人脐带间充质干细胞分离、鉴定方法及其治疗难治性免疫性血小板减少症的有效性及安全性研究

Separation and Identification Methods of Human Umbilical Cord Mesenchymal Stem Cells and Its Effectiveness and Safety in Treating Refractory Immune Thrombocytopenia

目的探讨人脐带间充质干细胞(hUC-MSCs)分离、鉴定方法及其治疗难治性免疫性血小板减少症(ITP)的有效性及安全性。方法选取2016年12月—2017年7月西安高新医院收治的难治性ITP患者6例。脐带取自足月剖宫产健康产妇,采用酶消化法分离、培养hUC-MSCs,BD FACSCalibur流式细胞仪检测hUC-MSCs免疫表型,成骨、成脂诱导分化实验评估hUC-MSCs分化能力。6例患者均静脉输注hUC-MSCs(1×10~6/kg),输注后1个月如患者血小板计数≤50×10~9/L可重复输注1次。所有患者至少随访3个月,随访截至2017-11-01,观察患者临床疗效、复发情况及治疗期间不良反应发生情况,并采用流式细胞术检测外周血淋巴细胞亚群表达情况。结果采用酶消化法分离、培养hUC-MSCs,可见原代细胞贴壁生长并逐渐融合成片状,形态均一,倒置相差显微镜下观察细胞类似于成纤维细胞,成长梭形流水状贴壁生长,细胞形态良好,并随着细胞传代,细胞形态未发生变化。流式细胞术检测结果显示,hUC-MSCs高表达CD_(73)、CD_(90)、CD_(105),表达量均>98.0%;不表达或低表达CD_3、CD_(11b)、CD_(19)。成骨、成脂诱导分化实验结果显示,hUC-MSCs能向成骨细胞、脂肪细胞分化。输注hUC-MSCs后,6例患者完全反应3例,有效2例,无效1例,总有效率为5/6;随访期间复发1例,再次输注hUC-MSCs后仍有效。hUC-MSCs输注前后患者外周血CD_3^+CD_4^+T淋巴细胞比例、CD_3^+CD_8^+T淋巴细胞比例及CD^+_(19)B淋巴细胞比例比较,差异无统计学意义(P>0.05);hUC-MSCs输注后CD^+_(16)CD^+_(56)自然杀伤细胞(NK细胞)比例和CD_4^+CD^+_(25)CD^-_(127)辅助性T细胞(Treg细胞)比例均高于hUC-MSCs输注前(P<0.05)。本组患者治疗期间2例出现轻度发热,对症处理后完全缓解;1例患者出现激素依赖,经抗病毒治疗后痊愈。结论采用酶消化法能有效分离、培养hUC-MSCs,流式细胞术及成骨、成脂诱导分化实验能有效鉴定hUC-MSCs分化能力,且hUC-MSCs治疗难治性ITP的近期疗效较好,不良反应轻微,可能成为难治性ITP的新治疗方法。

Objective To investigate the separation and identification methods of human umbilical cord mesenchymal stem cells（hUC-MSCs）and its effectiveness and safety in treating refractory immune thrombocytopenia（ITP）.MethodsA total of 6 patients with refractory ITP were selected in Xi＇an Gaoxin Hospital from December 2016 to July 2017.Umbilical cord was collected from mature healthy parturients underwent caesarean section,enzymatic digestion method was used to separate and culture hUC-MSCs,BD FACSCalibur Flow Cytometer was used to detect the immunophenotyping of hUC-MSCs,and the differentiation ability of hUC-MSCs was evaluated by osteosynthesis and adipogenesis induced differentiation experiment.All of the6 patients received intravenous infusion of hUC-MSCs（1×10^6/kg）,thereinto patient with PLT equal or less than 50×10^9/L 1month after intravenous infusion of hUC-MSCs received repeat intravenous infusion of hUC-MSCs.All of the 6 patients were followed up for 3 months at least,the expiration date was 2017-11-01;clinical effect,relapse and incidence of adverse reactions during the treatment were observed,and flow cytometry was used to detect the expression of peripheral blood lymphocyte subsets.Results Enzymatic digestion results showed primarily cultured cells growth adheres to the wall and schistose conjugation little by little,with homogeneous form;inverted phase contrast microscope showed fibroblast-like cells with long fusiform and flowing water shape growth adheres to the wall,and the cellular morphology was good,meanwhile as the cells propagation,the cellular morphology did not occurred obvious change.Flow cytometry detection results showed that,expressions of CD73,CD90and CD105were significantly high in hUC-MSCs（all over 98.0%）,while expressions of CD3,CD11b and CD19were significantly low or negative.Osteosynthesis and adipogenesis induced differentiation experiment showed that,hUC-MSCs can differentiate into osteoblast and adipocyte.After intravenous infusion of hUC-MSCs,3 cases got complete response,2 cases were effective,1 case was invalid,the total effective rate was 5/6;1 case occurred relapse during the follow-up,but repeat intravenous infusion of hUC-MSCs was still effective.After intravenous infusion of hUC-MSCs,peripheral blood CD3^＋CD4^＋T-lymphocyte ratio,CD3^＋CD8^＋T-lymphocyte ratio or CD19^＋B-lymphocyte ratio was not statistically significantly different with that before intravenous infusion of hUC-MSCs,respectively（P〉0.05）,while CD16^＋CD56^＋natural killer cells ratio and CD4^＋CD25^＋CD127^-T helper cells ratio were statistically significantly higher than those before intravenous infusion of hUC-MSCs（P〈0.05）.During the treatment,2 cases occurred mild fever and completely relieved after symptomatic treatment;1 case occurred steroid dependence and cured after anti-virus therapy.ConclusionEnzymatic digestion method is effective to separate and culture hUC-MSCs;flow cytometry,osteosynthesis and adipogenesis induced differentiation experiment can effectively identify the differentiation ability of hUC-MSCs;hUC-MSCs has good recent clinical effect in treating refractory ITP with mild adverse reactions,may be a new therapeutic method for refractory ITP.