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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 compare the proliferation and differentiation of umbilical cord blood mesenchymal stem cells (MSCs) cultured in single and double cultures to neuron-like cells and observe the effect of clinical infusion. Methods Umbilical cord blood mononuclear cells were obtained by Ficoll-Hypaque lymphocyte isolation and density gradient centrifugation in multi-bag, and the number of CD133 + cells was detected. The prepared mononuclear cells were inoculated into DMEM / F12 flasks containing B27 and bFGF and cultured in an incubator with a volume fraction of 5% CO2 saturated humidity at 37 ° C. When cells were cultured for 3 days, 6 parts of umbilical cord blood mesenchymal stem cells (2/3) were randomly divided into group A and 6 parts of cord blood mesenchymal stem cells (2/3) The remaining one third of the cord blood mesenchymal stem cells mixed group C. 3 groups of cells were adjusted to 1.0 × 104 / ml. The numbers of proliferating cells in three groups were compared by the method of viable cell count. The expression of nestin in umbilical cord blood mesenchymal stem cells was detected by immunohistochemistry. The numbers of CD133 + cells were detected by flow cytometry after 10 days of culture. Umbilical cord blood mesenchymal stem cells (MSCs) in single culture and mixed culture for 7 days were infused intravenously into patients with stroke sequelae, each with 2 infusions. Each patient received an average of 6 infusions with an average interval of 4 days. Before treatment and after 3 months of treatment evaluation of neurological status, limb motor function and activities of daily living. Results The results of viable cell count showed that after mixed with 2 different umbilical cord blood mesenchymal stem cells, the cells adherent and proliferated faster than single cord blood mesenchymal stem cells (P <0.05). Nestin was expressed in all three groups after induced by bF-GF, the expression rate was not significantly different (P> 0.05). The number of CD133 + cells was detected by flow cytometry. The results showed that there was no significant difference between A and B2 groups (P> 0.05). However, there were significant differences between MSCsCs mixed with umbilical cord blood and single cord blood MSCs A and B (P <0.05). Compared with 3 months after treatment, the scores of neurological function, limb motor function and activities of daily living in two groups were significantly increased (P <0.01), but there was no significant difference between the two groups after treatment (P> 0.05), no immune rejection and adverse reactions were found. Conclusion The mixed culture of umbilical cord blood mesenchymal stem cells promote mutual proliferation, bFGF induced the expression of nestin in umbilical cord blood derived mesenchymal stem cells.