| Objective:Premenstrual studies have shown that rapamycin pretreatment of exosomes secreted by MDSC(bone marrow-derived immunosuppressive cells)can effectively delay corneal transplant rejection.But its mechanism of action is unknown.The objective of this study was to further explore the mechanism of immunorejection of corneal transplantation delayed by rapamycin pretreatment of MDSC-secreted exosomes.Method(1)Isolation and identification of MDSC-Exo: Normal 8-week-old BALB/c mice were selected,bone marrow cells were extracted,MDSCs in bone marrow cells were sorted by immunomagnetic bead sorting,the supernatant was collected by Rapa(100nmol/L)culture for 2 days,Exo in the culture supernatant was extracted by ultracentrifugation,and Exo was identified by transmission electron microscopy and western blotting(WB).(2)To verify the expression of miR-181d-5p in cells,exosomes and cornea:Normal 8-week-old BALB/c mice were selected,bone marrow cells were extracted,and MDSCs in bone marrow cells were sorted by immunomagnetic bead sorting,and MDSC was divided into Nor group and Rapa group.The Rapa group joins Rapa(100 nmol/L).The two groups of MDSCs were cultured for 2 days to collect cells and cell supernatants,and the cell supernatants were extracted from the culture supernatant by ultracentrifugation.RNA from two sets of cells and Exo was extracted.Paragonal transplantation models of homologous mouse penetrating cornea transplantation and allogeneic mouse penetrating cornea transplantation were established,and the eyeballs were removed after the cornea of the allotransplanted mice were rejected,and the corneal RNA was extracted from the eyes of normal mice of the same age as a control.The expression of miR-181d-5p in cell,exosome,and corneal tissues was detected by PCR.(3)Construction of penetrating corneal transplantation model and Rapa-Exo intervention with high expression of miR-181d-5p and evaluation of effect: A mouse allogeneic penetrating corneal transplantation model was established and randomly divided into Allo+PBS group,Allo+Rapa-Exo group,Allo+Rapa-Exo+miR-181d-5p antagomir group.Subconjunctival injection was started three days after surgery,and PBS solution was injected subconjunctivally in the Allo + PBS group;Allo+Rapa-Exo group subconjunctival injection of Rapa-Exo at a concentration of 100μg/ml;Allo+Rapa-Exo+miR-181d-5p antagomir group subconjunctival injection of Rapa-Exo and miR-181d-5p antagomir,Rapa-Exo concentration of 100μg/ml,miR-181d-5p antagomir concentration of 1000μg/ml,the dose is 10μl/time,once every 3 days,a total of3 injections.Every other day,the mouse eye was photographed with a slit lamp to observe the rejection of the mouse for 30 days,and the survival curve was drawn according to the rejection time of the transplant.Twenty-three days after surgery,the above three groups of corneas were taken and HE staining was performed to compare the inflammatory response of each group of implants.The expression of inflammatory factors IL-6,IL-12,IL-1β,TNF-α,Ccr7 and IL-17α in corneal tissues was detected by PCR.(4)Isolation of macrophages and co-culture with Exo: Bone marrow cells were extracted from normal 8-week-old BALB/c mice,and m-csf stimulatory factor was added to culture until day 6,and macrophages were divided into four groups,normal group(Nor group),lipopolysaccharide group(LPS group),lipopolysaccharide + Rapa-Exo group(LPS+Rapa-Exo group),lipopolysaccharide + Rapa-Exo + miR-181d-5p inhibitor group(LPS+Rapa-Exo+ miR-181d-5p inhibitor group).Cells and cell supernatants are collected after stimulation by adding cells to each group.Cellular RNA was extracted by Real time PCR to detect the expression levels of IL-6,IL-12,IL-1β and TNF-α in cells.Commercial ELISA kits were used to detect cultured supernatant IL-6 and TNF-α protein levels.(5)Bioinformatics analysis of target genes:At four different sites,miRDB,Target Scan,RNA22,and Starbase all predicted that KLF6 was the target gene for miR-181d-5p.Target Scan is the first miRNA target gene software to appear,and it is also the most used and accurate prediction software to obtain the binding sites and sequences of miR-181d-5p and KLF6 in rats,mice and humans.The diluciferase reporter gene detected whether mmu-miR-181d-5p could interact with KLF6.(6)Macrophages are co-cultured with si KLF6:According to the method in(2)above,the macrophages were cultured to the sixth day,and the cells were divided into 3groups,normal group(Nor group),lipopolysaccharide group(LPS group),lipopolysaccharide + si KLF6 group(LPS+si KLF6 group).After the stimulation of each group,each group of cells was cultured to collect cells(cell 1),cultured for 48 hours to collect cells(cell 2)and cultured supernatant.RNA from cell 1 was extracted and the expression levels of IL-12,IL-6,IL-1β,TNF-α and klf6 in cells were detected by Real time PCR.The protein in extracted cell 2 was detected by Western Blot(WB)to detect the expression of klf6 in each group.Expression levels of IL-6 and TNFα in cultured supernatants were detected with ELISA kits.(7)Construction of penetrating corneal transplantation model and intervention treatment and effect evaluation of si KLF6:A mouse model of heterogeneous penetrating corneal transplantation was established and randomly divided into Allo+PBS group,Allo+si KLF6 group.Subconjunctival injection of si KLF6(1000μg/ml)was started three days after surgery at a dose of 10μl once every 3 days,for a total of three injections.Every other day,the mouse eye was photographed with a slit lamp to observe the rejection of the mouse for 30 days,and the survival curve was drawn according to the rejection time of the transplant.20 days after surgery,the above two groups of corneas were taken and HE staining was performed to observe and compare the inflammatory response of the corneas in the two groups.The expression of inflammatory factors IL-6,IL-12,IL-1βand TNF-αin corneal tissues was detected by PCR.Results:(1)Identification of exosomes derived from MDSC:The extracted exosomes were detected and imaged by electron microscopy at 100 k V,and the form was disc-shaped vesicles with one or both depressions,the diameter was mainly concentrated at 180 nm,and the distribution range was between 50nm~200nm.WB results showed positive exosome CD81 and CD9.The results indicate successful exosome extraction.(2)Differences in the expression of Mi R-181d-5p in cells,exosomes and cornea:The results of Real time PCR showed that the expression of miR-181d-5p in Rapa-pretreated MDSCs and their secreted exosomes was higher than that in the untreated group of MDSCs and their secreted exosomes.In corneal tissue,the expression of corneal miR-181d-5p in the allogeneic rejection group was lower than that in the normal group and the homologous non-excluded cornea.(3)In vivo experimental observation of Mi R-181d-5p:In the three groups of mouse corneal transplantation models,the average survival time of corneal transplantation in each group was(18.9±5.8)d in Allo+PBS group,25.4±6.0)d in Allo+Rapa-Exo group,and 17.1±3.8)d in Allo+Rapa-Exo+miR-181d-5p antagomir group.The survival time of the Allo+Rapa-Exo group was significantly higher than that of the other two groups,and the difference was statistically significant(P<0.05).The inflammatory response of Allo+PBS group and Allo+Rapa-Exo+miR-181d-5p antagomir group was heavier,and the corneal inflammatory response was lighter in the Allo+Rapa-Exo group.Real time PCR detected the expression of inflammatory factors IL-6,IL-12,IL-1β and TNF-α in each group and found that the Allo+PBS group and the Allo+Rapa-Exo+miR-181d-5p antagomir group were significantly higher than those in the Allo+Rapa-Exo group(P<0.05).(4)Upregulation of miR-181d-5p in macrophages:The expression level of inflammatory factors in the Nor group without any treatment was the lowest in the 4groups of macrophages,and there was no significant difference between the LPS group and the LPS+Rapa-Exo+miR-181d-5p inhibitor group,and the expression level of inflammatory factors was significantly higher than that in the LPS+Rapa-Exo group(P<0.05).ELISA detected cell culture supernatant,and the expression level of inflammatory factors was consistent with the results of Real time PCR.Immunofluorescence showed that the expression level of klf6 was lowest in the Nor group,and the expression in the LPS group was the highest with the LPS+Rapa-Exo+miR-181d-5p inhibitor group,and there was no significant difference,and it was higher than that in the LPS+Rapa-Exo group.The results showed that upregulation of miR-181d-5p could significantly inhibit the expression of macrophage inflammatory factors,that is,miR-181d-5p had the effect of inhibiting the inflammatory response of macrophages.Mi R-181d-5p regulates macrophage inflammatory response with klf6 as a target.(5)Bioinformatics analysis of target genes : At four different sites,miRDB,Target Scan,RNA22,and Starbase all predicted that KLF6 was the target gene for miR-181d-5p.Target Scan is the first miRNA target gene software to appear,and it is also the most used and accurate prediction software to obtain the binding sites and sequences of miR-181d-5p and KLF6 in rats,mice and humans.Dual luciferase reporter detection mmu-miR-181d-5p can bind to the promoter of KLF6.(6)In vivo experimental observation of Mi R-181d-5p:In the three mouse corneal transplantation models,the average survival time of corneal transplantation in each group was(18.9±5.8)d in the Allo+PBS group,25.4±6.0)d in the Allo+Rapa-Exo group,and17.1±3.8)d in the Allo+Rapa-Exo+miR-181d-5p antagomir group.The survival time of the Allo+Rapa-Exo group was significantly higher than that of the other two groups,and the difference was statistically significant(P<0.05).The inflammatory response of Allo+PBS group and Allo+Rapa-Exo+miR-181d-5p antagomir group was heavier,and the corneal inflammatory response was lighter in the Allo+Rapa-Exo group.Real time PCR detected the expression of inflammatory factors IL-6,IL-12,IL-1β,TNF-α and KLF6 in each group,and found that the Allo+PBS group and the Allo+Rapa-Exo+miR-181d-5p antagomir group were significantly higher than those in the Allo+Rapa-Exo group(P<0.05).(7)Si KLF6 transfection of macrophages:The expression levels of cellular inflammatory factors IL-6,IL-12,IL-1β and TNF-α were detected by Real time PCR,and the expression levels of LPS,LPS+si KLF6 and Nor were detected in descending order.The detection of IL-6 and TNF-α protein levels in cell supernatant by ELISA and the protein levels of KLF6 with Western Blot were consistent with Real time PCR.(8)In vivo experiments with si KLF6:In the two groups of mouse corneal transplantation models,the survival time of corneal transplantation in each group was(18.2±7.0)d in Allo+PBS group and(27.7±6.4)in Allo+si KLF6 group.Real time PCR detected the expression of inflammatory factors IL-6,IL-12,IL-1β,TNF-α and KLF6 in each group,and the Allo+si KLF6 group was significantly lower than that in the Allo group.Conclusion:(1)Exosomes secreted by MDSC pretreated with rapamycin have the effect of delaying the immune rejection of corneal transplantation.(2)Mi R-181d-5p targets klf6 to inhibit macrophage inflammation and delay corneal transplant rejection. |
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