四逆散、六味地黄丸诱导神经干细胞增殖及对c-myc mRNA,CyclinD1 mRNA表达的影响

Expression of c-myc mRNA,CyclinD1 mRNA and Proliferation of Neural Stem Cells Induced by Sini San or Liuwei Dihuang Wan

摘要:

目的: 观察四逆散、六味地黄丸对体外培养神经干细胞(neural stem cells,NSCs)增殖的影响. 方法: 选取第3代NSCs作为分组造模对象.空白对照组:采用DMEM/F12(1:1)培养.四逆散低剂量组:采用DMEM/F12(1:1)培养基加入四逆散低浓度煎剂(终浓度含生药量0.133 g·L^-1).四逆散高剂量组:采用DMEM/F12(1:1)培养基加入四逆散高浓度煎剂(终 质量浓度含生药量0.267 g·L^-1).六味地黄丸低剂量组:采用DMEM/F12(1:1)培养基加入六味地黄丸低浓度煎剂(终质量浓度含生药量0.208 g·L^-1).六味地黄丸高剂量组:采用DMEM/F12(1:1)培养基加入六味地黄丸高煎剂(终质量浓度含生药量0.416 g·L^-1). 各组细胞隔天30%换液1次,培养4 d.采用5-Bromo-2-deoxy Uridine(Brdu)荧光免疫细胞化学技术标记法检测细胞增殖及Real time-PCR法检测各组细胞c-myc及CyclinD1 mRNA表达. 结果: 各组细胞在1~4 d内均处于增殖状态,其中六味地黄丸高、低剂量组增殖最明显,均比其他各组高(P<0.01);而六味地黄丸低、高剂量组之间没有显著差异;空白对照组、四逆散低剂、高剂量组之间比较没有显著差异.c-myc,CyclinD1 mRNA在六味地黄丸低、高剂量组表达最高,与空白对照组、四逆散低、高剂量组比较有显著差异(P<0.01);而六味地黄丸低、高剂量组之间没有差异;空白对照组、四逆散低、高剂量组之间比较没有差异. 结论: 与四逆散比较,六味地黄丸对体外培养NSCs增殖有明显促进的效应,可上调NSCs c-myc,CyclinD1 mRNA表达.

Abstract:

Objective: To observe the effects of Sini San and Liuwei Dihuang Wan on the proliferation and the expression of c-myc mRNA and CyclinD1 mRNA of neural stem cells(NSCs) in vitro. Method: NSCs of the thirrd generation in good condition were resuspended, blew, struck and cultured in medium with the density of 1×10⁵/mL and in incubator with 5% CO₂ and 95% humidity at 37℃.The cultured NSCs were divided into five groups: the normal control group, the low doses of and the high doses of Sini San group, the low doses of and the high doses of Liuwei Dihuang Wan group. All NSCs in five groups were cultivated with DMEM/F12(1: 1) medium for 4 days. The culture medium was changed 30% every other day. The low concentration of Sini San decoction with 0.133 g crude drug per liter, the high concentration of Sini San decoction with 0.267 g crude drug per liter, the low concentration of Liuwei Dihuang Wan decoction with 0.208 g crude drug liter and the high concentration of Liuwei Dihuang Wan decoction with 0.416 g crude drug per liter were added respectively into the medium in the low doses of Sini San group, the high doses of Sini San group, the low doses of Liuwei Dihuang Wan group and the high doses of Liuwei Dihuang Wan group. The proliferation of cells was detected by 5-bromo-2-deoxy uridine(BrdU) labeling fluorescence immunocytochemistry every day. The relative expression of c-myc mRNA and cyclinD1 mRNA was detected by fluorescence real-time quantitative PCR in each group. Result: The results of BrdU labeling fluorescence immunocytochemistry:the proliferation of cells in the low and high doses of Liuwei Dihuang Wan group was more obvious than that in the low doses of Sini San group and the high doses of Sini San group(P<0.01).There was no obvious difference in the proliferation of cells between the low doses of Sini San group, the high doses of Sini San group and normal control group. Results of fluorescence real-time quantitative PCR:the relative expression of c-myc mRNA and CyclinD1 mRNA in the low doses of Liuwei Dihuang Wan group and the high doses of Liuwei Dihuang Wan group was higher obvious than that in the low doses of Sini San group and the high doses of Sini Powder group(P<0.01).There was no obvious difference in the relative expression of c-myc mRNA and CyclinD1 mRNA between the low doses of Sini San group, the high doses of Sini San group and normal control group. Conclusion: Compared with Sini San, Liuwei Dihuang Wan has a significant stimulative effect on the proliferation of NSCs or the expression of c-myc mRNA and CyclinD1 mRNA.