人参皂苷在神经干细胞中的干预机制及对HIF-1a-VEGF表达水平的影响研究

Intervention Mechanism of Ginsenosides in Neural Stem Cells and Its Effect on HIF-1a-VEGF Pathway Expression

目的探讨人参皂苷在神经干细胞中的干预机制及对HIF-1a-VEGF通路的影响。方法选择C57BL/6小鼠25只作为对象,采用颈椎脱臼法处死小鼠,完成NSCs的分离、培养、传代及鉴定。将最终获得的NSCs细胞分为观察组与对照组。对照组中常规加入DMEM/F12培养基培养,观察组细胞分别盛放在3个试管中,分别加入0.1、1、10.0μmol/L的人参皂苷,连续进行48 h培养。采用一步法TUNEL染色测定两组NSCs凋亡情况;采用CCK法检测两组NSCs增殖能力;采用半定量逆转录-聚合酶链反应(RT-PCR)法测定HIF-1a、VEGF通路。结果和结论①原代细胞倒置显微镜下细胞呈分散的圆球状,且悬浮于培养液中,细胞具有良好的折光性;细胞培养24 h后部分细胞开始分裂,形成小克隆神经球;连续进行48 h培养后NSCs增殖活力旺盛,细胞直径大、规则、饱满,具有较强的折光性;在EGF与bFGF等生长因子培养基培养下,多数细胞显示小胞体,且伴有双极或多极态具有巢蛋白(绿色为巢蛋白阳性标记,蓝色为DAPI细胞核染);细胞分化培养5 d后NSCs细胞成功分化为Tui-1标记神经元和GFAP标记的星形胶质细胞;②人参皂苷对于NSCs细胞凋亡率具有明显的抑制作用,且浓度越高抑制效果越明显;人参皂苷干预后NSCs细胞不同时间点凋亡率,均低于DMEM/F12培养基(P<0.05);③不同浓度人参皂苷均能促进NSCs细胞增殖,且人参皂苷浓度越高,对NSCs细胞增殖能力越强;④人参皂苷不同浓度下NSCs细胞HIF-1a-VEGF通路,均高于DMEM/F12培养基(P<0.05);人参皂苷浓度越高,NSCs细胞HIF-1a-VEGF通路水平越高,不同浓度下NSCs细胞HIF-1a-VEGF水平具有统计学意义(P<0.05);⑤人参皂苷用于NSCs中能促进能促进细胞增殖、抗凋亡,且呈药物剂量依赖性,可能与提高HIF-1a-VEGF通路有关,能为NSCs细胞分离、制备提供理论依据。

Objective To investigate the intervention mechanism of ginsenosides in neural stem cells and its effect on HIF-1 a-VEGF pathway. Methods Twenty-five C57 BL/6 mice participated in the experiment from April 2018 to June 2018 were selected as subjects. The mice were killed by cervical dislocation. The isolation, culture, passage and identification of NSCs were completed. The NSCs were divided into observation group and control group. In the control group, DMEM/F12 medium was added regularly. In the observation group, cells were left in three test tubes, and ginsenosides of 0.1 μmol/L, 1 μmol/L and 10.0 μmol/L were added respectively. The cells were cultured continuously for 48 h. One-step TUNEL staining was used to detect the apoptosis of NSCs in the two groups. CCK method was used to detect the proliferation of NSCs in the two groups. Semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR) was used to detect the HIF-1 a and VEGF pathway. Results and Conclusion ①The cells under the inverted microscope were scattered and spherical, and suspended in the culture medium. The cells had good refraction. After 24 h cell culture, some cells began to divide and form small cloned neurospheres. After culture, NSCs proliferate vigorously, and the cell diameter was large, regular, full, and had strong refraction. Under the culture medium of growth factors such as EGF and bFGF, most cells showed small cell bodies with bipolar or multipolarity. Nestin(the green color was a nestin-positive marker, the blue color was DAPI-nuclear staining). NSCs differentiated into Tui-1 labeled neurons and GFAP-labeled astrocytes after 5 days of cell differentiation. ②Ginsenosides had obvious inhibitory effect on NSCs apoptotic rate and the higher the concentration, the more obvious the inhibitory effect. The apoptosis rate of NSCs at different time points after ginsenoside intervention was lower than that of DMEM/F12 medium(P<0.05). ③Different concentrations of ginsenosides all can promote the proliferation of NSCs cells, and the higher the concentration of ginsenosides, the stronger the proliferation ability of NSCs cells. ④HIF-1 a-VEGF of NSCs cells at different concentrations of ginsenosides were higher than DMEM/F12 medium(P<0.05). The higher the concentration of ginsenoside, the higher the level of HIF-1 a-VEGF pathway in NSCs. The level of HIF-1 a-VEGF in NSCs cells at different concentrations was statistically significant(P<0.05). ⑤Ginsenosides can promote cell proliferation and anti-apoptosis in NSCs, and it is dose-dependent. It may be related to the increase of HIF-1 a-VEGF pathway, which can provide theoretical basis for the isolation and preparation of NSCs.