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

[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、40μmol/L浓度PQ处理m NSCs 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40μmol/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)。m NSCs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。

[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 C57 BL/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 40 μmol/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 3 d 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 cellsdisplayed 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.