补肾益精方对外周血BMSCs在干性ARMD小鼠中视网膜分化及CNTF表达的影响

Effect of Bu Shen Yi Jing Fang on differentiation of peripheral blood stem cells and expression of ciliary neurotrophic factor in retina of dry ARMD mouse model

目的:观察补肾益精方对外周血移植骨髓间充质干细胞(BMSCs)在视网膜分化及睫状神经营养因子(CNTF)表达的影响,探讨补肾益精方治疗干性年龄相关性黄斑变性(ARMD)的作用机制。方法:将24只C57BL/6小鼠以碘酸钠(NaIO3)尾静脉注射的方法建立干性ARMD模型。造模后第1d,尾静脉注射3×106个绿色荧光蛋白标记的骨髓间充质干细胞(GFP-BMSCs),按随机数字表法分为蒸馏水组(12只,给予蒸馏水灌胃)及补肾益精方组(12只,给予补肾益精方灌胃)。另选12只健康C57BL/6小鼠常规饲养作为正常组。治疗14d时,采用免疫荧光染色观察GFP-BMSCs在视网膜的分化情况;免疫荧光染色及实时荧光定量PCR检测CNTF在视网膜中的表达情况。结果:免疫荧光染色显示补肾益精方组胶质纤维酸性蛋白(GFAP)及GFP双染细胞阳性率高于蒸馏水组(P<0.01);两组视网膜色素上皮细胞特异蛋白65(RPE65)及GFP双染细胞阳性率无差异(P>0.05);两组均未见视紫红质(Rhodopsin)与GFP双染阳性细胞。免疫荧光染色及实时荧光定量PCR显示补肾益精方组CNTF的表达高于蒸馏水组(P<0.05)。结论:补肾益精方能够促进外周血BMSCs在视网膜分化为胶质细胞,并促进CNTF的表达,这可能是补肾益精方治疗干性ARMD的作用机制之一。

AIM:To explore the differentiation of bone marrow-derived mesenchymal stem cells from peripheral blood to the retina and the expression of ciliary neurotrophic factor(CNTF).We also investigate the mechanism by which Bu Shen Yi Jing Fang could treat dry age-related macular degeneration(ARMD).METHODS:C57BL/6 mice were administered with sodium iodate(NaIO3)by tail intravenous injection.One day after modeling,3×106 green fluorescent protein labeled bone marrow-derived mesenchymal stem cells(GFP-BMSCs)were injected into the tail vein.The injected mice were randomly divided into distilled water group and Bu Shen Yi Jing Fang group according to random number table,and 12 mice in each group.The mice were intragastrical administrated with either Bu Shen Yi Jing Fang solution or distilled water every day.Twelve healthy C57BL/6 mice were fed regularly as the normal group.At 14d after the treatment,the differentiation of GFP-BMSCs in retina was determined by immunofluorescence,and the expression of CNTF in the retina was detected by immunofluorescence and quantitative real-time PCR.RESULTS:Immunofluorescence staining showed that there were more glial fibrillary acidic protein(GFAP)and GFP double-stained positive cells in the Bu Shen Yi Jing Fang group than in the distilled water group(P<0.01),and the positive rate of retinal pigment epithelium 65(RPE65)was not significantly different between two groups(P>0.05).There were no Rhodopsin and GFP double-stained positive cells in the two groups.Immunofluorescence and quantitative real-time PCR showed that the expression of CNTF in the Bu Shen Yi Jing Fang group was higher than which in the distilled water group(P<0.05).CONCLUSION:Bu Shen Yi Jing Fang facilitated the differentiation of peripheral blood stem cells into glial cells in the retina and the expression of CNTF,which might be one of the mechanisms of Bu Shen Yi Jing Fang in the treatment of dry ARMD.