【作者】 董旻岳；

【导师】 黄荷凤；

【作者基本信息】 浙江大学 ， 妇产科学， 2008， 博士

【摘要】 研究目的:观察人胎盘滋养细胞对T淋巴细胞增殖与细胞因子产生的影响,探讨滋养细胞对淋巴细胞辅助T细胞细胞因子(Th)1/Th2平衡的影响,探讨滋养细胞源性影响T细胞的效应分子。材料与方法:分离早孕胎盘细胞滋养细胞,制备滋养细胞条件培养基,供淋巴细胞培养用。分离、纯化健康未孕妇女T淋巴细胞。以植物血凝素刺激T淋巴细胞,~3H—胸腺嘧啶掺入法测定T淋巴细胞的增殖反应,酶联免疫吸附分析法测定淋巴细胞产生的细胞因子,放射免疫分析法测定培养基雌二醇、孕酮、人绒毛膜促性腺激素和人胎盘生乳素浓度。高效液相分析测定培养基中色氨酸浓度。建立早孕绒毛组织培养,制备绒毛组织条件培养基,经浓缩、电泳、切胶、胶内酶解和脱盐等处理,鸟枪策略串联质谱分析蛋白质谱。质谱测序数据查人蛋白质数据库,查库结果进行GOA分类,并查PUBMED医学文献库、复习文献。结果:(1)、滋养细胞条件培养基中检测到人绒毛膜促性腺激素、人胎盘生乳素、雌二醇和孕酮,而对照培养基中未检测到4种激素。对照培养基的色氨酸浓度为19.01±1.04μmol/L,条件培养基为3.79±0.45μmol/L,当制备条件培养基时添加特异性吲哚胺2,3双氧化酶(IDO)抑制物1-甲基色氨酸,条件培养基中色氨酸浓度为15.40±2.66μmol/L。(2)、滋养细胞条件培养基显著抑制T淋巴细胞~3H—胸腺嘧啶掺入率,随着淋巴细胞培养液中条件培养基含量的增加,淋巴细胞~3H—胸腺嘧啶掺入率逐步降低;制备条件培养基时添加特异性抑制剂1-甲基色氨酸抑制IDO活性或者向条件培养基添加色氨酸至生理浓度均显著减弱条件培养基对T细胞增殖的抑制作用,淋巴细胞~3H—胸腺嘧啶的掺入率得到部分恢复。(3)、滋养细胞条件培养基显著减少淋巴细胞产生肿瘤坏死因子(TNF)α,并显著增加淋巴细胞产生白介素(IL)-10。辅助T细胞1(Th1)/Th2比值即TNF-α/IL-10比值显著降低。(4)、根据质谱数据检索人类蛋白质数据库得到1869个肽段,对应548个蛋白质。经GOA分类分析,在总共548个蛋白质中,细胞内/膜蛋白质285个,占52%:细胞外蛋白质72个,占13%;细胞定位不明的蛋白质127个,占23%;其它蛋白质64个,占12%。(5)、在总共72个细胞外蛋白质中,血浆蛋白质31个,占43%;细胞外基质蛋白21个,占29%;滋养细胞源性蛋白质7个,占10%;来源不明的蛋白质13个,占18%。(6)、10种蛋白质对T淋巴细胞具有抑制作用:人绒毛膜促性腺激素、人胎盘生乳素、妊娠特异性β1糖蛋白、甲胎蛋白、glycodelin、转化生长因子β2、thrombospondin-1、色素上皮衍生因子、巨噬细胞迁移抑制因子和galectin-1。结论:滋养细胞产物抑制T淋巴细胞增殖,增加T淋巴细胞IL-10产生,减少TNF-α产生,降低Th1/Th2比值。IDO介导的选择性快速降解色氨酸是滋养细胞抑制T淋巴细胞增殖的机制之一。滋养细胞条件培养基是多种免疫调节物的混合体,其免疫抑制作用是这些免疫调节物协同作用的结果。更多还原

【Abstract】 Objectives:To observe the effects of placental trophoblasts on T lymphocyte proliferation and cytokine production and cytokine balance after affected by trophoblast and to explore the possible trophoblast-derived effector molecules.Materials and Methods:Placental trophoblasts were isolated from chorionic villi of normal pregnancy and conditioned medium was made for the culture of T lymphocyte. Peripheral blood mononuclear cells(PBMC)were isolated from healthy female donors and T lymphocytes were purified.Thymidine incorporation test was used to detect the T cell proliferation after the stimulation of phytohemagglutinin(PHA). Radioimmunoassay(RIA)was used to detect estradiol(E2),progesterone(P),human chorionic gonadotropin(hCG)and human chorionic somatomammotropin(hCS)and enzyme-linked sorbent immune assay(ELISA)was used to detect cytokines,TNF-αand IL-10.High performance liquid chromatography(HPLC)was used to measure the concentration of trypotphan in medium.Explant culture of chorionic villi was established to make villi-conditioned medium that was used for proteomic analysis. The protein in the conditioned medium was concentrated,separated with electrophoresis and trypsinized in gel.Shotgun-strategy liquid chromatography-mass spectrometry/mass spectrometry(LC MS/MS)was used to analyze the trypsinized peptide.The raw data of LC MS/MS were searched against human protein database. The identified proteins were grouped according to GOA analysis and searched against the medical literature at Pubmed for their effects on T lymphocyte.Results:(1).hCG,hCS,E2 and P were detected in the conditioned medium,but these hormones were not detected in the un-conditioned medium.The concentration of tryptophan was 19.01±1.04μmol/L in unconditioned medium,3.79±0.45 mol/L in conditioned medium and 15.40±2.66μmol/L in medium when IDO was inhibited by 1-methyltryptophan during previous conditioning.IDO mRNA was expressed in chorionic villi,term placenta and isolated trophoblasts.(2).The proliferation of T lymphocyte was significantly inhibited by the conditioned medium in a dose-dependent manner.The inhibitory effect of conditioned medium on T cell proliferation was partly reversed by either the inhibition of IDO during previous conditioning or supplementation of tryptophan to the conditioned medium.(3).The production of TNF-αwas significantly decreased,while the production of IL-10 was increased,and thus the ratio of TNF-α/IL-10 was decreased by conditioned medium. (4).1869 peptides,corresponding to 548 proteins,were identified in villi-conditioned medium.Among these proteins,as the result of GOA anlysis,285 proteins(52%) were intracellular/membrane proteins,72(13%)extracellular,127(23%)cellular component unknown and 64(12%)miscellaneous.(5).Among the extracellular proteins,31(43%)were plasma proteins,21(29%)extracellular matrix proteins,7 (10%)trophoblast-derived and 13(18)origin-unknown.(6).Ten proteins were found to inhibit T lymphocyte.They were human chorionic gonadotropin(hCG),human chorionic somatomammotropin(hCS),pregnancy-specificβ1-glycoprotein(SP1), α-fetal protein(AFP),glycodelin,transforming growth factor-β2(TGF-β2), thrombospondin-1(TSP-1),pigment epithelium-derived factor(PEDF),macrophage migration inhibitory factor,(MIF)and galectin-1.Conclusions:The product of placental trophoblast inhibited T lymphocyte proliferation,increased the production of IL-10 but decreased TNF-αand subsequently decreased Th1/Th2 ratio.Placental trophoblasts express functional IDO-mediated selective and rapid degradation of tryptophan is one of the mechanisms by which trophoblasts inhibit T cell proliferation.The trophoblast-conditioned medium represented a mixture of several immuneregulatory substances and the inhibitory effect on T cells was the result of synergistic action.更多还原