【摘要】 目的:观察甲氨蝶呤(MTX)对体外培养的神经干细胞(NSC)毒性作用的特点,并初步探讨其毒性机制。方法:体外培养胎鼠大脑皮质NSCs,采用Nestin免疫荧光法进行鉴定;光镜下观察MTX干预后NSC形态变化;采用5-溴脱氧尿苷(BrdU)掺入法和Nestin/BrdU免疫组化双标记检测MTX对NSCs增殖的影响;流式细胞术分析细胞周期变化;高压液相法检测细胞内同型半胱氨酸(Hcy)的浓度。结果:400μg/LMTX可引起明显的细胞形态改变;免疫荧光双标记结果显示,与对照组相比,MTX干预组可明显抑制NSCs增殖(P<0.05);MTX干预48h后,细胞停留在G1期的比例比空白对照组明显增加;MTX还可引起细胞内Hcy浓度升高。结论:MTX可能通过改变细胞周期和升高细胞内Hcy浓度而对体外培养NSCs产生明显的毒性损伤作用。更多还原

【Abstract】 Objective:To study the toxic effects induced by methotrexate on primary cultured neural stem cells from fetal rats in vitro,and explore the possible mechanism for toxic effects.Methods:NSCs were identified by immunofluorescent staining using antibody against Nestin.Morphological observation were used to evaluate the toxicity of methotrexate on NSCs by light microscope.BrdU incorporating method and Nestin/BrdU double-label immunofluorescence technique were used to detect NSCs proliferation under the treatment of methotrexate.The process of cell cycle was analyzed by flow cytometry.High per-formance liquid chromatography(HPLC) was used to detect Hcy concentration in NSCs.Results:In the serum-free suspension medium,neurosperes that consisted of a great number of Nestin-positive cells could be obtained.Cell morphology was changed after being exposed to 400 μg/L methotrexate for 48 h.The number of Nestin positive cells in total cells in methotrexate group was significantly lower than that in control group(P < 0.05).Compared with control group,double-label immunofluorescence for Nestin/BrdU manifested that the growth tendency was decreased significantly after treated with 400 μg/L methotrexate for 48 h(P < 0.05).The proportion of MTX-treated NSCs rested on G1 phase was significantly increased after 48 h culture.The concentration of Hcy in MTX group was much higher than that in control group.Conclusion:NSCs with methotrexate treatment decreased the growth and viability.Methotrexate could produce an obvious toxic injury to NSCs by conducting a cell cyclespecific mechanism and increasing homocysteine concentration.更多还原