甲氨蝶呤对体外培养神经干细胞的毒性作用

Toxic Effects of Methotrexate on Primary Cultured Neural Stem Cells of Fetal Rats in Vitro

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。

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 fow cytometry. High performance 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 ug/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 ug/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.