摘要
目的 探讨远志皂苷元对神经干细胞（neural stem cells, NSCs）增殖分化的影响及其作用机制。方法 将原代培养的NSCs按随机数字表法分为空白对照组，远志皂苷元低、中、高剂量组。远志皂苷元低、中、高剂量组分别加入含10、20、40 μmol/L远志皂苷元的完全培养基，空白对照组常规培养。培养4 d后，采用CCK8实验检测细胞活力、显微镜观察并计算神经球数目；Western bolt法检测巢蛋白（Nestin）、神经特异性第三类微管蛋白（neuron-specific class III b-tubulin, TUJ1）、糖原合成酶激酶-3b （glycogen synthase kinase 3b, GSK-3b）、p-GSK-3b（Ser9）的表达，免疫荧光染色观察Nestin、TUJ1的表达。结果 与空白对照组比较，远志皂苷低、中、高剂量组NSCs神经球数量[（32.78±6.30）个、（40.93±8.34）个、（45.37±7.96）个比（26.48±5.19）个]、增殖率[（127.50±9.31）%、（138.13±6.88）%、（151.25±9.38）%比（100.00±5.63）%]增加（P＜0.05或P＜0.01），TUJ1蛋白[（2.21±0.14）、（3.10±0.16）、（3.30±0.15）比（1.00±0.00）]表达升高（P＜0.05），Nestin蛋白[（0.36±0.04）、（0.53±0.05）、（0.46±0.05）比（1.00±0.00）]表达降低（P＜0.05），p-GSK-3b（Ser9）/GSK-3b[（2.31±0.17）、（3.41±0.11）、（3.59±0.16）比（1.00±0.00）]表达增加（P＜0.01）,Nestin阳性细胞数[（50.29±3.18）个、（45.28±6.23）个、（38.72±5.31）个比（75.27±6.03）个]减少（P＜0.05或P＜0.01），TUJ1阳性细胞数[（32.23±4.36）个、（38.23±6.01）个、（46.23±4.36）个比（20.31±5.23）个]增加（P＜0.01）。结论 远志皂苷元可激活Wnt信号通路从而促进NSCs增殖和分化。
Objective To study the effect and mechanism of senegenin on proliferation and differentiation of neural stem cells （NSCs）. Methods The primary cultured NSCs were divided into the high-dose, medium-dose, low-dose group and normal control group （NC）. The complete medium containing 10, 20 and 40 μmol/L senegenin was added to senegenin low-, middle-, and high- dose groups, and the NC group was routinely cultured. After 4 days of culture, CCK8 assay was used to detect cell viability, and microscopy was performed and the number of neurospheres was counted. Western blot was used to detect the expression of Nestin, TUJ1, GSK-3b, and p-GSK-3b （Ser9）, and immunofluorescence staining was used to visualized Nestin and TUJ1. Results Compared with the control group, the number of NSCs neurospheres （32.78 ± 6.30, 40.93 ± 8.34, 45.37 ± 7.96 vs. 26.48 ± 5.19） and the proliferation （127.50% ± 9.31%, 138.13% ± 6.88%, 151.25% ± 9.38% vs. 100.00% ± 5.63%） in the low-, middle- and high-doses of senegenin group significantly increased （P〈0.05 or P〈0.01）. The expression of TUJ1 （2.21 ± 0.14, 3.10 ± 0.16, 3.30 ± 0.15 vs. 1.00 ± 0.00） in the low-, middle- and high-doses of senegenin group significantly increased （P〈0.05）; and the expression of Nestin （0.36 ± 0.04, 0.53 ± 0.05, 0.46 ± 0.05 vs. 1.00 ± 0.00） significantly decreased （P〈0.05）. The ration of p-GSK-3β （Ser9）/ GSK-3β （2.31 ± 0.17, 3.41 ± 0.11, 3.59 ± 0.16 vs. 1.00 ± 0.00） in the low-, middle- and high-doses of senegenin group significantly increased （P〈0.01）. The cell number of Nestin＋ （50.29 ± 3.18, 45.28 ± 6.23, 38.72 ± 5.31 vs. 75.27 ± 6.03） in the low-, middle- and high-doses of senegenin group significantly decreased （P〈0.05 or P〈0.01）, and the cell number of TUJ1＋ （32.23 ± 4.36, 38.23 ± 6.01, 46.23 ± 4.36 vs. 20.31 ± 5.23） significantly increased （P〈0.01）. Conclusions The senegenin may promote the proliferation and differentiation of NSCs through the activation of Wnt pathway.
作者
张荣
刘彬
甘荷霞
康涛生
吴晓伟
林俊
肖雁
Zhang Rong, Liu Bin, Gan Hexia, Kang Taosheng, Wu Xiaowei, Lin Jun, Xiao Yan(Neuromedicine Center, the 174th Hospital of People＇s Liberation Army of China （Affiliated Chenggong Hospital, Xiamen University）, Xiamen 361003, China)
出处
《国际中医中药杂志》
2018年第4期334-338,共5页
International Journal of Traditional Chinese Medicine
基金
国家白然科学基金（81302892）
广州自然科学基金（S2013040016226）
