| Objective:To study the effect and mechanism of human umbilical cord mesenchymal stem cells derived microvesciles(hUCMSCs-MVs)on the diabetic retinal neurodegeneration(DRN)in vivo and vitro.Methods:This research included three parts.The primary part of the research was to subculture the cryopreserved hUCMSCs and extract the hUCMSCs-MVs.HUCMSCs-MVs was isolated through the method of ultracentrifugation from the collected hUCMSCs supernatant.The morphological and immunological phenotypic identification of hUCMSCs-MVs were carried out by flow cytometry and transmission electron microscopy.In the part II,the retinal ganglion cells(RGCs)of the SD rats were separated and cultured by immunopanning in vitro.The characters of the cultuered RGCs were identified by immunofluorescent technique.The behavior of the internalization of hUCMSCs-MVs and RGCs were observed by fluorescence microscope.The RGCs were divided into 4 groups under the conditions below:normal control group(A),high glucose(GLU)condition group(B)(RGCs+GLU33mmol/L),normal RGCs co-cultured with hUCMSCs-MVs group(C)(RGCs+hUCMSCs-MVs),and RGCs co-cultured with hUCMSCs-MVs in high glucose condition group(D)(RGCs+hUCMSCs-MVs+GLU 33mmol/L).The cell activity was tested by CCK-8 test;AnnexinV-PI staining detected the cell apoptosis rate by flow cytometry;the relative expression levels of the mRNA and protein of Bcl-2,Bax and procaspase-3 were detected by RT-PCR and WB.Statistical analysis was performed by using One-way analysis of variance and SNK-q test was used for the comparison between groups.Significance was setted at P<0.05.The diabetes mellitus(DM)rats were induced by intraperitoneal injection of streptostatin(STZ).The rats were divided into 4 groups:normal control group(A),DM group(B),normal control rats received the intravitreal injection of hUCMSCs-MVs group(C),DM rats received the intravitreal injection of hUCMSCs-MVs group(D).The intravitreal injections of hUCMSCs-MVs were carried out at 8 weeks after the station of the DM was successfully induced by STZ.The time point of the observation was 4weeks.The changes of the retinal structure were observed by hematoxylin-eosin(HE)staining,the apoptosis of the retinal cells was tested by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay(TUNEL),the location and the expression of the phosphorylated-c-Jun amino terminal kinase(p-JNK)in the retina was determined by immunofluorescence staining.The relative expressions of JNK,p-JNK,c-Jun,p-c-Jun,Bcl-2,Bax,and Cleaved Caspase-3 werw measured by WB.Results:The hUCMSCs were successfully subcultured.hUCMSCs-MVs were isolated which were characterized as the form of single or cluster of circular membranous vesicle-like structure.The diameter of hUCMSCs-MVs ranged from 10~2to 10~3 nm.The surface of the hUCMSCs-MVs expressed the hUCMSCs-specific marker CD44,CD29,CD73,and CD105,which negatively expressed the integrin(CD49f),HLA class II(HLA-DR),and hematopoietic stem cell-specific marker protein CD34,CD45.300 ml of the collected supernatant of P2-P4 hUCMSCs with the cell dencity of 1×10~5/ml could extracted the amount of(342.65?1.32)μg hUCMSCs-MVs.The RGCs of the SD rats were isolated and cultured in vitro by using the immunopanning antigen screening and separation method.The expression of Thy-1 and Brn3a in primary rat RGCs were positive.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 the other groups where as the cell activity of group B was lower than that the others(p<0.05).The results of RT-PCR and Western Blot showed that there were significant differences in the relative expression of Bcl-2,Bax,Caspase-3 mRNA and protein among the groups,the expression of Bax and Caspase-3 in group B was significantly higher than that of the other groups(P<0.05).SNK-q test was performed to the difference between the groups.The relative expression of Bcl-2 mRNA and protein of group D was significantly higher than that of group B.The relative expression of Bax,Caspase-3mRNA and protein of group B was higher than that of group D.The relative expression of Caspase-3 protein of group B and D was higher significantly than that of group A and C.The relative expression of Cleaved Caspase-3 protein of group B was higher significantly than that of group D.After 8 weeks of STZ-induced diabetic rats,the character of the DRN was observed by H-E staining with the atrophy of the inner retina,the apoptosis of RGCs and the atrophy of the INL.Statistical analysis showed that there were significant differences in the inner retinal thickness between the four groups(P<0.05).SNK-q test showed that there were significant differences among groups.The inner retinal thickness of group B was lower than that of the other three groups,and that of group D was higher than that of group B.The positive staining of TUNEL(TUNEL~+)cells in the inner retina mainly concentrated in GCL,the rates of TUNEL~+cells in the inner retina of rats among groups were significantly different(P<0.05).The rate of TUNEL~+cells of group B was higher than that in other groups.The rate of TUNEL~+cells of group D was lower than that of group B.The activation of JNK was mainly localizated in the cytoplasm of RGCs in early DR rats by immunofluorescence.The positive staining rate of p-JNK(p-JNK~+)cells of each group was significantly different(P<0.05).SNK-q test showed that the rate of p-JNK~+cells in GCL of group B was higher than that of the other groups where as the rate of group D was lower than group B.WB results showed that there were significant differences in the relative expression of the target protein among four groups(p<0.05).SNK-q test showed that the relative expression of Bcl-2 of group B was lower than that of the others.The relative protein expression of JNK,p-JNK,c-Jun,p-c-Jun,Bax,Cleaved Caspase-3 of group B was higher than that in the other three groups where as the aboves of group D were lower than that of group B.Conclusion:The hUCMSCs-MVs could protect the cultured rat RGCs from the damage of the hyperglycemia condition by 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 Caspase-3 into the activity form of Cleaved Caspase-3.The intravitreal injection of hUCMSCs-MVs could protecte the RGCs from apotosis,increase the thinkness of inner retina,alleviate DRN in early stage of DR rats by decreasing the expression and activation of JNK in the inner retina,reducing the expression of Bax、Cleaved Caspase-3 and increasing the expression of Bcl-2. |
