Effect of liquiritin on the proliferation of neural stem cells from the brain of mouse embryos

doi:10.3969/j.issn.2095-4344.2017.21.009

Abstract

Abstract:

BACKGROUND: Liquiritin has the protective and nutritive effects on neural stem cells. However, the effect of liquiritin on neural stem cells from the brain of mouse embryos remains unclear.
OBJECTIVE: To investigate the effects of different concentrations of liquiritin on the proliferation of neural stem cells from the brain of mouse embryos.
METHODS: Neural stem cells were separated from the embryonic brain of Kunming white mice at the gestational age of 14 days. The identification of embryonic neural stem cells was performed by immunocytochemistry method. The expression of neural stem cells-special genes was determined by qRT-PCR. The cell growth curve was drawn and proliferation of embryonic neural stem cells treated with 0, 1, 2, 4 or 8 g/L liquiritin for 48 hours was measured by MTT assay.
RESULTS AND CONCLUSION: (1) When cultured at day 5, all individual neural stem cells gathered together into neurospheres; with the extension of time, the neurospheres were enlarged, and gathered together into larger cell masses. (2) Results from immunocytochemistry showed that all the floating neurospheres was nestin-positive. Data from qRT-PCR revealed a higher expression of nestin mRNA, but there was no expression of neuron-specific enolase and glial fibrillary acidic protein in the neural stem cells. (4) The growth of neural stem cells was slow at the beginning. After 2-3 days, the cell proliferation quickly entered the exponential phase. After 4 days, the cell proliferation gradually slowed down, and the overall cell growth entered into the platform period. (5) The cell proliferation after treatment with 2, 4 or 8 g/L liquiritin was faster than that in the control group (0 g/L). To conclude, 2-8 g/L liquiritin could increase the proliferation of neural stem cells from the brain of mouse embryos.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Neural Stem Cells, Drugs, Chinese Herbal, Cell Proliferation, Tissue Engineering

CLC Number:

R394.2

Li She-fang, Miao Ling-juan, Li Ning, Liang He, Ren De-qi, Guo Jian. Effect of liquiritin on the proliferation of neural stem cells from the brain of mouse embryos[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(21): 3332-3337.

Figures/Tables 1

2.1 小鼠胚胎脑神经干细胞分离及培养结果将分离后的细胞使用无血清培养基培养，1 d后少部分细胞开始聚集成球，形成的神经球大小不等，形状不规则。生长至2-5 d后，发现神经球数目不断增多，体积也不断增大，大多数神经球呈圆形或椭圆形，并且分散悬浮，大部分神经球外围细胞大小较均匀，生长状态良好。如图1所示，第5天时，几乎所有单个的脑神经干细胞全部聚集成球。随着时间的延长，细胞球的体积越来越大，细胞球融合成更大的细胞团。 2.2 小鼠胚胎脑神经干细胞的鉴定利用免疫细胞化学技术，以巢蛋白为检测标记，对分离得到的小鼠脑神经干细胞进行鉴定，结果显示神经干细胞克隆球呈现巢蛋白阳性，且100%的神经干细胞球呈阳性表达。另外，运用qRT-PCR技术又进一步对小鼠胎脑神经干细胞相关基因的表达进行了验证，结果显示分离出来的神经干细胞巢蛋白阳性表达较强，几乎不表达神经元特异性烯醇化酶及神经胶质纤维酸性蛋白(图2)。 2.3 小鼠胚胎脑神经干细胞的生长曲线如图3所示，分离到的神经干细胞在刚开始生长缓慢，处于生长缓慢期。第二三天起，细胞增殖迅速，细胞进入指数生长期，第4天后细胞增殖逐步减缓，细胞整体进入生长平台期。 2.4 甘草苷对小鼠胚胎脑神经干细胞增殖的影响选用1，2，4，8 g/L的甘草苷处理小鼠胚胎脑神经干细胞，48 h后观察其对干细胞增殖情况的影响。MTT法检测结果显示，与对照组相比，1 g/L的甘草苷对胚胎神经干细胞的增殖无明显影响，而随着甘草苷质量浓度的升高，神经干细胞的增殖速度明显升高，且高于对照组(P < 0.05)，见图4，说明甘草苷能够促进小鼠胚胎脑神经干细胞的增殖。"

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