神经管畸形胎鼠中神经干细胞增殖分化及H3K27me3表达的变化

Proliferation and Differentiation of Neural Stem Cells and Change of H3K27me3 Expression in Neural Tube Defects Rats

目的建立全反式维甲酸(all-trans retinoic acid,ATRA)诱导神经管畸形(neural tube defects,NTDs)鼠神经干细胞无血清培养法,了解神经管发育过程中神经干细胞的变化规律及叶酸的干预作用,并观察H3K27me3在NTDs中表达的变化。方法分别取妊娠第15.5天的正常、实验及叶酸干预后的胎鼠神经管分离培养神经干细胞,采用免疫细胞化学鉴定;用神经球计数、直径测量及MTT法检测各组神经干细胞生长情况,免疫细胞化学法分析各组细胞分化情况,Western blot法测定H3K27me3在各组细胞中的表达情况。结果正常组、实验组和叶酸干预组,200倍镜下取5个视野计数神经球数,其平均值分别为(29.4±2.07)、(17.8±2.17)和(23.4±1.52)个,3组间差异有统计学意义(P<0.05);正常组、实验组和叶酸干预组神经干细胞直径的平均值分别为(140.2±4.82)、(106.6±3.85)和(121.8±4.09)μm,3组间差异有统计学意义(P<0.05);正常组、实验组和叶酸干预组神经干细胞诱导分化后Nestin表达量分别为(6±0.9)%、(32±3)%、(21±4)%(P<0.05);实验组中H3K27me3明显高表达。结论 ATRA致NTDs的发生与神经干细胞的数目减少、增殖分化抑制及H3K27me3的高表达密切相关。

Objective To establish all- trans retinoic acid( all- trans retinoic acid,ATRA) induction of neural tube defects( neural tube defects,NTDs) serum- free culture method of the rat neural stem cells,to investigate the changing rule of the neural stem cells in the development of neural tube and the intervention effect of folic acid and to observe H3K27me3 expression changes in NTDs. Methods E15. 5d normal,deformity and folic acid intervention fetal rat neural tube cultivation of neural stem cells were taken respectively and identified using immunocytochemistry Neural stem cell growth was detected with neural ball count,diameter measurement and determined by MTT method. Differentiation of cells was analyzed with immunocytochemical and H3K27me3 expression in cells of each group was determined with Western blot. Results The average count neurosphereswere( 29. 4 ± 2. 07) cells,( 17. 8 ± 2. 17) cells,( 23. 4 ± 1. 52) cells,in normal,deformity,folic acid intervention group and the difference among three groups was statistically significant( P <0. 05). The average diameter of neural stem cells in three groups were( 140. 2 ± 4. 82) μm,( 106. 6 ±3. 85) μm and( 121. 8 ± 4. 09) μm,respectively and the difference among three groups was statistically significant( P < 0. 05). Nestin expression after induction of differentiation of three groupswere respectively( 6 ±0. 9) %,( 21 ± 4) %,( 32 ± 3) %( P < 0. 05). H3K27me3 expression in defects group was significantly higher than that in other groups. Conclusion The NTDs induced by the ATRA and reduced numbers of neural stem cells proliferation differentiation inhibiting and high expression of H3K27me3 are closely related.