补肾益精方对外周血干细胞在RCS大鼠视网膜上的募集及睫状神经营养因子表达的影响

Bu Shen Yi Jing Fang Recruitment of Peripheral Blood Stem Cells to the Retina of RCS Rats and Expression of Retinal Ciliary Neurotrophic Factor

目的:观察补肾益精方对外周血骨髓间充质干细胞在视网膜变性模型(RCS)大鼠视网膜募集及睫状神经营养因子(CNTF)分泌的影响,探讨补肾益精方治疗视网膜退行性病变的作用机制。方法:实验研究。采用尾静脉注射方式将1×107个GFP标记的骨髓间充质干细胞(GFP-MSCs)注射至3周龄RCS大鼠外周血,然后按随机数字表法分为蒸馏水组及补肾益精方(BSYJ)组,每组39只。注射后第1天开始分别采用蒸馏水及补肾益精方药液灌胃,正常SD大鼠作为对照组常规饲养。给药7d、14d及28d时分别采用视网膜铺片、免疫荧光显微镜观察GFP-MSCs在视网膜的募集情况;实时荧光定量PCR及免疫荧光双染技术检测视网膜表达CNTF的情况;TUNEL法检测视网膜细胞凋亡情况;采用单因素方差进行数据分析。结果:给药7d、14d和28d时,蒸馏水组GFP-MSCs的数目少于BSYJ组,其中给药28d时,2组差异有统计学意义(t=2.71,P=0.001)。给药7d、14d和28d时,BSYJ组CNTF表达较蒸馏水组均明显增加,3组差异有统计学意义(F=5.763,P=0.003;F=3.165,P=0.042;F=4.839,P=0.004)。给药7d和28d时,BSYJ组视网膜细胞凋亡率明显少于蒸馏水组,3组差异有统计学意义(F=0.002,P=0.001;F=0.307,P=0.004)。结论:补肾益精方能够促进外周血干细胞在视网膜上的募集以及促进CNTF的表达,这可能是补肾益精方延缓RCS大鼠视网膜细胞凋亡和病变进程的作用机制之一。

Objective:To measure recruitment of bone marrow-derived mesenchymal stem cells from peripheral blood to the retina of royal college of surgeon(RCS)rats and the expression of ciliary neurotrophic factor(CNTF).We also investigated the mechanism by which Bu Shen Yi Jing Fang(BSYJ)could treat retinal degenerative diseases.Methods:This was an experimental study.RCS rats were injected with 1×107 green fluorescent protein-(GFP-)labeled bone marrow-derived mesenchymal stem cells(GFP-MSCs).The injected rats were assigned to a distilled water group or a BSYJ group according to selection from a random number table,and 39 rats in each group.Each rat in the respective groups was gavaged with either distilled water or BSYJ solution one day after injection.Normal SD rats were treated routinely as the control group.At 7,14,and 28 days after treatment,retina flat mounts were prepared to assess the recruitment of GFP-MSCs to the retina.Quantitative polymerase chain reaction(Q-PCR)and immunofluorescence double labeling were used to determine the effect of BSYJ on expression of ciliary neurotrophic factor(CNTF)in the retina.Apoptosis of retinal cells was measured by the TUNEL method.Data were analyzed using ANOVA.Results:At 7,14,and 28 days after treatment,the numbers of GFP-MSCs in the distilled water group were less than in the BSYJ group,and the difference was significant at 28 days(t=2.71,P=0.001).At 7,14,and 28 days after treatment,CNTF mRNA expression in the BSYJ group was significantly higher than in the distilled water group(F=5.763,P=0.003;F=3.165,P=0.042;F=4.839,P=0.004).At 7 and 28 days after treatment,the apoptosis rate of retinal cells in BSYJ group was significantly less than in the distilled water group(F=0.002,P=0.001;F=0.307,P=0.004).Conclusions:BSYJ improved recruitment of peripheral blood stem cells to the retina and increased CNTF expression.The recruitment of stem cells by BSYJ may delay progression of retinal cell apoptosis and degeneration in RCS rats.