人脐带间充质干细胞源外泌体对高糖环境下RPE细胞自噬和VEGF表达的影响

Effect of human umbilical mesenchymal stem cells derived exosomes on autophagy and expression of VEGF of RPE cells under a high-glucose condition

目的:探讨人脐带间充质干细胞(hUMSC)源外泌体对高糖环境下ARPE-19细胞自噬及VEGF表达情况的影响。方法:原代培养hUMSC,分离纯化hUMSC外泌体,Western blot鉴定hUMSC外泌体标志蛋白CD63表达,透射电镜观察外泌体形态。将体外培养的ARPE-19细胞随机分为对照组、高糖组和外泌体+高糖组(75μg/mL外泌体预处理)。培养48h后,电镜观察三组细胞内自噬体的超微结构,MTT法检测各组细胞增殖情况,Western blot检测各组细胞中自噬标志性蛋白LC3B、Beclin-1及p62的表达,ELISA法检测细胞上清液中VEGF的质量浓度。结果:分离纯化后的外泌体为直径30~100nm的球状膜性囊泡,表达特异性标识蛋白CD63。外泌体+高糖组中自噬体的数量明显少于高糖组。外泌体+高糖组的细胞增殖率较高糖组明显升高(P<0.01)。对照组、高糖组、外泌体+高糖组细胞中LC3B-Ⅱ/Ⅰ、Beclin-1和p62值分别为(0.214±0.019、0.461±0.067和0.332±0.079),(0.186±0.029、0.615±0.044和0.464±0.046)和(0.771±0.051、0.364±0.016和0.547±0.039)。高糖组细胞中LC3B-Ⅱ/Ⅰ值和Beclin-1值均明显高于对照组和外泌体+高糖组(均P<0.05),高糖组细胞中p62值明显低于对照组和外泌体+高糖组(均P<0.01),外泌体+高糖组VEGF的质量浓度较高糖组明显降低(P<0.01)。结论:高糖环境激活ARPE-19细胞自噬,并促进其VEGF的表达。hUMSC外泌体可有效抑制高糖环境下ARPE-19细胞自噬水平,并下调VEGF的表达。

AIM:To investigate the effect of human umbilical mesenchymal stem cells(hUMSC)exosomes on the autophagy and expression of vascular endothelial growth factor(VEGF)of ARPE-19 cells under the condition of high glucose.METHODS:Tissue explants adherent culture was adopted to cultivate umbilical cord mesenehymal stem cells.The ultrastmcture of exosomes was observed under transmission electron microscope and the expression of surface specific marker proteins CD63 on exosomes were determined by Western blot.ARPE-19 cells were divided into the control group,high-glucose group and exosomes+high-glucose group.The cells in the exosomes+high-glucose group were pretreated with 75μg/mL exosomes.The ultrastructure of autophagosomes formed in all cells of the three groups was detected under transmission electron microscopy.The viability of ARPE-19 cells was measured by MTT assay.Western blot was applied to detect the expression of autophagy-associated proteins in different groups,including microtubule related protein 1 light chain 3B(LC3B),Beclin-1 and p62.The expression of VEGF in the cell supernatants was examined by ELISA assay.RESULTS:Transmission electron microscopy showed that the shape of hUMSC exosomes was spherical with membranous structure.The diameter of exosomes ranged from 30-100nm.Western blot indicated that the surface marker protein CD63 of exosomes showed positive expression.The number of autophagosomes in high glucose group was more than that in control group,while it was less in exosome+high-glucose group than in high glucose group.Compared with the high-glucose group,the cell proliferation rate of exosomes+high-glucose group was significantly increased(P<0.01).In the control group,high-glucose group and exosomes+high-glucose group,the LC3B-Ⅱ/LC3B-Ⅰratio in ARPE-19 cells was 0.214±0.019,0.461±0.067 and 0.332±0.079,respectively;the relative expression of Beclin-1 protein in the cells was 0.186±0.029,0.615±0.044 and 0.464±0.046,respectively;the relative expression of p62 protein in the cells was 0.771±0.051,0.364±0.016 and 0.547±0.039,respectively.Compared with the control group and exosomes+high-glucose group,the ratio of LC3B-Ⅱ/LC3B-Ⅰand the protein expression levels of Beclin-1 were significantly increased in the high-glucose group(all P<0.05).Compared with the control group and exosomes+high-glucose group,the protein expression level of p62 were significantly decreased in the high-glucose group(all P<0.01).Compared with the high-glucose group,the expression levels of VEGF were significantly decreased in the exosomes+high-glucose group(P<0.01).CONCLUSION:High glucose conditions activated autophagy of ARPE-19 cells and promoted the expression of VEGF.hUMSC exosomes could effectively inhibit the autophagy level of ARPE-19 cells and the expression of VEGF in high-glucose environments.