骨髓间充质干细胞移植对缺血-再灌注损伤视网膜中Fas／FasL及caspases-3表达的影响

背景视网膜缺血-再灌注损伤（RIRI）严重影响视力，其损伤机制是视网膜细胞的凋亡，治疗效果不佳。研究证实骨髓间充质干细胞（BMSCs）视网膜下移植后可显著减轻RIRI，而BMSCs视网膜移植所产生的神经保护机制是否与其抑制凋亡作用有关目前尚不清楚。目的观察BMSCs视网膜下移植对RIRI大鼠视网膜中凋亡相关因子Fas／FasL和caspases-3蛋白表达的影响，探讨BMSCs移植治疗RIRI的神经保护机制。方法无菌条件下分离SD大鼠的股骨和胫骨骨髓，离心收集细胞后用DMEM低糖培养液制成骨髓细胞悬液，体外培养大鼠BMSCs，并以1×10^6～2×10^6个／ml的细胞密度和1：2进行传代。64只清洁级健康成年SD大鼠按随机数字表法随机分为正常对照组、模型对照组、BMSCs移植组及PBS对照组，每组16只。用视神经线栓法制备大鼠视网膜RIRI模型，BMSCs移植组大鼠于造模后24h视网膜下注入5×10^4个活BMSCs，PBS对照组以同样的方式注入PBS。用颈椎脱臼法处死大鼠，制备16μm厚视网膜切片。采用免疫组织化学法动态观察BMSCs移植后6、24、48、72h各组大鼠视网膜中Fas、FasL及caspase-3蛋白表达的变化。结果BMSCs视网膜下移植后72h，荧光显微镜观察可见视网膜下Hoechst33324阳性细胞。BMSCs视网膜下移植后6、24、48、72h，正常对照组大鼠视网膜仅见微量Fas、FasL和caspase3的阳性表达；BMSCs视网膜下移植后各时间点，模型对照组与BMSCs移植组大鼠视网膜均可见Fas、FasL、caspase-3阳性表达细胞，免疫组织化学染色强度和细胞数量值均明显高于正常对照组，差异均有统计学意义（均P〈0．01）。随着BMSCs移植时间的延长，各组大鼠视网膜中Fas、FasL及caspase-3阳性细胞数均逐渐下降，各时间点BMSCs移植组大鼠视网膜中Fas、FasL、caspase-3的阳性表达值均明显低于模型对照组和PBS对照组大鼠，差异均有统计学意义（P〈0．05），但各时间点模型对照组与PBS对照组间大鼠视网膜中Fas、FasL、caspase-3阳性细胞数变化的差异均无统计学意义（均P〉0．05）。结论视网膜下移植BMSCs可下调RIRI大鼠视网膜中凋亡相关蛋白Fas、FasL及caspase-3的表达，从而减轻RIRI大鼠视网膜神经节细胞的凋亡。

The effect of bone marrow mesenchymal stem cells transplantation on the expressions of retinal Fas/FasL and caspase-3 after ichemic reperfusion injury

Background Retinal ichemic-reperfusion injury （RIRI） affects vision and life quality and has no ideal treatment outcome up to now. The mechanism of RIRI is associated with apoptosis. Researches showed that subretinal transplantation of bone mesenchymal stem cells （BMSCs） can relieve RIRI,but its mechanism is unclear. Objective This study was to observe the influence of BMSCs subretinal transplantation on the expressions of apoptosis protein Fas/FasL, caspases-3 after RIRI, and to explore the neuroprotecitve mechanism of BMSCs transplantation to treat RIRI. Methods Bone marrow was isolated and extract form femur and tibia of SD rat by the density gradient centrifugation method and then the BMSCs were collected. BMSCs suspension was prepared using DMEM containing low-glucose with the cell density 1 ×10^6-2×10^6/ml. RIRI models were established by ligation of optical nerve （Daniel M method）. Sixty-four SD rats were randomly divided into the normal control group, model control group,BMSCs transplantation group and PBS control group. Twenty-four hours after RIRI, BMSCs suspension with the cells density 5×10^4 cells was subretinally injected in the rats of the BMSCs transplantation group, and equal volume of PBS was injected in the same way in the rats of the PBS control group. The expressions of Fas/FasL and caspase-3 were dynamically detected using immunohistochemistry at 6,24,48 and 72 hours after BMSCs transplantation. Results The positive response cells for Hoechst33324 were seen in subretlna 72 hours after BMSCs transplantation. A few of positive cells for Fas, FasL, easpase-3 proteins were expressed in the rats of the normal control group at 6,24,48 and 72 hours after BMSCs transplantation. However,the positive cells for Fas, FasL, caspase-3 protein were significantly increased in the model control group and the BMSCs transplantation group compared with the normal control group at various time points after injection （ all at P〈0.01 ） , and the number of positive cells was significantly less in the BMSCs group than those of the RIRI group and the BMSCs transplantation group （P〈0. 05, P〈0. 01 ）. No significant differences were found in the numbers of positive cells of Fas, FasL and easpase-3 proteins at various time points between the model control group and the PBS control group （ all at P〉 0. 05）. Conclusions The subretinal transplantation of BMSCs down-regulate the expression of Fas/FasL and caspases-3 proteins in RIRI rats,and thus alleviate the apoptosis of retinal neural cells.