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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Cyclin D1 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,Cyclin D1 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,Cyclin D1 mRNA表达。
Objective: To observe the effects of Si - Ni - San and Liu Wei Di Huang Wan on the proliferation of neural stem cells (NSCs) in vitro. Methods: The third generation of NSCs was selected as a grouping model. Blank control group: cultured with DMEM / F12 (1: 1). Sini powder low dose group: DMEM / F12 (1: 1) medium was added Sini Powder low concentration decoction (final concentration containing crude drug 0.133 g · L-1). Sini powder high dose group: DMEM / F12 (1: 1) medium was added to the Sini powder high concentration decoction (final concentration of crude drug containing 0.267 g · L-1). Liuweidihuangwan low-dose group: DMEM / F12 (1: 1) medium was added to low concentration of Liuweidihuangwan decoction (final concentration of crude drug containing 0.208 g · L-1). Liuwei Di Huang Wan high-dose group: DMEM / F12 (1: 1) medium was added to Liu Wei Di Huang Wan Gao decoction (final concentration of crude drug 0.416 g · L-1). Each group of cells 30% fluid replacement 1 day, cultured 4 d. Cell proliferation was detected by 5-Bromo-2-deoxy Uridine (Brdu) fluorescent immunocytochemistry and mRNA expressions of c-myc and Cyclin D1 in each group were detected by Real time-PCR. Results: The cells in all groups proliferated within 1 ~ 4 days. Among them, Liuweidihuangwan high and low dose groups had the most obvious proliferation (P <0.01), while those of Liuweidihuangwan low and high dose groups There was no significant difference between the blank control group, Sini Powder and high-dose group no significant difference between the groups. The expression of c-myc and Cyclin D1 mRNA in Liuweidihuangwan low and high dose groups was the highest, which was significantly lower than that in the blank control group and Siniosan low and high dose groups (P <0.01) No difference between groups; blank control group, Sini scattered low, high-dose group was no difference between. CONCLUSION: Compared with Sini Decoction, Liuweidihuang Pill can significantly promote the proliferation of NSCs in vitro and up-regulate the expressions of c-myc and Cyclin D1 mRNA in NSCs.