人参皂苷Rg3对体外培养小鼠神经干细胞增殖的影响
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摘要
目的研究人参皂苷Rg3对体外培养小鼠神经干细胞(NSCs)增殖的影响.方法分离培养胎鼠NSCs,使用神经球悬浮培养法传代,特异性抗体Nestin和Sox2鉴定NSCs.采用Brd U标记法和CCK-8法检测不同浓度Rg3(20 n M、50 n M、250 n M和500 n M)对NSCs增殖的影响.结果传代2次后的神经球几乎全为Nestin/Sox2免疫荧光双阳性细胞,表明其为纯度较高的NSCs.Brd U标记法表明与对照组、20 n M组、250 n M组和500 n M组相比,50 n MRg3组Brd U阳性细胞相对比率最高,差异有统计学意义(P<0.01),但其他各组之间差异无统计学意义(P>0.05).CCK-8法检测,结果显示50 n M Rg3在450 nm处吸光值最高,且与对照组(P<0.01)、20 n M组(P<0.05)和500 n M组相比,差异有统计学意义(P<0.01),但其他各组之间无统计学差异(P>0.05).结论人参皂苷Rg3能促进体外培养小鼠NSCs的增殖,且其最佳浓度是50 n M.
Objective To investigate the effect of ginsenoside Rg3 on mouse neural stem cells(NSCs)proliferation in vitro.Me thods Fetal cortices of embryonic 14 d(E14) C57 BL/6 mouse were isolated for culturing primary NSCs.After being passaged twice, NSCs specific antibodies, Nestin and Sox2 were identified by immunocyto chemistry staining. CCK-8 assay and Brd U labelling assay were used to measure NSCs proliferation in response to different ginsenoside Rg3 concentrations(20 n M, 50 n M, 250 n M and 500 n M). Re s ults Primary and passaged NSCs were successfully cultured and almost all the cells were both Nestin and Sox2 positive NSCs,which indicated high purity of NSCs. Brd U labelling assay showed the group of 50 n M Rg3 had the highest relative ratio of Brd U positive percentage, and the difference with other groups was statistically significant(P<0.01).CCK-8 assay also showed 50 n M Rg3 group had the highest OD value, which was significantly higher than the control group(P<0.01), 20 n M group(P<0.05) and 500 n M group(P<0.01), while other groups had no significant difference between each other(P >0.05). Conclus ion 50 n M ginsenoside Rg3 could significantly promote mouse NSCs proliferation in vitro.
Objective To investigate the effect of ginsenoside Rg3 on mouse neural stem cells(NSCs)proliferation in vitro.Me thods Fetal cortices of embryonic 14 d(E14) C57 BL/6 mouse were isolated for culturing primary NSCs.After being passaged twice, NSCs specific antibodies, Nestin and Sox2 were identified by immunocyto chemistry staining. CCK-8 assay and Brd U labelling assay were used to measure NSCs proliferation in response to different ginsenoside Rg3 concentrations(20 n M, 50 n M, 250 n M and 500 n M). Re s ults Primary and passaged NSCs were successfully cultured and almost all the cells were both Nestin and Sox2 positive NSCs,which indicated high purity of NSCs. Brd U labelling assay showed the group of 50 n M Rg3 had the highest relative ratio of Brd U positive percentage, and the difference with other groups was statistically significant(P<0.01).CCK-8 assay also showed 50 n M Rg3 group had the highest OD value, which was significantly higher than the control group(P<0.01), 20 n M group(P<0.05) and 500 n M group(P<0.01), while other groups had no significant difference between each other(P >0.05). Conclus ion 50 n M ginsenoside Rg3 could significantly promote mouse NSCs proliferation in vitro.
引文
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[16]MORTE M I,CARREIRA B P,MACHADO V,et al.Evaluation of proliferation of neural stem cells in vitro and in vivo[J].Curr Protoc Stem Cell Biol,2013,2(14):1-14.
[2]WALKER T,HUANG J,YOUNG K.Neural stem and progenitor cells in nervous system function and therapy[J].Stem Cells Int,2016,6(2016):1890568.
[3]LEE R,KIM IS,HAN N,et al.Real-time discrimination between proliferation and neuronal and astroglial differentiation of human neural stem cells[J].Sci Rep,2014,9(4):6319.
[4]石海杉,吴文,徐建兰.内源性神经干细胞的研究进展[J].实用医学杂志,2013,29(19):3252-3254.
[5]赵瑜,袁婧,鲁琳.小鼠胚胎脑源与脊髓源神经干细胞增殖能力的比较[J].昆明医科大学学报,2017,38(9):19-23.
[6]胡炜彦,于浩飞,张荣平.人参皂苷Rg3对H2O2导致海马神经元损伤的保护作用研究[J].中成药,2014,36(4):670-674.
[7]KIM H S,LEE E H,KOSR,et al.Effects of ginsenosides rg3 and rh2 on the proliferation of prostate cancer cells[J].Arch Pharm Res,2004,27(4):429-435.
[8]孙建忠,刘新伟,管华鹏,等.人参皂苷Rg1与神经干细胞共培养的促增殖和保护作用[J].中国组织工程研究,2015,19(10):1580-1584.
[9]郑玉芹,姜正林,徐美玉,人参皂苷Rb1对体外培养胎鼠神经干细胞增殖及分化的影响[J].神经解剖学杂志,2014,30(3):273-279.
[10]BABU H,CLAASEN J H,KANNAN S,et al.A protocol for isolation and enriched monolayer cultivation of neural precursor cells from mouse dentate gyrus[J].Front Neurosci,2011,5(89):1-10.
[11]XIONG F,GAO H,ZHEN Y,et al.Optimal time for passaging neurospheres based on primary neural stem cell cultures[J].Cytotechnology,2011,63(6):621-631.
[12]SINGEC I,KNOTH R,MEYER RP,et al.Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology[J].Nat Methods,2006,3(10):801-806.
[13]ZHANG J,JIAO JW.Molecular Biomarkers for Embryonic and Adult Neural Stem Cell and Neurogenesis[J].Biomed Res Int,2015,2015:1-14.
[14]XU R,WU C,TAO Y,et al.Description of distributed features of the nestin-containing cells in brains of adult mice:a potential source of neural precursor cells[J].J Neurosci Res,2010,88(5):945-956.
[15]ZHANG S,CUI W.Sox2,a key factor in the regulation of pluripotency and neural differentiation[J].World J Stem Cells,2014,6(3):305-311.
[16]MORTE M I,CARREIRA B P,MACHADO V,et al.Evaluation of proliferation of neural stem cells in vitro and in vivo[J].Curr Protoc Stem Cell Biol,2013,2(14):1-14.