促进大鼠视网膜神经节细胞轴突再生的中药筛选研究

Screening out of traditional Chinese drugs on the effect of promoting regeneration of axon of the retinal ganglion cell in rat

目的比较葛根素、川芎嗪、银杏内酯B和西红花苷-I四种中药单体促进大鼠视网膜神经节细胞轴突(ARGC)再生的作用。方法将1~3日龄新生SD大鼠的视网膜切成0.5mm×0.5mm大小的小片,培养于体外三维立体培养系统,实验组分别加入不同浓度的葛根素、川芎嗪、银杏内酯B和西红花苷-I(0.125g/L、0.25g/L、0.5g/L、1.0g/L),对照组只加培养液。通过倒置的相差显微镜动态观察第3、6、9天ARGC再生数目及长度,比较不同药物及浓度对ARGC再生的作用,用抗大鼠Thy1.1和GAP-43抗体于第6天进行免疫组化染色检测再生轴突。结果与对照组相比,各实验组ARGC数目均明显增多、长度明显增加,其中银杏内酯B组作用更为明显(P<0.05)。免疫组化染色再生轴突标记物Thy1.1和GAP-43阳性,证明培养生长物为再生的大鼠ARGC。结论葛根素、川芎嗪、银杏内酯B和西红花苷-I均能促进体外培养的大鼠ARGC再生和伸长,其中银杏内酯B活性最强。

Objective To compare the effect of herb monomers (puerarin, tetramethylpyrazine, bilobalide-B and saffron-I ) on promoting regeneration of axons of retinal ganglion cell (ARGC) in rat. Methods The retina of SD rats aged 1-3d were cut into pieces of 0.5mm×0.5mm, and cultured in three-dimensional culture system. The tissues of experimental group were cultured in culture medium with different concentration of puerarin, tetramethylpyrazine, bilobalide-B and saffron-I (0.125g/L, 0.25 g/L, 0.5 g/L, 1.0 g/L), while control group only cultured with normal medium. The growth of RGCs axons were investigated by fluorescence inverted microscopy and immunohistochemical staining. The number and length of outgrowing RGC axons were calculated in the 3rd, 6th and 9th day. The effect of different herb monomers and concentrations on regeneration of ARGC were compared. The regeneration axons with Thy-1.1 and GAP-43 by immunohistochemical staining in the 6th day were tested. Results The number of regrowing axons of all experimental groups were larger and the length was longer than which in the control group, among which the effect of bilobalide-B group was the most obvious (P<0.05). Thy-1.1 and GAP-43 double positive fibers shown to be regenerated axons. Conclusion Puerarin, tetramethylpyrazine, bilobalide-B and saffron-I could improve the regeneration and elongation of cultured RGCs axons, and bilobalide-B could be the most effective one.