目的：探讨人脐带间充质干细胞（ human umbilical cord mesenchymal stem cells， hUC-MSCs）对难治性免疫性血小板减少症（ refractory immune thrombocytopenia， rITP）患者B淋巴细胞分化及分泌功能的影响。方法酶消化法提取健康胎儿hUC-MSCs；密度梯度离心法分离2012年6月至2013年10月浙江省中医院26例rITP患者和6名健康供者的外周血单个核细胞；美洲商陆（ Pokeweed， PWM）刺激外周血B细胞分化；酶联免疫吸附测定法（ enzyme-linked immuno sorbent assay， ELISA）检测rITP患者和健康供者外周血单个核细胞和hUC-MSCs共培养时IgG、 IgM的分泌量； EnVision免疫组化法测B细胞诱导的成熟蛋白-1（B lymphocyte-induced maturation protein1， Blimp-1）表达。结果（1）流式细胞技术测健康胎儿脐带提取的hUC-MSCs表面CD44表达为99.8％。（2） PWM浓度为30 mg／L，作用4 d时rITP患者组IgG、 IgM分泌量分别为（7.39±1.42）和（5.44±0.17）μg／L，明显高于健康供者组（ P ＝0.014、0.039）。（3） hUC-MSCs和rITP患者外周血单个核细胞以2∶10共培养时IgG、 IgM分泌量分别为（4.98±1.63）μg／L和（3.78±0.82）μg／L，明显低于无hUC-MSCs时IgG、 IgM分泌量（P＝0.005、0.003），且和健康供者组差异无统计学意义（P＝0.266、0.543）；此比例下rITP患者组Blimp-1蛋白表达率为33.51％，较无hUC-MSCs时明显下降（P＝0.026），与健康供者组比差异无统计学意义（P＝0.076）。结论 hUC-MSCs和rITP患者外周血单个核细胞以2∶10比例共培养，可明显减少rITP患者IgM、 IgG的分泌量，抑制Blimp-1蛋白的表达，提示hUC-MSCs对rITP患者B细胞分化为浆细胞分泌抗体有一定的抑制作用，为临床hUC-MSCs治疗rITP提供一定的实验依据。%Objective To explore the impacts of hUC-MSCs on the secretion and immune function of B lymphocytes in refractory idiopathic thrombocytopenic purpura patients.Methods Enzyme digestion was used to extract healthy fetus umbilical cord mesenchymal stem cells; The 26 rITP patients who were in the Zhejiang Provincial Hospital of TCM from June 2012 to October 2013 and six healthy donors were included into this study, The peripheral blood mononuclear cells ( PBMCs) of them were extracted by density gradient centrifugation; Pokeweed ( PWM) was used to stimulate B lymphocyte transferring to plasmocytes for secretion of IgG and IgM.The concentration of IgG and IgM of the rITP patients were tested by enzyme-linked immuno sorbent assay ( ELISA) when their which PBMCs were co-cultured with hUC-MSCs, so did for healthy donors. The epression of Blimp-1 was measured by the EnVision immunohistochemistry.Results ( 1 ) The cells extracted from healthy fetus umbilical cord tested by flow cytometry labeled for CD44 was 99.8%. ( 2 ) The antibody IgG and IgM secreted from the rITP patients was ( 7.39 ±1.42 ) μg/L and ( 5.44 ±0.17 ) μg/L respectively, that was significantly increased than healthy doners, when the concentration of PWM was 30 mg/L and the cells were stimulated for 4 days (P=0.014, 0.039) .(3) When the hUC-MSCs and the PBMCs was at a 2∶10 ratio, the IgG and IgM concentration of rITP patients was (4.98 ±1.63) μg/L and (3.78 ±0.82) μg/L respectively, which was declined sharply than non-hUC-MSCs'( P =0.005, 0.003 ) , and not much difference with healthy donors ( P=0.848) .The rate of Blimp-1 protein of rITP patients was 33.51%, that apparently declined at the ratio 2∶10 of hUC-MSCs/peripheral blood mononuclear cells ( P =0.026), but no significant difference when compared to healthy donors (P=0.076).Conclusions The hUC-MSCs co-cultured with rITP patiens'peripheral blood mononuclear cells at 2∶10 ratio could decline IgG and IgM secretion and inhibit the expression of Blimp-1 protein.We suggest that hUC-MSCs affect on B lymphocytes of rITP patients and provides basis for the clinical application in rITP.However, this needs to be further studied before it is applied in clinical practice.
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