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目的:探讨垂体同源盒家族因子3(Pitx3)基因在SD大鼠脊髓不同年龄阶段的表达变化以及细胞定位。方法:采用半定量RT-PCR和Western Blot检测不同年龄组大鼠脊髓中Pitx3 mRNA及蛋白水平表达变化;采用免疫荧光和免疫组化染色显示Pitx3在脊髓中的细胞定位。结果:(1)RT-PCR和Western Blot检测显示Pitx3基因在各年龄组脊髓各段均有表达;大鼠脊髓发育过程中Pitx3 mRNA及其蛋白水平出生前逐渐升高,在出生后(P0 d)达高锋,以后维持在一定的生理水平,且出生以后随年龄的增长表达呈下调趋势;(2)免疫荧光双标证实Pitx3蛋白在P0 d大鼠脊髓主要定位于脊髓灰质,与神经元的标记物NeuN存在共定位;免疫组化检测显示Pitx3蛋白集中分布于各年龄组脊髓灰质腹角运动神经元胞质中,少量分布于背角和侧角神经元胞质中。结论:在大鼠脊髓发育和生存维持过程中,Pitx3在基因和蛋白水平呈现明显的时相变化;提示Pitx3基因的表达在脊髓腹角运动神经元的发育和生存维持中可能发挥一定的调控作用。
Objective: To investigate the expression and location of Pitx3 gene in different age stages of SD rat spinal cord. Methods: The expression of Pitx3 mRNA and protein in the spinal cord of different age groups were detected by semi-quantitative RT-PCR and Western Blot. The cell localization of Pitx3 in the spinal cord was detected by immunofluorescence and immunohistochemistry. Results: (1) The expression of Pitx3 gene in all stages of spinal cord was detected by RT-PCR and Western Blot. The level of Pitx3 mRNA and protein in spinal cord during the development of the spinal cord increased gradually at birth, ) Up to Gao Feng, maintained at a certain physiological level after birth, and with the increase of age after birth, the expression showed a downward trend; (2) double immunofluorescence confirmed that Pitx3 protein in spinal cord of P0 d locates mainly in the spinal cord gray matter, and neurons Immunohistochemistry showed that Pitx3 protein was localized in the cytoplasm of spinal motor neurons in the gray matter of the gray matter in various age groups and was localized in the cytoplasm of dorsal horn and lateral horn neurons. CONCLUSION: Pitx3 gene and protein levels show significant time-phase changes in spinal cord development and survival in rats. It suggests that the expression of Pitx3 gene may play a regulatory role in the development and survival of motor neurons in the ventral horn of spinal cord .