 3: under the condition of cell contact, high dose BMSCs could inhibit the proliferation of CD4 T cells, induce the production of Treg cells, promote the production of TGF- 尾 1 and IL-10 and inhibit the secretion of IFN- 纬 in POF mice. The immunosuppressive effect of co-culture of Transwell and spleen cells was more obvious than that of cell contact. TGF- 尾 1 neutralizing antibody and PGE2 inhibitor could block the effect. High dose BMSCs could induce CD4 CD25-T cells to differentiate into CD4 CD25 T cells. 4th% BMSCs mainly migrated to the ovary stroma of POF mice. Serum E2 of POF mice was increased and decreased, the number of follicles and corpus luteum was increased, the expression of ovarian AMH was up-regulated, the pregnancy rate and the average number of mice were improved, the percentage of Treg cells in spleen and the expression of Foxp3TGF- 尾 1 and IL-10 in ovary were increased. Conclusion BMSCs can induce the production of TGF- 尾 1 and IL-10 in Treg cells and inhibit the secretion of IFN-y in POF mice, which can restore the immune tolerance and improve the ovarian function of immune injury.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R711.75

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