【作者】 赵霞；

【导师】 郭成山；

【作者基本信息】 山东大学 ， 内科学， 2008， 硕士

【摘要】 目的:体外观察骨髓间充质干细胞(Mesenchymal stem cells,MSCs)对特发性血小板减少性紫癜(Idiopathic thrombocytopenic purpura,ITP)患者CD4~+CD25~+T细胞比例以及T细胞分泌IL-2、IFN-γ、IL-4、IL-10功能的影响,并探讨上述两种调控作用之间的相关性,旨在探究MSCs调节ITP免疫平衡及诱导自身免疫耐受的机制,从而为ITP的临床治疗奠定初步的实验基础。研究方法:采用Ficoll分离骨髓单个核细胞,通过体外培养,扩增出MSCs,并通过形态学特征及流式细胞术检测其表面标志加以鉴定;通过Ficoll分离法和尼龙棉柱法获取正常人及ITP患者外周血T淋巴细胞,并应用流式细胞术检测T细胞中CD4~+CD25~+T细胞比例;MSCs经丝裂霉素MMC处理后按不同数量(2×10~3、1×10~4、5×10~4个细胞/孔)接种培养板作为基底层细胞,然后分别接种体外分离纯化的异体ITP及正常人T淋巴细胞,于2d、4d、6d后各自收集T淋巴细胞及培养上清,采用ELISA测定T细胞分泌的细胞因子IL-2、IFN-γ、IL-4、IL-10水平;并用流式细胞术检测接种于骨髓MSCs的ITP患者CD4~+CD25~+T细胞比例。结果:分离培养的骨髓MSCs贴壁生长呈梭形,原代细胞呈集落分布,10d左右达到融合,传代细胞保持原代细胞的形态且增殖速度加快,6～7d即可达到融合;经流式细胞仪检测骨髓MSCs高表达CD29、CD105、CD166,低表达CD34、CD45、HLA-DR,3代以后的细胞中MSCs纯度达到95%以上;尼龙棉柱法分离的T淋巴细胞纯度可达到70%～80%左右。流式细胞仪检测结果,ITP患者外周血CD4~+CD25~+T细胞数量及CD4~+CD25~+/CD4~+比值均明显低于正常对照组(P＜0.05);在PHA作用下,数量＞1×10~4的骨髓MSCs与T淋巴细胞共培养4d后,与正常对照组相比,MSCs可显著上调ITP患者及正常人T淋巴细胞中CD4~+CD25~+T淋巴细胞比例及CD4~+CD25~+/CD4~+比值(P＜0.05),且随MSCs量的增加,作用增强(P＜0.05)。本研究未检测到骨髓MSCs本身分泌IL-2、IFN-γ、IL-4及IL-10。在PHA作用下,ITP患者T淋巴细胞分泌细胞因子IL-2、IFN-γ较正常人高(P＜0.05),IL-4、IL-10较正常人低(P＜0.05)。骨髓MSCs与T淋巴细胞共培养后,与对照组相比,MSCs可显著抑制ITP患者或正常人T淋巴细胞分泌IL-2、IFN-γ(P＜0.05),且随MSCs数量增加,抑制作用增强(P＜0.05),共培养4d、6d其抑制作用明显强于2d(P＜0.05);MSCs可促进ITP患者T淋巴细胞分泌IL-4、IL-10(P＜0.05),且随MSCs数量增加,促进作用增强(P＜0.05);MSCs对IL-4的作用与培养时间无显著相关性(P＞0.05),但对IL-10促进作用随时间延长而增强(P＜0.05);当MSCs数量＞1×10~4可促进正常人T淋巴细胞分泌IL-4(P＜0.05),且随MSCs数量增加,促进作用增强(P＜0.05),共培养4d、6d作用明显强于2d(P＜0.05);MSCs可促进正常人T淋巴细胞分泌IL-10(P＜0.05),且随MSCs数量增加,促进作用增强(P＜0.05),共培养4d、6d其促进作用明显强于2d(P＜0.05)。体外骨髓MSCs对ITP患者T淋巴细胞分泌IL-2、IFN-γ、IL-4、IL-10具有调控作用,同时对CD4~+CD25~+T淋巴细胞具有上调作用,以上这种机制可使ITP患者的细胞因子及CD4~+CD25~+T淋巴细胞逐渐接近于正常人但仍达不到正常人水平(P＜0.05)。结论:①ITP患者存在T细胞分泌细胞因子的异常,表明ITP是一种Th1优势性的自身免疫性疾病,CD4~+CD25~+T细胞比例低于正常亦提示调节性T细胞在ITP发病中可能起重要作用。②MSCs在体外可调节ITP患者T淋巴细胞Th1/Th2反应平衡,并能上调CD4~+CD25~+调节性T细胞,进而诱导ITP患者免疫耐受形成,这一特性将为寻求免疫调节治疗新途径提供实验依据。更多还原

【Abstract】 Objectives:By analyzing the effect of human bone marrow-derived mesenchymal stem cells(MSCs)on ratio of CD4~+CD25~+ T cells and secreting interleukin-2 (IL-2),interferon-γ(IFN-γ),IL-4 and IL-10 from T lymphocytes of ITP,and furthermore the relationship between the two types of effect in vitro,we could study the mechanism of correcting immune balance and inducing autoimmune tolerance of ITP by MSCs,so as to offer preliminary experimental data for clinical treatment of ITP.Methods:Human bone marrow- derived MSCs were isolated by Ficoll Hypaque and cultured for proliferating to passage cells,and the purity of MSCs was identified with the spindle-fibroblastic morphology characterized by microphotograph and the phenotypes tested by flow cytometry,respectively.Allogeneic T lymphocytes of health adults and ITP patients were isolated from peripheral blood by Ficoll Hypaque and nylon cotton column,and their ratio of the CD4~+CD25~+ T cells was detected by flow cytometry.Then the stromal feeder layers of different amount(2×10~3,1×10~4, 5×10~4)per well of MSCs dealed with mitomycin were cocultured with above-mentioned T lymphocytes,and T lymphocytes and supernatant were collected on day 2,4 and 6 after cocultivation,respectively,then the level of IL-2,IFN-γ,IL-4, IL-10 secreted by T lymphocytes was measured by enzyme linked immunosorbent assay(ELISA),and the ratio of CD4~+CD25~+ T cells in every experimental group is detected by flow cytometry.Results:Mononuclear cells isolated from bone marrow adhered the bottom of the flask and were fusiform shape.The primary cell grew as many colonies,and reached confluence in about 10 days.The passage cells had the same morphology feature as the primary and grew quickly that reached confluence in 6 or 7 days.According to the flow cytometry assay,MSCs were uniformly positive for CD29,CD105 and CD166, while negative for CD34,CD45 and HLA-DR,and the purities of MSCs were above 95%from the third passage on;furthermore,the purities of the T lymphocytes isolated by nylon cotton column could reach 70%～80%.The result determined by flow cytometry assay as follows:the amount of CD4~+CD25~+ T cells and the ratio of CD4~+CD25~+/CD4~+ in the peripheral blood of ITP patients was obviously lower than normal control(P＜0.05,respectively).While the amount of MSCs exceeded 1×10~4 and cocultured with T lymphocytes with PHA-activating for 4 days,MSCs could obviously up-regulate the ratio of CD4~+CD25~+ T cells and significantly increase CD4~+CD25~+/CD4~+ in the peripheral blood of ITP patients and health adults compared to control(P＜0.05,respectively)in a dose dependent way(P＜0.05,respectively).IL-2,IFN-γ,IL-4 and IL-10 had not been secreted by MSCs themselves detected by ELISA.The level of IL-2 and IFN-γ,secreted by PHA-activating T cells from ITP patients was higher than normal control(P＜0.05,respectively),inversely,IL-4 and IL-10 was lower than normal control(P＜0.05,respectively).After cocultured with PHA-activating T lymphocytes,MSCs could significantly inhibited the cytokine level of IL-2 and IFN-γsecreted by T lymphocytes of ITP patients or health adults(P＜0.05, respectively)in a dose dependent way(P＜0.05,respectively),and the effect was higher when co-cultured for 4 days and 6 days than that for 2 days(P＜0.05, respectively);however,MSCs could significantly promoted IL-4 and IL-10 secreted by T lymphocytes of ITP patients(P＜0.05,respectively)in a dose dependent way (P＜0.05,respectively),while the effect on IL-4 had no obvious correlation with cocultivation-time(P＞0.05),but the effect on IL-10 in a time dependent way(P＜0.05). While their amount exceeded above 1×10~4,MSCs could obviously promoted IL-4 level secreted by T lymphocytes of health adults(P＜0.05)in a dose dependent way(P＜0.05),also they could obviously promoted IL-10 level secreted by T lymphocytes of health adults(P＜0.05)in a dose dependent way(P＜0.05).And both of the effects were higher when co-cultured for 4 days and 6 days than that for 2 days (P＜0.05,respectively). As for ITP patients,whose T lymphocytes and CD4~+CD25~+T cells regulated by MSCs,though the levels of IL-2,IFN-γ,IL-4 and IL-10,and even the the amount of CD4~+CD25~+ T cells gradually tended to normal level but not reached that yet in vitro (P＜0.05,respectively).Conclusions:①ITP is a kind of Th1-dominant autoimmune diseases having abnormal T cytokines profile,and the lower quantity of CD4~+CD25~+ T cells also suggests that CD4~+ CD25~+ T cells could play an important role in the course of ITP.②By regulating the balance between Th1 and Th2 reaction as well as up-regulating the expression of CD4~+CD25~+T cells to induce immunotolerance of ITP in vitro, MSCs could play a part in use of potential immunotherapy for ITP.更多还原