体外培养胚胎神经干细胞的增殖及中药有效成分对其增殖能力的影响

Effects of active extractions from Chinese medicines on the proliferation of neural stem cells cultured in vitro and related mechanisms study of proliferation

关键词:  胚胎神经干细胞  增殖  人参皂苷Rg1  黄芪皂苷ASIV  周期蛋白D1  Hes1  Hes5

Key words:  embryonic neural stem cells  proliferation  ginsenoside Rg1  astragaloside IV  cyclin D1  Hes1  Hes5

摘要:

[目的]探讨中药有效成分对神经干细胞(NSCs)增值能力的影响并探讨其作用机制。[方法]从16天SD大鼠胚胎脑体外培养原代NSCs,增生并传代。用Nestin免疫细胞-化学染色(ICC)鉴定NSCs,用BrdU染色鉴定NSCs增生。限量稀释法观察了几种中药有效成分对NSCs增生的作用,如人参皂苷Rg1和黄芪皂苷(ASIV)。[结果]不同剂量的人参皂苷Rg1和黄芪皂苷ASIV能促进神经干细胞的体外增殖。与对照组比较,Rg1和AS的3种剂量可使神经球生成数目增加(P<0.05或P<0.01)。其中两种药物成分低剂量组比其他组效果更显著。Hes1,Hes5和细胞周期蛋白D1的实时定量逆转录-聚合酶链(RT-PCR)分析显示Rg1高剂量组的Hes1基因表达和AS高剂量组Hes5基因表达增长明显。同时,Rg1中剂量组的Hes5基因表达轻微增加(P>0.05)。两药各剂量组的周期蛋白D1(CyclinD1)基因表达可见有增加,但无统计学意义(P>0.05)。[结论]Rg1和ASIV可明显促进神经干细胞体外增殖,实时定量检测结果显示Hes1、Hes5和周期蛋白D1与Rg1和AS促进神经干细胞增殖有一定关系。

Abstract:

[Objective] To explore the effective Chinese medicine extractions that have the ability of protecting neural stem cells (NSCs). Rat embryonic were cultured in vitro. The proliferation of NSCs was observed and identified. Different Chinese medicine extractions were applied,whose effect and function mechanism were investigated. [Methods] The NSCs cultures were generated from the brain of embryonic day (E) 16 SD rat. Primitive NSCs were cultured,proliferated and passaged. The NSCs were identified by the immunocyto-chemical (ICC) staining of Nestin. The ICC staining of BrdU was adopted to characterize the proliferation of NSCs. According to limited dilution method,the effect of different Chinese medicine extractions on the proliferation of NSCs was observed,such as Ginsenoside Rg1 and AS-tragaloside IV. [Results] The NSCs were successfully cultured and proliferated in vitro. Different dosages of Ginsenoside Rg1 and As-tragaloside IV couldpromote the proliferation of NSCs in vitro. Compared with the control group,the numbers of nerurospheres of the three dosage Ginsenoside Rg1 groups (40,4,0.4 μM) were increased obviously (P<0.05,P<0.01). The neurospheres numbers of all of three Astragaloside IV Groups (6,0.6,0.06 μM) were increased significantly(P<0.05,P<0.01). The results showed that the lower dosage groups of both medicines have more outstanding effects than other groups. Real time RT-PCR analysis of Hes1,Hes5 and cyclinD1 deemonstrated that the Hes1 expression of high dosage group of Ginsenoside Rg1 (40 μM) and Hes5 expression of high dosage group of Astragaloside IV (6 μM) increased significantly (P<0.05). Simultaneously,Hes5 expression of middle dosage froup of Ginsenoside Rg1 (4 μM) increased slightly (P>0.05). CyclinD1 expression increased in different dosage groups of Ginsenoside Rg1 and Astragaloside IV (P>0.05). [Conclutions] Ginsenoside Rg1 and Astragaloside IV significantly promote the proliferation of NSCs in vitro. The real-time quantitative data suggest that Hes1,Hes5 and cyclinD1 might be involved in the proliferation of NSCs induced by Ginsenoside Rg1 and Astragaloside IV.