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目的:利用甲氨蝶呤(methotrexate,MTX)干预孕鼠,探讨MTX对早期神经胚基因表达的影响。方法:用MTX(4.5 mg/kg体重)干预孕鼠,通过NimbleGene表达谱芯片、Real time-PCR及免疫组化等方法进行差异表达基因的筛选和验证。结果:MTX处理后神经管畸形(NTDs)发生率为32.1%。表达谱芯片筛选出166个差异表达基因,其中4个凋亡相关基因(Endog,Trp53,Casp3,Bax)均表现为上调(fold change>1.5,P<0.05),3个增殖相关基因(Ptch1,Pla2g4a,Foxg1)均表现为下调(fold change<0.67,P<0.05);NTDs胚胎神经上皮Caspase-3表达显著升高(P<0.05),phospho-histone H3(pH3)表达显著降低(P<0.05)。结论:MTX影响了早期神经胚的基因表达,尤其是引起了凋亡、增殖相关基因表达的异常,这可能在叶酸缺乏引起NTDs发生的相关机制之一。
OBJECTIVE: To use methotrexate (MTX) to interfere pregnant rats and explore the effect of MTX on the gene expression of early neural germ. METHODS: Pregnant mice were treated with MTX (4.5 mg / kg body weight), and the differentially expressed genes were screened and validated by NimbleGene microarray, Real time-PCR and immunohistochemistry. Results: The incidence of neural tube defects (NTDs) after MTX treatment was 32.1%. 166 differentially expressed genes were screened by expression profiling. Four of the four apoptosis related genes (Endog, Trp53, Casp3, Bax) were upregulated (fold change> 1.5, P <0.05) Pla2g4a and Foxg1 were down-regulated (fold change <0.67, P <0.05). Caspase-3 expression was significantly increased in NTDs (P <0.05) and phospho-histone H3 ). CONCLUSION: MTX affects the gene expression of early neural embryos, especially apoptosis and proliferation-related gene expression, which may be one of the mechanisms involved in the development of NTDs induced by folic acid deficiency.