Effects of drug-contained sera of Naoluo Xintong versus Zuogui pill on the proliferation and differentiation of in vitro cultured neural stem cells

doi:10.3969/j.issn.2095-4344.2016.32.020

Abstract

Abstract:

BACKGROUND: Because of limited source and a relatively weak ability of differentiation and proliferation, how to take positive and effective measures to promote neural stem cell proliferation, differentiation has become the focus of research.
OBJECTIVE: To investigate the effects of drug-contained sera of Naoluo Xintong versus Zuogui pill on the proliferation and differentiation of in vitro cultured rat neural stem cells.
METHODS: Embryonic neural stem cells of Sprague-Dawley rats were isolated and cultured in vitro, and then were co-cultured with the serum medium containing 10% Naoluo Xintong and 10% Zuogui pill, respectively. Comparative observations were performed between two groups by inverted microscope and immunofluorescence staining.
RESULTS AND CONCLUSION: Under the inverted microscope, the cells began to grow in cluster and gather into a ball, but the diameter was relatively small after 24-hour culture; the neurospheres expended further, with relatively regular shape, but no neurosphere differentiation appeared after 48 hours of culture. The average prominent length of the neurospheres in the Zuogui pill group was significantly greater than that in the Naoluo Xintong group after 5 days of culture (P < 0.05). The rate of rat neural stem cells differentiating into MAP-2-positive cells in the Zuogui pill group was significantly lower than that in the Naoluo Xintong group (P < 0.05), but the rate of differentiated cells positive for glial fibrillary acidic protein in the Zuogui pill group was significantly higher than that in the Naoluo Xintong group (P < 0.05). After 48 hours of culture, neurospheres were cultured in the different drug-contained media, and 12 hours later, the neurospheres adhered to the wall, and a small amount of cell migration occurred. Then, cell migration began to increase with time. Under the immunofluorescence staining: prominent neurons with long protrusions were increased in both two groups, but there were no significant differences in the proportion of neurons and astrocytes between two groups (P > 0.05). These findings suggest that drug-contained sera of Naoluo Xintong and Zuogui pill can both not only promote the proliferation and differentiation of in vitro cultured rat neural stem cells, but also provide a suitable microenvironment for neural cell proliferation. Additionally, there are significant differences between the two drugs. Consequently, it is feasible to induce neural cell proliferation and differentiation by Naoluo Xintong and Zuogui pill.

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: Stem Cells, Cell Proliferation, Cell Differentiation, Neural Stem Cells, Cell Movement

CLC Number:

R394.2

Chen Li-ming, Pei Lei. Effects of drug-contained sera of Naoluo Xintong versus Zuogui pill on the proliferation and differentiation of in vitro cultured neural stem cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(32): 4845-4851.

Figures/Tables 2

2.1 大鼠神经干细胞增殖的检测结果剥离出SD大鼠大脑皮质并接种在无血清培养基中，倒置显微镜下在200倍镜下拍摄神经干细胞图像。24 h后倒置显微镜下观察细胞开始团生长，并聚集成球，但是细胞直径相对较小，36 h神经干细胞增大。48 h后神经球进一步增大，形态相对规则，但是未见神经球分化，见图2。 2.2 大鼠神经干细胞5 d后细胞球突起平均长度比较加入左归丸药物血清培养5 d后SD大鼠神经干细胞细胞球突起平均长度显著大于脑络欣通组(P < 0.05)，见图3。 2.3 大鼠神经干细胞分化为MAP-2、胶质纤维酸性蛋白阳性细胞率比较左归丸药物血清条件下SD大鼠神经干细胞分化为MAP-2阳性细胞率显著低于脑络欣通组(P < 0.05)；左归丸药物血清条件下SD大鼠神经干细胞分化为胶质纤维酸性蛋白阳性细胞率，显著高于脑络欣通组(P < 0.05)。左归丸药物血清条件下大鼠神经干细胞MAP-2阳性细胞率较低，而胶质纤维酸性蛋白阳性细胞率较高。见图4。 2.4 两种不同含药血清对体外培养的大鼠神经干细胞增殖分化的影响将培养48 h的神经球放入含有脑络欣通和左归丸药物血清的培养皿中培养，12 h后发现神经球贴壁生长，少量细胞迁移出来。随着时间的延长，迁移的细胞数量开始增多，48 h后可见大量细胞迁移，免疫细胞荧光染色下显示：脑络欣通和左归丸药物血清下神经元突出均相对较多，且突起的长度较长，见图5。 2.5 两种不同含药血清培养的大鼠神经干细胞神经元以及星形胶质细胞比例比较 2种不同含药血清培养的大鼠神经干细胞神经元以及星形胶质细胞比例差异无显著性意义(P > 0.05)，见表1。由此看出：2种不同含药血清均能促进SD大鼠神经干细胞的增殖、分化。"

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