课题背景：目前课题组在间充质干细胞的分离、培养及定向诱导分化为神经干细胞等方面已取得一定成果，“无菌塑料袋分离单个核细胞的研究”（专利号200610033008.3）获河南省医药科技成果奖，现已具备完整的干细胞基础研究体系。

应用要点：①建立了应用四联袋结合无菌接口技术分离脐血干细胞的方法，效果满意，优于试管法。②脐血中加入适量羟乙基淀粉，改良了密度梯度离心法，使混入单个核细胞的红细胞明显减少。③脐血间质干细胞在没有传代培养情况下加入适当细胞因子进行诱导，仍然可得到神经元样细胞，缩短了脐血间质干细胞向神经元样细胞诱导分化的时间，节约了成本，减少细胞培养费用。

偏倚或不足：①四联袋还需要进一步改进以提高实验的稳定性。②单个核细胞的接种密度及脐血干细胞诱导分化为神经元样细胞的其他影响因素，培养获得的贴壁细胞是否含有其他异质细胞成分，以及诱导分化的神经元样细胞何时适宜临床移植等问题均有待进一步分析。

摘要
背景：研究证明脐血间质干细胞可向神经元样细胞分化，要同时保证脐血间质干细胞的扩增能力与分化潜能，其培养和诱导分化条件的优化显得尤为重要。
目的：分析筛选人脐血间质干细胞向神经元样细胞体外诱导分化过程的影响因素。
设计、时间及地点：随机对照实验，于2006-08/2007-05在河南省红十字血液中心血液成分应用研究所完成。
材料：脐血来自郑州市妇幼保健院正常足月分娩的胎儿，平均采血量95 mL。重组人表皮生长因子、碱性成纤维细胞生长因子购自Sigma公司；分离细胞所用的四联袋为山东威高集团公司产品。
方法：取采集6 h内的脐血，采用四联袋密度梯度离心法分离脐血单个核细胞，以5×109 L-1接种于DMEM/F12培养基中。①细胞因子实验：单独细胞因子组加入20 μg/L B27、10 μg/L碱性成纤维细胞生长因子；细胞因子联合组在此基础上加入5 μg/L重组人表皮生长因子。②红细胞混入量实验：裂解组加入红细胞裂解液1 mL，裂解后红细胞混入量为(0.44±0.13)×108/份；少红细胞组红细胞混入量为（0.51±0.21）×108/份，多红细胞组红细胞混入量为（2.65±1.28）×108/份。③首次换液时间实验：分别于接种后48 h、7 d首次换液。④诱导分化：将碱性成纤维细胞生长因子、重组人表皮生长因子共诱导7 d的细胞爬片进行巢蛋白免疫组织化学染色。
主要观察指标：观察不同因素对脐血间质干细胞增殖分化的影响。检测诱导分化后神经干细胞巢蛋白的表达。
结果：培养7 d后，表皮生长因子与碱性成纤维细胞生长因子联合诱导促细胞增殖的效果优于单独应用碱性成纤维细胞生长因子（t=2.880，P < 0.05）；红细胞裂解液组、多红细胞组贴壁细胞数明显低于少红细胞组（t=7.332~7.550，P < 0.05），即混入的红细胞总量不大于108/份对细胞增殖无影响；接种后7 d首次换液所获贴壁细胞数明显多于接种后48 h首次换液(P < 0.05)。两种细胞因子共诱导后，神经干细胞巢蛋白呈阳性表达。
结论：在脐血间质干细胞向神经元样细胞诱导分化过程中，细胞因子、红细胞混入量及首次换液时间都是重要的影响因素。
关键词：脐血间质干细胞；神经元样细胞；诱导分化；影响因素

赵林娜，李建斌，单泓，段艳丽，焦红亮，宁晓琳，马会娟，杨朋辉，龚桂玲.人脐血间质干细胞向神经元样细胞分化的诱
导因素[J].中国组织工程研究与临床康复，2008，12(21):4049-4053
[www.zglckf.com/zglckf/ejournal/upfiles/08-21/21k-4049(ps).pdf]

Influential factors of human umbilical cord blood-derived mesenchymal stem cells differentiating into neuron-like cells

Abstract

BACKGROUND: Studies have identified that, human umbilical cord blood-derived mesenchymal stem cells (HUCB-derived MSCs) may differentiate into neuron-like cells, and simultaneously requires to maintain the amplification ability and differentiating potency of HUCB-derived MSCs. It is extremely important to optimize the conditions of culture and differentiation.
OBJECTIVE: To analyze and screen the factors inducing HUCB-derived MSCs to differentiate into neuron-like cells in vitro.
DESIGN, TIME AND SETTING: A randomized control experiment was carried out in the Blood Components Application Institute of Red-cross Blood Center of Henan Province (Zhengzhou, Henan, China) from August 2006 to May 2007.
MATERIALS: Umbilical cord blood was from the term-delivery fetus in Zhengzhou Hospital of Maternal and Child Health (China), the average blood volume was 95 mL. Recombinant human epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were purchased from Sigma Company (USA); Quadruple blood bag for the isolation was produced by Shandong Weigao Group (China).
METHODS: Umbilical cord blood samples were collected within 6 hours, and umbilical blood mononuclear cells were separated with quadruple bags by means of density gradient centrifugation. Cytokine trial: single cytokine group was added with 20 μg/L B27 and 10 μg/L bFGF; combined cytokine group was added with 20 μg/L B27, 10 μg/L bFGF and 5 μg/L recombinant human EGF.Erythrocyte interfusion trial: cell lysis group was added with 1 mL erythrocyte lysis liquid, and the quantity of lyzed erythrocyte was (0.44±0.13)×108, while the quantity of few erythrocyte group and many erythrocyte group was (0.51±0.21)×108 and (2.65±1.28)×108, respectively. First medium change trial: the first medium changing time was 48 hours and 7 days following the incubation. Induced differentiation: induced by bFGF and recombinant human EGF, cells were stained with nestin immunohistochemistry.
MAIN OUTCOME MEASURES: The influence of variant factors on the proliferation and differentiation of HUCB-derived MSCs; expression of nerve stem cells nestin after induce differentiation.
RESULTS: At day 7 of the culture, EGF and bFGF contributed to more perfect function than only bFGF in the process of HUCB-derived MSCs proliferating (t=2.880, P < 0.05); the quantity of adherent cells in cell lysis group and many erythrocyte group was obviously lower than that in the few erythrocyte group (t=7.332-7.550, P < 0.05), which indicated that the interfused erythrocytes with quantity of less than 108 had no obvious influence on the proliferation of stem cells. The first medium changing at seven days achieved remarkably more adherent cells compared with at 48 hours (t=3.680, P < 0.05). Induced by two cytokines, the MSCs expressed nestin.
CONCLUSION: Cytokines, quantity of interfusion erythrocytes and first medium changing time are the important factors in the process of HUCB-derived MSCs differentiating into neuron-like cells.

Zhao LN, Li JB, Shan H, Duan YL, Jiao HL, Ning XL, Ma HJ, Yang PH, Gong GL.Influential factors of human umbilical cord blood-derived mesenchymal stem cells differentiating into neuron-like cells.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(21):4049-4053(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-21/21k-4049(ps).pdf]