熊贵娅, 赵利娜, 左真子, 周志俊, 常秀丽. 体外百草枯染毒后神经干细胞的分化特点[J]. 环境与职业医学, 2019, 36(10): 903-908. DOI: 10.13213/j.cnki.jeom.2019.19328
引用本文: 熊贵娅, 赵利娜, 左真子, 周志俊, 常秀丽. 体外百草枯染毒后神经干细胞的分化特点[J]. 环境与职业医学, 2019, 36(10): 903-908. DOI: 10.13213/j.cnki.jeom.2019.19328
XIONG Gui-ya, ZHAO Li-na, ZUO Zhen-zi, ZHOU Zhi-jun, CHANG Xiu-li. Differentiation characteristics of neural stem cells after paraquat exposure in vitro[J]. Journal of Environmental and Occupational Medicine, 2019, 36(10): 903-908. DOI: 10.13213/j.cnki.jeom.2019.19328
Citation: XIONG Gui-ya, ZHAO Li-na, ZUO Zhen-zi, ZHOU Zhi-jun, CHANG Xiu-li. Differentiation characteristics of neural stem cells after paraquat exposure in vitro[J]. Journal of Environmental and Occupational Medicine, 2019, 36(10): 903-908. DOI: 10.13213/j.cnki.jeom.2019.19328

体外百草枯染毒后神经干细胞的分化特点

Differentiation characteristics of neural stem cells after paraquat exposure in vitro

  • 摘要: 背景 百草枯(PQ)作为一种广泛使用的除草剂,其神经发育毒性及机制尚未完全明确,且针对PQ对神经干细胞分化影响的研究较少。

    目的 研究PQ对神经干细胞分化趋势的影响,并探究神经干细胞分化结局良好的观察时点。

    方法 分离提取新生C57BL/6小鼠侧脑室下区原代神经干细胞(mNSCs)进行体外培养,使用0、10、20、40 μmol/L浓度PQ处理mNSCs 24 h后,采用MTT法检测细胞活力,并用DCFH-DA荧光探针检测细胞内活性氧(ROS)生成。PQ处理mNSCs 24 h后,更换不含PQ的分化培养基持续培养5 d,分别在培养1、3、5 d后镜下观察细胞分化状态,用免疫荧光检测PQ处理对mNSCs分化成神经元及星形胶质细胞的影响。

    结果 当PQ浓度为40 μmol/L时,细胞活力下降到对照组的86%,且ROS水平升高至对照组的1.67倍(P < 0.05)。mNSCs分化3 d后出现明显的细胞分层,经免疫荧光鉴定可知上层主要为神经元细胞。分化5 d后,上层细胞已经长出明显的神经突起样结构,而细胞数量较分化3 d后检测结果无肉眼可见差异。采用免疫荧光方法计数分化后的各种细胞数量,发现随着PQ染毒剂量的增加,神经元细胞比例呈现逐渐下降的趋势,且神经元与星形胶质细胞生成比也呈现逐渐降低的趋势,当PQ浓度为20、40μmol/L时差异有统计学意义(P < 0.05)。

    结论 PQ可抑制原代培养的mNSCs向神经元方向的分化,这种作用可能与PQ诱导细胞氧化应激相关。体外培养的mNSCs分化5 d可作为良好的研究干细胞分化的时点。

     

    Abstract: Background Paraquat (PQ) is a widely used herbicide. Its neurodevelopmental toxicity and relevant mechanism are not completely clear. Furthermore, few studies focus on the effects of PQ on neural stem cell differentiation.

    Objective This experiment is conducted to study the effects of PQ on the differentiation of neural stem cells and explore the appropriate observation time point of neural stem cell differentiation outcome.

    Methods The primary murine neural stem cells (mNSCs) were isolated from subventricular zone of neonatal C57BL/6 mice and cultured in vitro. The mNSCs were treated with different concentrations of PQ (0, 10, 20, and 40 μmol/L) for 24 h. Cell viability was detected by MTT assay; intracellular reactive oxygen species (ROS) was detected using DCFH-DA probe. After treatment with PQ for 24 h, the mNSCs were maintained in differentiation medium without PQ for 5 d, and observed for differentiation outcome under light microscope after 1, 3, and 5 d, respectively. Immunofluorescence was used to detect the effects of PQ on differentiated neurons and astrocytes.

    Results When being treated with 40μmol/L PQ, the cell viability decreased to 86% of the control group, and the ROS level increased to 167% of the control group (P < 0.05). After 3d of differentiation, the cells showed obvious cell stratification. The immunofluorescence identification showed that the upper layer was mainly neuron cells. After 5 d of differentiation, the upper-layer cells displayed a distinct neurite-like structure; however, the number of cells was not visually different from that of the differentiation after 3 d. By immunofluorescence method, it was found that PQ decreased the proportions of neuron cells in a dose-dependent manner; moreover, the ratio of neurons to astrocytes also decreased, and the difference was statistically significant between the 20 μmol/L and the 40 μmol/L PQ groups (P < 0.05).

    Conclusion PQ could inhibit the differentiation of primary mNSCs into neurons, which may be related to PQ-induced cellular oxidative stress. Five days could be a good time point to study mNSCs differentiation in vitro.

     

/

返回文章
返回