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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)分化为神经元的影响,并探讨其作用机制。方法选取第3代NSCs作为分组造模对象。空白对照组:DMEM/F12(1∶1)培养基加入10%胎牛血清。四逆散低剂量组:DMEM/F12(1∶1)培养基加入10%胎牛血清和四逆散低浓度煎剂(终浓度含生药量0.133 g·L-1)。四逆散高剂量组:DMEM/F12(1∶1)培养基加入10%胎牛血清和四逆散高浓度煎剂(终浓度含生药量0.267 g·L-1)。六味地黄丸低剂量组:DMEM/F12(1∶1)培养基加入10%胎牛血清和六味地黄丸低浓度煎剂(终浓度含生药量0.208 g·L-1)。六味地黄丸高剂量组:DMEM/F12(1∶1)培养基加入10%胎牛血清和六味地黄丸高煎剂(终浓度含生药量0.416g·L-1)。隔天换液1次,培养7 d。采用免疫细胞荧光化学技术检测各组神经元特异性烯醇酶单克隆抗体(neuron-specific enolase,NSE)阳性细胞数。采用荧光实时定量PCR法检测各组细胞的Wnt1、Wnt3a、β-catenin mRNA表达相对量。结果神经元分化率:六味地黄丸低剂量组(12.15±2.32)%;六味地黄丸高剂量组(12.58±1.70)%;四逆散低剂量组(12.28±1.01)%;四逆散高剂量组(11.84±1.01)%;空白对照组(6.16±1.42)%;空白对照组神经元分化率最低,与六味地黄丸低、高剂量组、四逆散低剂量组及四逆散高剂量组比较(P<0.01)。Real time-PCR检测:Wnt1、Wnt3a、β-catenin在空白对照组表达最低,与六味地黄丸低、高剂量组及四逆散低、高剂量组比较有显著差异(P<0.01)。结论四逆散、六味地黄丸可促进NSCs向神经元分化,其作用机制可能与上调Wnt1、Wnt3a及β-catenin表达,激活Wnt/β-catenin信号转导通路有关。
Objective To observe the effect of Si Ni San and Liu Wei Di Huang Wan on differentiation of neural stem cells (NSCs) into neurons in vitro and its mechanism of action. Methods The third generation of NSCs was selected as a grouping model. Blank control group: DMEM / F12 (1: 1) medium added 10% fetal bovine serum. Sini powder low dose group: DMEM / F12 (1: 1) medium was added 10% fetal calf serum and Sini Powder low concentrations of decoction (final concentration containing crude drug 0.133 g · L-1). Sini powder high dose group: DMEM / F12 (1: 1) medium was added 10% fetal calf serum and Sini powder high concentration decoction (final concentration of crude drug containing 0.267 g · L-1). Low dose of Liuweidihuangwan group: DMEM / F12 (1: 1) medium was added with 10% fetal bovine serum and Liuweidihuangwan low concentration decoction (final concentration of crude drug 0.208g · L-1). Liuweidihuangwan high-dose group: DMEM / F12 (1: 1) medium was added 10% fetal bovine serum and Liu Wei Di Huang Wan Gao decoction (final concentration containing crude drug 0.416g · L-1). Change the fluid every other day for 7 days. The number of neuron-specific enolase (NSE) -positive neurons in each group was detected by immunocytochemistry. Real-time quantitative PCR was used to detect the relative amount of Wnt1, Wnt3a and β-catenin mRNA expression in each group. RESULTS: The rate of neuron differentiation was significantly lower in Liuwei Dihuang Pill group (12.15 ± 2.32)%, in Liuwei Dihuang Pill group (12.58 ± 1.70)%, in Sini Powder low dose group (12.28 ± 1.01%), Group (11.84 ± 1.01)%; control group (6.16 ± 1.42)%; control group neurological differentiation rate lowest, and Liu Wei Di Huang Wan low, high dose group, Sini Powder low dose group and Sini Powder high dose group (P <0.01). Real time-PCR assay showed that Wnt1, Wnt3a andβ-catenin had the lowest expression in the blank control group, which was significantly lower than that of the Liuwei Dihuang Pill low, high dose group and Siniosan low and high dose groups (P <0.01). Conclusion Siniosan and Liuweidihuang Pill can promote the differentiation of NSCs into neurons. The mechanism may be related to up-regulating the expression of Wnt1, Wnt3a and β-catenin and activating Wnt / β-catenin signal transduction pathway.