色素上皮衍生因子基因修饰的人脐带间充质干细胞对大鼠视网膜缺血-再灌注损伤的保护作用

Protective effects of PEDF gene modified human umbilical cord mesenchymal stem cells in rat with retinal ischemia-reperfusion injury

目的观察色素上皮衍生因子色素上皮衍生因子(pigment epithelial-derived factor,PEDF)基因修饰的人脐带间充质干细胞(pigment epithelial-derived factor gene-odified human umbilical cord mesenchimal stem cells,PEDF-MSCs)对大鼠视网膜缺血-再灌注损伤(retinal ischemia-reperfusion injury,RIRI)模型中PEDF、血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的影响及对视网膜神经节细胞的保护作用。方法绿色荧光蛋白(green fluorescent protein,GFP)标记的慢病毒(lentivirus,LV)为载体,将PEDF基因以感染复数(multiplicity of infection,MOI)为1、10、20、50体外转染至人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs)中,应用RT-PCR及ELISA法分别检测以最佳MOI值转染细胞中PEDF的表达情况。选取健康SD雄性大鼠,采用高眼压的方法制成RIRI模型,随机分为正常对照组(N组)、磷酸盐缓冲液(phosphate bufferedsaline,PBS)治疗组(PBS组)、hUCMSCs治疗组(M组)及PEDF基因转染的hUCMSLs治疗组(P组);N组不予特殊处理,其余三组大鼠造模成功后立即进行干预治疗,PBS组给予PBS治疗,P组与M组分别给予PEDF-MSCs、hUCMSCs治疗。5 d后处死大鼠,尼氏染色计数视网膜神经节细胞数目,计算视网膜内层厚度,RTMPCR检测大鼠视网膜PEDF和VECGF的mRNA表达情况。结果荧光显微镜下观察到转染率高达75.8%。RT-PCR结果显示,转染后PEDF-MSCs的上清液中PEDF mRNA相对表达量(4.34±0.29)明显高于hUCMSCs(1.08±0.15),差异有统计学意义(P<0.05)。ELISA检测结果显示,转染后PEDFMSCs上清液中PEDF蛋白表达量[(83.09±7.58)μg·L^(-1)]明显高于hUCMSCs[(12.30±1.24)μg·L^(-1)],差异有统计学意义(P<0.05)。尼氏染色结果显示造模后PBS组大鼠神经节细胞数量变少,治疗5 d后,P组、M组大鼠神经节细胞数量均高于PBS组,差异均有统计学意义(均为P<0.05);P组高于M组,差异有统计学意义(P<0.05);治疗5 d后,P组、M组大鼠视网膜厚度均厚于PBS组,差异均有统计学意义(均为P<0.05);P组厚于M组。RT-PCR检测结果显示,P组PEDF mRNA表达水平显著升高,VEGF mRNA表达水平下降,与PBS组、M组相比差异均有统计学意义(均为P<0.05)。结论玻璃体内注射PEDF-MSCs能上调RIRI模型大鼠视网膜PEDF表达,并下调VEGF表达,对RIRI大鼠视网膜神经节细胞有保护作用。

Objective To observe the effects of pigment epithelial-derived factor gene-modified human umbilical cord mesenchymal stem cells(PEDF-MSCs)on the expression of pigment epithelial-derived factor(PEDF)and vascular endothelial growth factor(VEGF)in a rat model of retinal ischemia-reperfusion injury(RIRI)and its protection on retinal ganglion cells.Methods Lentivirus(LV)labeled with green fluorescent protein(GFP)was used as a vector to transfect the PEDF gene into human umbilical cord mesenchymal stem cells(hUCMSCs)at a multiplicity of infection(MOI)of 1,10,20,and 50 in vitro,and then the expression of PEDF gene and protein in cells transfected with the best MOI value was detected by RT-PCR and ELISA.The healthy male SD rats were randomly divided into normal group(N group)and experimental group.The RIRI model was made by high intraocular pressure in the experimental group,and the RIRI rats with PBS treatment were allocated as the PBS group,with hUCMSCs treatment as M group and LV-PEDF-MSCs treatment as P group,and the N group was left untreated.All rats were sacrificed on day 5,and the number of retinal ganglion cells were counted by Nissl staining,the thickness of the retina was calculated,as well as the expression of PEDF and VEGF mRNA in rat retina was detected by RT-PCR.Results The transfection efficiency was as high as 75.8%under fluorescence microscope.The results of RT-PCR showed that the relative expression of PEDF mRNA in PEDF-MSCs supernatant(4.34±0.29)was significantly higher than that in hUCMSCs(1.08±0.15),and the difference was statistically significant(P<0.05);moreover,the results of ELISA showed that PEDF protein expression in PEDF-MSCs supernatant[(83.09±7.58)μg·L-1]was significantly higher than that in hUCMSCs[(12.30±1.24)μg·L-1],and the difference was statistically significant(P<0.05).Nissl staining results showed that the number of ganglion cells in group PBS decreased after model establishment.After 5 days of treatment,the number of ganglion cells in P group and M group was higher than that in PBS group,and the difference was statistically significant(both P<0.05);and P group was higher than M group,with the significant difference(P<0.05).And this was true of the thickness of the retina.RT-PCR showed that the expression of PEDF mRNA in P group was significantly up-regulated,but VEGF mRNA expression was significantly down-regulated,and the differences were statistically significant when compared with PBS and M group(both P<0.05).Conclusion Intravitreal injection of PEDF-MSCs can up-regulate the expression of PEDF but down-regulate the expression of VEGF in the retina of RIRI rats,which can protect the retinal ganglion cells against RIRI