Yan Xiao-hua, Huang Rui-bin
Department of Pediatrics and Hematology, The First Affiliated Hospital, Nanchang University, Nanchang 330006, China.
Zhonghua Er Ke Za Zhi. 2006 Mar;44(3):214-9.
To study the possibility and mechanism by which the human umbilical blood mesenchymal stem cells (MSCs) are induced to differentiate into neuron in vitro by baicalin, a kind of flavonoid isolated from an important medicinal plant Scutellariae Radix.
Human cord blood was obtained via sterile procedure with 20 U/ml of preservative-free heparin. MSCs were isolated by the two-step assay of gelatin sedimentation plus Ficoll centrifugation separation, purified and amplified in the liquid culture medium. According to the kind of antioxidants, the experiment was conducted in five groups: induction group, control group I, control group II, control group III and control group IV. Five subgroups of MSCs amplificated ex vivo for 2 weeks in each group were induced by the media containing baicalin (BC, 200 - 400 micromol/L) or baicalin-free for seven days. The media consisted of induction medium (DMEM plus 200 - 400 micromol/L of baicalin) and post-induction medium (DMEM plus 200 - 400 micromol/L of baicalin, B27). The expression of neuronal or glia specific markers was evaluated by using indirect immunofluorescence cytochemistry staining. The percentage of differentiated cells and living cells was measured by Hoechest 33,258 staining assay.
After induction for 7 days, MSCs displayed neuronal morphologies, such as pyramidal cell bodies and processes formed extensive network. The undifferentiated cells did not exhibit a neuronal or glial phenotype, while the differentiating cells expressed NSE and MAP-2, the specific markers of neuron, but did not express GFAP, the specific marker of glia on the seventh day after induced by baicalin. The percentage of NSE and MAP-2 expressed on the seventh day after induced by baicalin was (76.3 +/- 9.2)% and (78.5 +/- 5.5)%, respectively, which was significantly higher compared with control group I, control group II and control group III (P < 0.01), respectively. In addition, the ratio of living cells after induced for seven days in the BC group was (85.3 +/- 4.8)%, which increased significantly compared with control group I, control group II and control group III (P < 0.01), respectively.
Baicalin may induce the umbilical blood MSCs to neuron or neuron-alike cells in vitro in a moderate and stable manner. The mechanism of such an induction may be related with its controlling the activity of NF-kappaB which regulates the production of many kinds of cytokines, such as brain-derived neurotrophic factor (BDNF), etc.
研究从重要药用植物黄芩中分离得到的黄酮类化合物黄芩苷在体外诱导人脐血间充质干细胞（MSCs）分化为神经元的可能性及其机制。
通过无菌操作采集人脐带血，加入20 U/ml无防腐剂肝素。采用明胶沉降加Ficoll梯度离心两步法分离MSCs，在液体培养基中进行纯化和扩增。根据抗氧化剂种类，实验分为五组：诱导组、对照组I、对照组II、对照组III和对照组IV。每组将体外扩增2周的MSCs分为五个亚组，分别用含黄芩苷（BC，200 - 400 μmol/L）或不含黄芩苷的培养基诱导7天。培养基包括诱导培养基（DMEM加200 - 400 μmol/L黄芩苷）和诱导后培养基（DMEM加200 - 400 μmol/L黄芩苷、B27）。采用间接免疫荧光细胞化学染色法评估神经元或神经胶质细胞特异性标志物的表达。用Hoechest 33258染色法检测分化细胞和活细胞的百分比。
诱导7天后，MSCs呈现神经元形态，如锥体细胞体和形成广泛网络的突起。未分化细胞未表现出神经元或神经胶质细胞表型，而分化细胞在黄芩苷诱导7天后表达神经元特异性标志物NSE和MAP-2，但不表达神经胶质细胞特异性标志物GFAP。黄芩苷诱导7天后NSE和MAP-2的表达百分比分别为（76.3±9.2）%和（78.5±5.5）%，与对照组I、对照组II和对照组III相比显著升高（P < 0.01）。此外，BC组诱导7天后活细胞比例为（85.3±4.8）%，与对照组I、对照组II和对照组III相比显著升高（P < 0.01）。
黄芩苷可能在体外以适度且稳定的方式诱导脐血MSCs分化为神经元或类神经元细胞。这种诱导机制可能与其调控NF-κB的活性有关，NF-κB可调节多种细胞因子如脑源性神经营养因子（BDNF）等的产生。
