摘要
【目的】研究观察间充质干细胞(MSC)在体外培养体系对B细胞增殖和免疫调节作用的影响。【方法】采用密度梯度离心法分离健康捐献者来源的骨髓30 mL。用含100 mmol/L胎牛血清的L-DMEM培养基对分离出的MSC进行贴壁培养。培养至第6代时,进行多向分化和细胞表型鉴定。获取健康供者外周血单个核细胞,并通过流式细胞仪分选出CD19^+B细胞,进行荧光染料CFSE标记,然后分成单独培养组(CD19^+B细胞)和共培养组(按CD19^+B∶MSC:=5∶1的比例共培养),两组均采用CpG+CD40L刺激,96 h后检测B细胞的增殖情况,并通过流式细胞荧光分选技术(FACS)检测CD19^+CD5^+B细胞的百分比及其分泌的IL-10水平;进一步观察单独培养组和共培养组的CD19^+B细胞对CD4^+T细胞增殖以及分泌IFN-γ的影响,最后加入IL-10中和抗体,再次验证B细胞对CD4^+T细胞增殖以及分泌IFN-γ的影响。【结果】健康捐献者来源的MSC具有多向分化能力,并表达CD29、CD44、CD73、CD90、CD105、CD166,不表达CD45和CD34。与单独培养组比较,共培养组中的CD19^+B细胞与MSC在体外共培养3 d后,CD19^+B细胞增殖比例明显提高,并显著抑制CD4^+T细胞分泌IFN-γ的功能。进一步分析CD19^+B细胞的亚群,发现与单独培养组比较,共培养组中的CD5^+B细胞数量的百分比由(21.31±1.22)%增加至(31.27±0.92)%(P=0.014);而且其IL-10的分泌水平由(1.09±0.08)%增加至(2.44±0.06)%(P<0.001)。当在共培养组中加入IL-10中和抗体后,发现与MSC共培养的B细胞抑制CD4^+T细胞增殖和分泌IFN-γ的功能的能力明显减弱。【结论】MSC能促进CD19^+B细胞的增殖,同时具有增强CD19^+B细胞抑制CD4^+T细胞分泌IFN-γ,这可能与分泌IL-10的CD19^+CD5^+B细胞的表达增加相关。
【Objective】To study the effect of mesenchymal stem cells(MSC)on proliferation and immunomodulation of B cells in vitro.【Methods】Bone marrow 30 mL from healthy donors was isolated by density gradient centrifugation.Isolated MSC were cultivated adherently with L-DMEM medium containing 10%fetal bovine serum(FBS).Phenotype and differentiation capacity of MSC was detected after the sixth passage.Peripheral blood mononuclear cells(PBMC)of healthy donors were also obtained by density gradient centrifugation.CD19^+B cells were sorted by fluorescence activatedcell sorting(FACS).CD19^+B cells labeled with CFSE were divide into two groups:CD19^+B cultured individually group and co-cultured with MSC(CD19^+B∶MSC=5∶1)group.Stimulated by CpG+CD40L,the proliferation of B cells of two groups were checked 96 hours after co-culture respectively.The CD19^+CD5^+B cells percentage and its secretion of IL-10were detected by FACS.The effects of CD19^+B cells on the proliferation of CD4^+T cells and the secretion of IFN-γwere continually observed.Anti-IL-10 was used to confirm the effect of B cells on proliferation and IFN-γsecretion of CD4^+Tcells.【Results】MSC collected from healthy donors remained differentiation capacity.MSC derived from bone marrow expressed CD29,CD44,CD73,CD90,CD105,and CD166,but did not express CD45 and CD34.Compared with control group,the proportion of CD19^+B cells proliferation in co-cultured group was significantly increased after 3 days,meanwhile,the level of IFN-γ,which secreted by CD4^+T cells was significantly restrained.To make a further analysis of the subsets of CD19^+B cells,the percentage of CD5^+B cells in co-cultured group increased from(21.31±1.22)%to(31.27±0.92)%(P=0.014),and its IL-10 secretion increased from(1.09±0.08)%to(2.44±0.06)%(P<0.001)compared with control group.After anti-IL-10 was used,the B cells co-cultured with MSC showed a decreased capacity of inhibiting the proliferation and IFN-γsecretion of CD4^+T cells.【Conclusions】MSC could promote the proliferation of CD19^+B cells to inhibit the secretion of IFN-γby CD4^+T cells,which may be related to the increasing expression of IL-10 by CD19^+CD5^+B cells.
作者
刘秋莉
肖崇珺
黄牡丹
黄里
陈小湧
郑海清
LIU Qiu-li;XIAO Chong-jun;HUANG Mu-dan;HUANG Li;CHEN Xiao-yong;ZHENG Hai-qing(Biotherapy Center,The Third Affiliated Hospital of Sun Yat-sen University,Guangzhou 510630,China;Department of Rehabilitation,The Third Affiliated Hospital of Sun Yat-sen University,Guangzhou 510700,China;Center for Stem Cell Biology and Tissue Engineering,Key Laboratory for Stem Cells and Tissue Engineering,Ministry of Education,Sun Yat-Sen University,Guangzhou 510080,China)
出处
《中山大学学报（医学版）》
CAS
CSCD
北大核心
2019年第2期195-203,共9页
Journal of Sun Yat-Sen University:Medical Sciences
基金
国家自然科学基金(81572228
81760112)
广东省自然科学基金(2015A030313015
2016A030310131)
中山大学校高校青年教师培育项目(15ykpy26
17ykpy50)
关键词
间充质干细胞
B淋巴细胞
免疫调节
mesenchymal stem cells
B lymphocytes
immunomodulation
