【作者】 严晓晔；

【导师】 李彤；

【作者基本信息】 天津医科大学 ， 外科学， 2010， 硕士

【摘要】 目的探讨缺氧及非缺氧脐带来源的间充质干细胞(UC-MSCs)是否能有效抑制脐血CD34+祖细胞免疫反应,影响其反应过程中免疫细胞因子的分泌。方法通过胶原酶及胰酶消化法从脐带中分离获得MSCs,观察其生长形态,取传代培养至第3代的UC-MSCs流式细胞术检测其表面标志；淋巴细胞分离液分离外周血单个核细胞(PBMC);免疫磁珠分离脐血CD34+祖细胞；将不同数量的第三代UC-MSCs5×103、2×104、8×104分别加入脐血CD34+祖细胞、外周血单个核细胞共培养体系中,不加UC-MSCs的CD34+祖细胞、PBMC共培养体系为阴性对照；另设用Transwel1悬挂式小室将UC-MSCs和脐血CD34+祖细胞、PBMC共培养体系分隔培养；培养96 h后,分别收集上清液,ELISA检测IFN-y和IL-10分泌水平。氯化钴(COCl2)进行化学模拟缺氧,在UC-MSCs中加入氯化钴浓度为0、50、100、150、200、250μmol/L的细胞培养液,分别于12 h、1d、2 d、3d、4d、5d、6 d通过MTT法检测各组的OD值,从而确定各组细胞的增殖情况；将氯化钻浓度为150μmol/L的培养液缺氧处理脐带MSCs48h后,加到脐血CD34+祖细胞、PBMC共培养体系中,培养96 h后,收集上清液,ELISA检测IFN-y和IL-10分泌水平。结果脐带UC-MSCs高表达CD 105、CD90、CD29、CD49,不表达CD40、CD80、CD86、HLA-DR、CD34、CD45;加UC-MSCs组IFN-γ的分泌量减少(与阴性对照组比,P<0.05),IL-10的分泌量增多(与阴性对照组比,P<0.05),而且具有数量依赖性,即数量越多免疫抑制作用越强；Transwell分隔培养组IFN-γ的分泌量减少(与阴性对照组相比,P<0.05),IL-10的分泌量增多(与阴性对照组相比,P<0.05)；但与接触培养组相比,IFN-y的分泌量增多,IL-10的分泌量减少。经氯化钴模拟缺氧处理的UC-MSCs(150μmol/L)其对数生长期提前,对数期缩短,增殖速度加快,第3天之后减慢,提前达平台期,4-5 d后呈逐渐降低趋势,而经较高浓度(200、250μmol/L)CoCl2处理的UC-MSCs生长曲线趋于低平。缺氧的脐带MSCs能抑制共培养体系中淋巴细胞IFN-y的分泌(与阴性对照组比,P<0.05),同时促进IL-10分泌(与阴性对照组比,P<0.05),但与非缺氧脐带MSCs组相比,IFN-y的分泌量增多,IL-10的分泌量减少。结论通过酶消化法可从脐带中成功分离获得MSCs;氯化钴(浓度≤150μmol/L)对UC-MSCs增殖影响微弱,可用于其模拟缺氧预处理；缺氧／非缺氧UC-MSCs都能抑制脐血CD34+祖细胞的免疫反应中IFN-γ的分泌,同时促进IL-10分泌,并且这种作用部分依赖于细胞间的相互接触；缺氧处理后的UC-MSCs免疫调节功能下调；UC-MSCs与脐血CD34+祖细胞联合移植可有效减轻同种异体干细胞移植后的免疫排斥反应,提高干细胞的生存率,为治疗缺血性心肌病提供有效的方法。更多还原

【Abstract】 Objective To investigate wheather the hypoxic/non-hypoxic umbilical cord-derived mesenchymal stem cells can inhibit the immune responses of umbilical cord blood (UCB)CD34+ progenitor cells, affecting the secretion of immunomodulattory cytokines.Methods UC-MSCs were isolated and cultured, the growth morphology were observed. Surface markers of UC-MSCs were detected by flow cytometry. The CD34 +progenitor cells in umbilical blood were isolated by immunomagnetic beads. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers using Ficoll-Histopaque density gradients from heparinized samples.The different number of UC-MSCs(5×103、2×104、8×104)were added to the co-culture system of UCB CD34+ progenitor cells and peripheral blood mononuclear cells(PBMC),and cultivation of CD34+ cells and PBMC as negative group, in addition, we mixed this three kinds of cells using Transwell,UBC CD34+ cells and PBMC were cultured in the upper separated from UC-MSCs, after culturing for 96 h, supernatants were collected and detected of the IFN-y and IL-10 level by ELISA. Third passage (P3) UC-MSCs were separately cultivated in DMEM/F12 with cobalt chloride 0、50、100、150、200、250μmol/L at 37℃and 5% CO2 for 12h、1d、2d、3d、4d、5d、6d, detected the proliferation and vitality of every groups by MTT assay. The hypoxic UC-MSCs which were cultivated with CoCl2150μmol/L for 48h were added to the co-culture system of UBC CD34+ cells and PBMC, culturing for 96 h, detected cytokines using above method.Results UC-MSCs did not express CD40, CD80, CD86, HLA-DR, CD34.It can inhibit IFN-y secretion (compare with controls,P<0.05), promote IL-10 secretion(compared with controls,P<0.05) and had a dose-depended. Using Transwell,UC-MSCs have this immunomodulation too,but the effects decreased. The platform stage of UC-MSCs was in advance and decurtated after culturing with proper concentration of CoCl2(≤150μmol/L), and the proliferation decreased after 3 days,while the higher concentration of CoCl2 (200、250μmol/L) had a negative impact on the vitality of UC-MSCs.The hypoxic UC-MSCs could inhibit the secretion of IFN-y(compared with controls, P<0.05), while enhanced the secretion of IL-10(compared with controls, P<0.05), however, compared with non-hypoxia group, the secretion of IFN-y increased, IL-10 decreased. Conclusions The concentration of CoCl2(≤150μmol/L) hardly affected the vitality of UC-MSCs, so it can served as a method of hypoxia.UC-MSCs can inhibited the secretion of IFN-γand promoted the secretion of IL-10 in the immune response of UCB CD34+ progenitor cells, and partly depended on the contacts between cells. Hypoxic UC-MSCs also can inhibit the immune responses of UCB CD34+ progenitor cells, however, the effects decrease compared with normoxia. Co-transplantation of UC-MSCs and UBC CD34+ could can serve as alternative of stem cells transplantation for ischemic cardiomyopathy.更多还原