Experimental Study On The Effect Of IL-10-hAMSCS On IL-10?TNF-? And IL-6 During Wound Healing In Mice

Objective:Through detecting the changes of inflammatory factors IL-10,TNF-? and IL-6 in skin wound tissue of mice,investigate the effect of IL-10 gene modified hAMSCs(IL-10-hAMSCs)injection on the ability of inflammatory regulation and clinical transformation after transplantation of full-thickness skin defect models in mice.Methods:The lentiviral vector LV5-IL-10 containing the mouse IL-10 gene and the empty plasmid vector LV5 were transfected into hAMSCs respectively.After 72 hours,the content of IL-10 in the supernatant of the 2 kinds of transfected cells and non transfected hAMSCs cells was detected by ELISA method.The 60 C57BL/6 mice aged 8 weeks to prepare the full-thickness skin defect model,and randomly divided into control group(normal mice did not die without any intervention),PBS transplantation group(model group),hAMSCs transplantation group,IL-10-hAMSCs transplantation group(IL-10-hAMSCs group),hAMSCs group(transfected with empty plasmid the empty plasmid-hAMSCs group),8 rats in each group.The model was prepared by the back of the2 side of the clipping 1cm x 1cm size of full-thickness skin wounds,then according to different grouping,on each side of each wound from the midpoint of a margin of 1mm injection at the total 100 L PBS suspension groups of cells(cell number is 1 * 106),the model group was injected with PBS.On the first day,the third day seventh days,fourteenth days after transplantation,to collect the wound tissue samples,and were used to observe the inflammatory cell infiltration tissue by HE staining;detection of IL-10,ELISA method to detect IL-10,TNF-alpha,IL-6 content factors changes;real-time fluorescence quantitative polymerase chain reaction(Q-PCR)method to detect IL-10,TNF-alpha,IL-6factor the relative content of mRNA changes.In the experiment,CM-Dil labeled hAMSCs transplantation,along with IL-10-hAMSCs transplantation and empty plasmid transfection of hAMSCs,were used to analyze the colonization and survival of the transplanted cells in different groups.Results:IL-10 lentivirus and empty plasmid transfected hAMSCs 72 hours later,under the inverted microscope,the green fluorescence was detected in the visual field,indicating that the transfection rate was high after infection.To detect IL-10 content factors of the medium hAMSCs,IL-10-hAMSCs,empty plasmid-hAMSCs cell supernatant with ELISA after seventy-second hours,the results showed that hAMSCs,IL-10-hAMSCs,empty plasmid-hAMSCs can secrete IL-10,IL-10-hAMSCs compared with hAMSCs group and empty plasmid-hAMSCsgroup was secreting more IL-10 content factor(P < 0.05).The results of HE showed that under the microscope,the epithelium of the normal mouse skin was intact,with no inflammatory cells,accompanied by hair follicles and arranged in an orderly and reticular shape;At the first day,HE result show the wound that a lot of inflammatory cell infiltration was observed under the microscope from model group,hAMSCs group,empty plasmid-hAMSCs group,IL-10-hAMSCs group,the model group inflammation and necrosis tissue was the most,followed by hAMSCs group and empty plasmid-hAMSCs group,inflammatory reaction degree of IL-10-hAMSCs group was lightest and the necrotic tissue was the least.At third days,The results of HE under the microscope showed that the model group,hAMSCs group,empty plasmid-hAMSCs group,IL-10-hAMSCs group,there was a large number of inflammatory cells infiltration in the wound margin The inflammatory cell count in each group showed that the inflammatory cell infiltration in the model group was the most,and the inflammatory cell infiltration in the IL-10-hAMSCs group was the least.Compared with the other three groups,the inflammatory cell count was statistically different(P < 0.05);At seventh day,inflammatory cells in each group was decreased,inflammatory cells in the model group were the most,and inflammatory cell infiltration in group IL-10-hAMSCs was the least,and the difference was statistically significant(P < 0.05);The content of TNF-a and the relative expression ofmRNA: from the first day to the third day,the content of the model group,hAMSCs group,empty plasmid-hAMSCs group,IL-10-hAMSCs group increased gradually,reached the peak after third days,and gradually declined;At day 14 d,there was almost no inflammatory cell infiltration in hAMSCs group,the empty plasmid-hAMSCs group and the IL-10-hAMSCs group,and there was still a small amount of inflammatory cell infiltration in the model group,the difference was statistically significant(P < 0.05).ELISA method and qPCR method were used to detect the back wound and wound tissue in mice,and the content of IL-10 and the relative expression of m RNA were observed:The results showed that the skin tissue IL-10 of the normal mice in the blank group was very low,from first days to third days,and the content of IL-10 at the model group,the hAMSCs group,the empty plasmid-hAMSCs group and the IL-10-hAMSCs group increased gradually,and reached the peak at third days and then decreased gradually;At 1th day,3th day,7th day and 14 th day,the content of IL-10 and the relative expression of mRNA in IL-10-hAMSCs group were higher than those in other groups,the difference was statistically significant(P < 0.05).The results suggest that IL-10-hAMSCs can up regulate the expression of IL-10.TNF-alpha content and relative expression of mRNA: from first days to third days,and the content of TNF-alpha at the model group,the hAMSCs group,the empty plasmid-hAMSCs group and the IL-10-hAMSCs group increased gradually,and reached the peak at third days and then decreased gradually;At 1th day,3th day,7th day and 14 th day,the content of TNF-alpha and the relative expression of TNF-alpha mRNA in model group were higher than those of other groups,compared with the other groups was statistically significant(P<0.05).the content of TNF-alpha and the relative expression of TNF-alpha mRNA in IL-10-hAMSCs group were lower than those of other groups,compared with statistical difference(P<0.05).IL-6 content and relative expression of mRNA: At 1th day,3th day,7th day and 14 th day,the content of IL-6 and the relative expression of TNF-alpha mRNA in model group were higher than those of other groups,compared with the other groups was statistically significant(P<0.05).the content of IL-6and the relative expression of IL-6 m RNA in IL-10-hAMSCs group were lower than those of other groups,compared with statistical difference(P<0.05).The results showed thatIL-10-hAMSCs could down regulate the expression of TNF-alpha and IL-6.The expressions and patterns of cytokines above were basically consistent with the pathological results of HE staining.14 days after transplantation,fluorescence tracing showed that the transfected hAMSCs the empty plasmid transfected hAMSCs and IL-10-hAMSCs still survived.Conclusion:1.IL-10-hAMSCs can highly express and secrete IL-10 in vitro.2.Transplantation of IL-10-hAMSCs in part of wound can increase the expression of IL-10 and down regulate the expression of TNF-? and IL-6,and reduce the degree of inflammatory reaction,which may promote wound healing.