ABSTRACT
Objective To observe the effect and mechanism of human umbilical cord blood mesenchymal stem cells-derived microvesicles (hUMSCs-MVs) on the injury of the primary rat retinal ganglion cells (RGCs) by high glucose environment.Methods The primary RGCs of Sprague Dawley rats were cultured in vitro,hUMSCs-MVs were isolated and extracted by ultra-centrifugation.hUMSCs-MVs were internalized with RGCs.The RGCs were divided into 4 groups under the conditions below:normal control group (group A),high glucose condition group (group B,RGCs+glucose 33 mmol/L),normal RGCs co-cultured with hUMSCs-MVs group (group C,RGCs+hUMSCs-MVs),and RGCs co-cultured with hUMSCs-MVs in high glucose condition group (group D,RGCs+hUMSCs-MVs+glucose 33 mmol/L).The cell activity was detected by CCK-8 test.Annexin V/PI staining detected the cell apoptosis rate by flow cytometry.And the relative expression levels of the genes such as Bcl-2,Bax and Caspase-3 were detected by fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot.Statistical analysis was performed by using One-way analysis of variance and SNK-q test was used for the comparison between groups.Results The hUMSCs-MVs were extracted by ultra-centrifugation,which were characterized as single or cluster of circular membranous vesicle-like structure with diameter ranging from 100 nm to 1000 nm.The flow cytometry analysis showed that hUMSCS-MVs were highly positived by the surface markers of CD44,CD29,CD73,and CD105 whereas been poorly expressed the integrin (CD49f),HLA class Ⅱ,CD34,CD45.There were significant differences in the cell activity and the apoptosis rate among 4 groups,the cell apoptosis rate of group B was higher significantly than that of group A and group D (F=107.92,P=0.000),the cell activity of group B was lower than that of group A and group D (F=382.11,P=0.000).The results of RT-PCR and Western blot showed that the relative mRNA (F=219.79,339.198,1 071.21;P=0.000,0.000,0.000) and protein (F=544.28,295.79,224.75;P=0.000,0.000,0.000) expression of Bcl-2,Bax,Caspase-3 and the protein expression of cleaved Capspase-3 (F=533.18,P=0.000) in group B and D were higher significantly than those in group A and C.The relative expression of Bcl-2 mRNA and protein in group B was significantly lower than that of in group D (P＜0.05).The relative expression of Bax,Caspase-3 mRNA and protein in group B was higher than that in group D (P＜ 0.05).The relative expression of cleaved Caspase-3 protein in group B was higher significantly than that in group D (P＜0.05).Conclusion The hUMSCs-MVs can protect the cultured rat RGCs from the damage of the high glucose condition through increasing the cell activity and reducing the apoptosis rate of RGCs by promoting the Bcl-2 expression,decreasing the expression of Bax and Caspase-3 and inhibiting the Caspase-3 into the activity form of cleaved Caspase-3.