反式维甲酸诱导显性脊柱裂胎鼠骶尾部神经前体细胞凋亡和增殖的变化规律研究

Disturbed apoptosis and cell proliferation in developing neuroepithelium of lumbo-sacral neural tubes in retinoic acid-induced spina bifida aperta in rat

目的观察大鼠胚胎脊柱裂发生早期,细胞凋亡与细胞增殖的变化规律。方法孕鼠随机分为对照组和实验组。胚胎10天时,实验组1次性经胃管注入致畸量反式维甲酸诱导产生脊柱裂动物模型,对照组胃饲等量溶剂,分别在妊娠11、12、13天(E11,E12,E13)时剖宫取胚胎,一部分胚胎固定后进行全胚胎TUNEL染色观察其整体凋亡情况;另一部分胚胎常规制作石蜡切片,采用TUNEL切片染色和免疫荧光染色技术,检测胚胎脊部神经管组织中细胞凋亡和细胞增殖的变化。结果与对照组相比,细胞凋亡于多个发育部位明显增多,集中表现在颅面原基、颅部神经管的背外侧、骶尾部神经管的背部中线。免疫荧光染色显示,与对照组相比,脊柱裂组胚胎畸形发生部位的神经前体细胞的凋亡率升高[E11(2.02±0.52)%与(0.57±0.23)%,E12(3.56±0.33)%与(0.93±0.14)%,E13(3.76±0.37)%与(1.24±0.21)%,P<0.001]。而细胞增殖降低[E11(65.17±2.30)%与(81.76±2.17)%,E12(63.97±3.03)%与(76.98±5.14)%,E13(56.86±2.80)%与(73.43±1.99)%,P<0.001]。结论反式维甲酸诱导的大鼠脊柱裂胚胎骶尾部神经管中神经前体细胞凋亡增多,而细胞增殖减少,这可能是脊柱裂胚胎神经元发育异常的主要原因之一。

Objective To investigate the cell apoptosis in the whole embryosand proliferation of neural progenitor cells in the spinal neural tube during neurulation in all-trans retinoic acid (atRA)-induced spina biifda in fetal rats.MethodSpina biifda was induced by atRA in fetal rats. Cell apoptosis and cell proliferation were assessed using TUNEL labeling technique on both whole-mount and serially sectioned embryos and mitotic markers (Ki67) assay, respectively.ResultsAn excess of apoptosis in the neuroepithelium of embryos with spina bifida was found, which became more marked as embryos progress from E11 to E13[ E11: (2.02±0.52)% vs (0.57± 0.23)%; E12: (3.56±0.33)% vs (0.93±0.14)%; E13: (3.76±0.37)% vs (1.24±0.21)%;P<0.001]. Conversely, there was a reduction in cell proliferation in spina bifida embryos, with a progressively greater difference from controls with stage from E11 to 13 [ E11: (65.17±2.30)% vs (81.76± 2.17)%; E12: (63.97±3.03)% vs (76.98±5.14)%; E13: (56.86±2.80)% vs (73.43±1.99)%;P <0.001].Conclusions AtRA-induced spina biifda in rat embryos shows increased apoptosis and decreased proliferation in the neuroepithelium of the lumbo-sacral neural tube in rat, which might contribute to the pathogenesis of neuronal deifcits of spina biifda.