脐血间充质干细胞混合培养及临床实验研究

Cinical trails and test of mixed cultivation of human umbilical cord-derived mesenchymal stem cells

目的分析比较脐带血间质干细胞单份与双份混合培养细胞增殖诱导分化为神经样细胞及临床输注效果观察。方法应用Ficoll-Hypaque淋巴细胞分离液在多联袋中以密度梯度法无菌获取脐血单个核细胞,并检测CD133+细胞数。将制备的单个核细胞接种于含有B27和bFGF的DMEM/F12培养瓶中,置于37℃体积分数为5%CO2饱和湿度的培养箱内培养。当细胞培养3d时,随机取培养的6份脐血间充质干细胞(2/3量)为A组,6份脐血间充质干细胞(2/3量)为B组,将A、B组各剩余的1/3量脐血间充质干细胞混合为C组。3组细胞密度均调整为1.0×104/ml。以活细胞计数法比较3组细胞不同时间的扩增数量,免疫组织化学检测脐血间充质干细胞巢蛋白的表达,流式细胞仪检测分离后及培养10d后CD133+细胞数。将单份培养和混合培养7d的脐带血间充质干细胞分别经静脉输注入脑卒中后遗症患者体内,每次输注2份,每例患者平均输注6份,每份间隔时间平均4d。于治疗前和治疗3个月后评价患者神经功能状态、肢体运动功能及日常生活活动能力。结果活细胞计数检测显示,将2份不同脐血间充质干细胞混合后,细胞贴壁、增殖比单份脐血间充质干细胞快(P<0.05)。3组细胞经bF-GF诱导后均表达巢蛋白,表达率差异无统计学意义(P>0.05),流式细胞仪检测CD133+细胞数,结果显示A、B2组差异无统计学意义(P>0.05),而混合脐血MSCsC组与单份脐血MSCsA、B组差异均有统计学意义(P<0.05)。2组患者治疗前与治疗3个月后相比,神经功能状态、肢体运动功能及日常生活活动能力评分均明显升高(P<0.01),但治疗后2组之间无明显差异(P>0.05),均未发现免疫排斥及不良反应。结论混合培养时脐血间充质干细胞之间有互相促增殖作用,应用bFGF诱导的脐血间充质干细胞可以表达巢蛋白。

Objective To analyze and compare between single and mixed cultivation of the nerve cells differentiated from human umbilical cord-derived mesenchymal stem cells （HUMSCs）, then to observe the clinical effect after infusion. Methods Mononuclear cells of HUMSCs were collected by density gradient centrifugation with Ficoll-Hypaque lymphocyte separation, and then underwent B27^＋bFGF differentiation. After 3 days of cultivation, 12 portions of MSCs were randomly chosen, and 2/3 amount of MNC were taken from each portion and were divided into groups A and B, and the remaining 1/3 amount in the 12 portions were divided into group C. The cell concentration of each group was adjusted to 1.0×10^4/ml. The amplification of each group at different time points was observed by living cell count, and nestin was detected by immunohistochemistry, and CD133^＋ cell count after 10 d cultivation was detected by flow cytometry. Patients with stroke were infused with MSCs（7d-single cultivation and 7d -mixed cultivation, respectively） for 3 times （ 2 portions each time, and infusion every 4 days）. The neural function deficiency（NFD）, Fugl-Meyer assessment（FMA）, and Barthe（BI） of patients were assessed before and 3 months after treatment.Results The proliferation and adherence of MSCs was significantly better in mixed cultivation （P〈0.05）. The nestin express in each group showed no statistical differences （P〈0.05）. The CD133^＋ cell count showed no statistical differences between group A and B, and statistical differences were found between single and mixed cultivation of MSCs. NFD, FMA, and BI of patients after treatment were significantly improved （P〈0.01）, while there were no statistical significance（P〉0.05） in NFD,FMA, and BI between the two groups. No immune rejection and adverse reaction were observed. Conclusion The proliferation of MSCs were improve in mixed cultivation. Nestin express was detected in bFGF-induced MSCs.