| Objective(s): Through the treatment of diabetes wounds with endothelial progenitor cell exosomes(db/db EPC EXOs)and non diabetes wounds with endothelial progenitor cell exosomes(db/+EPC EXOs),the differences in angiogenesis,collagen deposition and final wound healing between the two in vivo were compared,providing direction for the study of its therapeutic mechanism,and providing theoretical basis for its clinical application.Methods:Endothelial progenitor cells were isolated from diabetes mice(db/db mice)and normal mice(db/+mice)by Ficoll density gradient centrifugation.Endothelial progenitor cells were cμLtured in vitro for a period of time to make them proliferate stably,and then the cell supernatant was collected.The exosome extraction kit and μLtrahigh speed centrifuge were used to extract the exosome from the cell supernatant.The exosome body weight was suspended in PBS.After its concentration was measured by BCA method,it was stored at-80 ℃.24 diabetes(db/db)mice were used to make 8mm round fμLl-thickness skin defect wounds.According to the method of random number table,they were divided into 3 groups with 8 rats in each group,namely group 1(transplanted db/+mouse EPC-EXO),group 2(transplanted db/db mouse EPC-EXO)and control group(with the same amount of PBS as the other two groups).After the wound was made,the exocrine body was injected subcutaneously at four sites on the wound surface of mice every two days(immediately after the end of modeling).On the 2nd,4th,6th,8th,10 th,12th and 14 th days after the injury,the wounds were observed and photographed,the wound healing rate was calcμLated.The wound tissue was taken on the 6th,10 th and 14 th days and various indexes were detected: real-time fluorescent quantitative reverse transcription-PCR and protein blotting were used to detect VEGFA,CD31 α-MRNA and protein expression of SMA,COL Ⅰ and COL Ⅲ;The vascμLar density of wound tissue and the proportion of mature blood vessels to total blood vessels(CD31 and α-SMA double staining)and the situation of vasc μLar proliferation potential(VEGFA and α-SMA double staining);HE and MASSON staining were used to observe the repair of wound tissue,the aggregation and infiltration of inflammatory cells,the deposition of collagen and the epidermal coverage.After collecting all data,conduct statistical processing,and use IBM SPSS STATISTICS statistical software to process all data.The measurement data is expressed by mean ± standard deviation.MμLti-group processing uses univariate analysis of variance and factorial analysis to calcμLate the F value,and perform Bonferroni correction on the p value(P value is less than 0.05,the difference between organizations has statistical significance).Results:The concentration of EPC-EXOS detected by BCA was 0.5 μg/μL.The three groups of db/db rats who used EPC-EXOS on the wound and after the wound were in good mental condition,drank and ate normally,had no significant change in weight,had bright hair,and had no accidental death.Under the general healing observation,it was found that there was no significant difference between the three groups of wounds at the time of wound healing to 2 and 4 days,and scabs slowly formed during this period;On the 6th day of wound healing,there was no significant difference in the wound healing rate between group 2 and the control group(P>0.05),while the wound healing rate of group 1 was significantly different from the other two groups(P<0.05),which was higher than the other two groups;From the 8th day to the 14 th day of wound healing,the wound healing rates of the three groups were significantly different(all P values were less than 0.05).The wound healing rate of Group 1 was the fastest,and the wound healing rate was the highest.The wound healing rate of Group 2 was between Group 1 and Control group,while the wound healing rate of Control group mice was slower,less skin coverage,and lower,which was basically consistent with the delayed healing of diabetes wounds in our preliminary experiment,The difference of wound healing rate among the three groups increased with time from the 8th day.The observation of HE staining showed that,on the whole,there was no obvious inflammatory cell aggregation and infiltration in group 1,group 2 and Ccontrol group,indicating that there was no obvious rejection reaction after transplantation of allogeneic EPCs-EXO.At the 6th day of the wound,the wound tissue of group 1 had initially formed a thin epidermal layer;Epidermis of group 2 was not covered;The control group has not yet formed a tissue with obvious structure,and the wound recovery is relatively slow.On the 10 th day of the wound,the wound tissue of group 1 had formed relatively complete skin tissue and subcutaneous tissue,and the wound recovery rate was faster;The epidermal layer of wound tissue in group 2 was relatively thin;Although the wound tissue in the Control group has become an observable tissue layer,the wound tissue is still relatively young,which is in line with the expected delayed healing of diabetes wounds.On the 14 th day of the wound,the wound surface of group 1 was close to normal skin tissue,with very thick subcutaneous tissue and skin visible;In group 2,the skin epidermal layer was covered but not completely,and the collagen deposition was still thin;In the control group,the wound epidermis was thinner and the epidermis was not firm.Under the observation of MASSON staining,it was found that on the 6th day of the wound,the wound surface of group1 had formed thin but dense collagen tissue;The collagen tissue in group 2 was thin and loose;There is not much collagen deposition in the tissue of Control.On the 10 th day of the wound,the amount of collagen tissue in group 1 was very rich and dense;The amount of collagen tissue in group 2 was less than that in group 1;In Control group,the amount of collagen was not only less,but also more loose.On the 14 th day of the wound,the amount of collagen tissue in group 1 was large,arranged neatly,and the structure was close to the normal tissue structure;Collagen tissue in group 2 was also disordered and thin;The amount of tissue collagen and its maturity in the Control group were not as good as those in the above two groups.From the collagen volume fraction(CVF)of the three groups,it can be seen that at the 6th,10 th and14th day,the relative amount of collagen is significantly different(P value is less than 0.05).The collagen deposition fraction of group 1 was the highest among the three groups,and that of group 2 was between the other two groups,while that of control group was the lowest,and the difference gradually increased after 6 days.On CD31 and α-In the case of blood vessels detected by SMA double staining,the density of blood vessels in group 1 of the three groups was higher,and the number of mature blood vessels accounted for a higher proportion of the total number of blood vessels;The total number of blood vessels in group 2 was between the two groups;In the Control group,the density of blood vessels was low,and the proportion of mature blood vessels was also small.In the detection of vascμLar proliferation by VEGFA and Ki67 double staining,there are more angiogenic factors in group 1 of the three groups and its vascμLar proliferation ability is more significant;The total number of blood vessels in group 2 was between the two groups;However,in the Control group,there are fewer angiogenic factors and the proliferation ability of blood vessels is also poor.It was observed from the detection of m RNA expression on the wound surface that at the 14 th day,CD31,VEGFA α-SMA,COL Ⅰ and COL Ⅲ showed significant differences in the relative expression of m RNA(P<0.05),which was similar to the general observation.The m RNA related to angiogenesis in group 1 was higher than that in control group;The m RNA related to angiogenesis in group 2 was higher than that in control group but lower than that in group 1.On this basis,COL Ⅰ and COL Ⅲ in the wound were higher in group 1,and lower in group 2,but both were higher than those in control group.CD31,VEGFA,α-actin.The three proteins related to angiogenesis of SMA in group 1 were higher than those in control group and group 2,and the difference was statistically significant,while the three proteins in group 2 were significantly lower than those in group 1 and higher than those in control group.COL Ⅰ and COL Ⅲ proteins related to collagen deposition were consistent with the resμLts of angiogenic protein in group 1,and were also higher than those in control group and group 2,while the collagen-related protein in group 2 was higher than that in control group and lower than that in group 1.Conclusion(s):(1)The EPCs EXO of db/+mice has a significant healing effect on diabetes wounds,while the EPCs EXOS of db/db mice has a weak healing effect on diabetes wounds,but it still has some corresponding functions.(2)The mechanism of action of exosomes is likely to be through increasing the production of VEGFA factor and increasing the surface protein CD31 of endothelial cells α-The amount of SMA increases the speed of proliferation and maturation of blood vessels,the speed of collagen deposition and the speed of skin covering the wound,thus promoting the healing of diabetes wounds.(3)The function of exosomes runs through the whole process of wound healing.From the generation of cytokines,to m RNA,to the expression of protein,its functional activity is crucial.Because its Antigenicity is very weak,the use of normal exosomes to treat diabetes wounds has good clinical application prospects. |
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