GAP-43在新生大鼠视网膜及其诱导的骨髓间充质干细胞向视网膜神经节样细胞分化过程中的表达

GAP-43 expression in retina of neonate rat and induced differentiation of bone marrow mesenchymal stem cells to retinal ganglion-like cells in vitro

目的探讨生长相关蛋白-43(growth associated protein,GAP-43)在新生大鼠视网膜发育过程中及在以新生大鼠视网膜细胞诱导体外培养的骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSC)向视网膜神经节样细胞定向分化中的作用。方法体外培养新生3d大乳鼠视网膜细胞,应用免疫细胞化学方法检测GAP-43的表达;提取新生大鼠视网膜细胞总RNA,应用RT-PCR方法检测GAP-43 mRNA的表达。体外培养大鼠BMSC至第3代,经流式细胞仪检测干细胞表面标志物进行鉴定,应用乳鼠视网膜细胞诱导BMSC分化,观察诱导后细胞的形态学变化,并应用免疫细胞化学方法对诱导后第5天的细胞进行神经元及视网膜神经节细胞标志物NSE、nestin、GAP-43、thy1.1鉴定,并应用RT-PCR的方法检测、分析GAP-43 mRNA的表达情况。结果体外培养的新生大乳鼠视网膜细胞应用免疫细胞化学方法可检测到GAP-43阳性表达;RT-PCR方法检测到新生大鼠的GAP-43 mRNA的表达;第3代BMSC经流式细胞仪检测CD71(+)、CD29(+)、CD34(-)、CD45(-),经与大乳鼠视网膜细胞共培养的方法可诱导BMSC分化为视网膜神经节样细胞,免疫细胞化学染色表明其表达特异性标志物NSE、nestin、GAP-43、thy1.1,分化后的神经节样细胞内可检测到GAP-43 mRNA阳性表达。结论本研究结果显示GAP-43不仅参与了大鼠视网膜发育过程,且GAP-43在以乳鼠视网膜细胞诱导BMSC定向分化为视网膜神经节样细胞这一过程中具有重要意义。

Objective To study the effect of growth associated protein -43（GAP-43） expression in retinal development of neonate rat and differentiation of bone marrow mesenchymal stem cells（BMSC） to retinal ganglion-like cells induced by retinal cells in vitro.Methods Retinal cells of neonate rat with 3 days old were cultivated to detect GAP-43 expression by immunocytochemistry,the total RNA of retinal cells were extracted and GAP-43 mRNA expression were detected by RT-PCR.BMSC were cultivated in vitro and passaged to the 3rd generation,the surface maker of BMSC were identified by flow cytometer.BMSC were induced and differentiated by co-cultivation with retinal cells of neonate rat.Morphological changes of induced cells were observed,and NSE,nestin,GAP-43,and thy1.1 of induced cells were detected by immunocytochemistry after co-cultivation on the 5th day.Results Positive expression of GAP-43 could be detected by immunocytochemistry in cultivated retinal cells of neonate rat,and GAP-43 mRNA expression also could be found in retina of neonate rat by RT-PCR.The 3rd generation of BMSC were identified with CD71（＋）,CD29（＋）,CD34（-）,and CD45（-） by flow cytometer.BMSC could be induced and differentiated into retinal ganglion-like cells by co-cultivation with retinal cells of neonate rat,which was proved that the expression of NSE,thy1.1,nestin and GAP-43 were positive.The positive expression of GAP-43 mRNA could be detected in retinal ganglion-like cells after differentiation.Conclusion GAP-43 not only participates in the retinal development of neonate rat,but also plays an important role in the differentiation of BMSC to retinal ganglion-like cells induced by the retinal cells of neonate rat.