The Establishment Of IL-10-hAMSCs And Its Effects On The Expression Of TGF-?1,MMP-1 And TIMP-1 In Wound Healing Of Mice

Objective:To investigate IL-10-h AMSCs about its efftcts on promting wound healing in the process of wound healing and the influence of related factors TGF-?1?MMP-1 ? TIMP-1on scar formation.In order to use the IL-10 transfected h AMSCs to promote wound healing and to provide an important basis for decreasing fibrosis healing in further clinical application.Methods:1?h AMSCs were extracted by mechanical separation and enzyme-collagenase digestion method in two steps from amnion.The h AMSCs were cultured in DMEM-F12 with10%FBS,put in 37??5%CO2 incubator.Spread to 3rd generations to stand by.2?By mechanical separation combined with trypsogen collagenase digestion method from human amniotic membrane two step extraction and separation of h AMSCs,culture medium containing 10%FBS DMEM-F12,cultured 37 DEG and 5%CO2 saturated humidity incubator,and spread to the 3 generation reserve.3?The cloned mouse IL-10 gene linked to the LV5 vector.The plasmid was transformed and amplified the Plasmid Extraction and identified by double enzyme digestion,packaged in 293 T cells LV5-IL-10 lentiviral vector,then transfected into h AMSCs,IL-10-h AMSCs,fluorescence microscope by green fluorescent protein reporter gene expression were observed and assessed transfection and transfection efficiency.4? C57BL/6 wild type male mice of 7 weeks old(n=100)were randomly divided into blank group,model group,h AMSCs group,empty plasmid transfected h AMSCs group and IL-10 transfected h AMSCs group,20 rats in each group.After intraperitoneal anesthesia,full-thickness skin defect model were made of 1cm * 1cm in the back of mice on both sides of the line,according to the different group,set up immediately in the wound around the wound on each multi point subcutaneous(a margin of each side from the midpoint of the wound 1mm)injection 100 L PBS suspension of different groups of h AMSCs cells(a total of 1*106),mice model group were injected with PBS and blank group without normal mice and intervention.In addition,3 mice were injected with IL-10-h AMSCs,empty plasmid-h AMSCs and CM-Dil labeled h AMSCs,which were used for fluorescence tracing of the engraftment and survival of the transplanted cells.5?The 1 day,the 3 day,the 7 day and the 14 day after the wound model of mices' back were established,obsevered the wound inflammation and wound healing progress,compared the wound healing rate between different groups,and cut wound tissue at the time point,observed the wound and wound infiltration of inflammatory cells by paraffin section and HE staining,observed the wound and wound the deposition of collagen by Masson staining,observed the MMP-1,TGF-beta 1 TIMP-1 expression by immunohistochemistry,observed the TGF-beta 1 MMP-1 and TIMP-1 content in wound tissues by ELISA,observed the relative expression of TGF-beta 1,MMP-1,TIMP-1mRNA in wound tissue by q PCR.Results:1?The adherent cells of h AMSCs were long spindle shaped,polygonal,and the cells were cross-linked with each other,in a nest or whirlpool arrangement.After subculture,the growth rate of cell adhesion was accelerated.After 24 h,the cells adhered to the wall,and the cells were spindle shaped,in a nest arrangement.2?IL-10 lentivirus recombinant plasmid was identified by double digestion showed the presence of gene positive clone,the virus titer was 5 *108TU/ml,72 h after the IL-10 lentivirus and empty plasmid infected h AMSCs,in the field of more than 80% cells under the inverted microscope were expressing green fluorescence,indicating infection cell survival rate and the infection rate are high.3?The results of ELISA showed that the secretion of TGF-beta1 in culture medium group 1in 1d?3d?7d?14d were as follows:0.47±0.34ng/L,0.33±0.22ng/L,0.27±0.21ng/L,0.25±0.15ng/L.the secretion of TGF-beta 1 in the h AMSCs group in 1d?3d?7d?14d were as follows: 3.26±0.26ng/L,6.37±0.34ng/L,9.86±0.43ng/L,9.08±0.50ng/L?the secretion of TGF-beta 1 in the IL-10-h AMSCs group in 1d ? 3d ? 7d ? 14 d were as follows:6.35±0.48ng/L,10.2±0.54ng/L,14.96±0.67ng/L,13.92±0.41ng/L?the empty plasmid group secrete TGF-beta1 in 1d ? 3d ? 7d ? 14 d were as follows: 3.23±0.30ng/L,6.30±0.31ng/L,9.30±0.22ng/L,9.13±0.43ng/L.It is suggested that h AMSCs can secrete TGF-beta 1 during the course of in vitro culture,and IL-10 can promote h AMSCs secretion of TGF-beta 1a bit.4?After the establishment of the model of full-thickness skin defect in the back of the mice,1d?3d,the red,swelling,exudation of IL-10-h AMSCs group was significantly lower than that of model control group,h AMSCs group,empty plasmid group.7d,h AMSCs group,empty plasmid group and IL-10-h AMSCs group healed faster than the model control group,the red,swelling,exudation was obviously weakened,and the healing rate of group IL-10-h AMSCs and the inflammatory reaction of reduced strength was better than h AMSCs group and empty plasmid group.14 d,in addition to the model control group,the wound healing was 100%,and the scar was smaller than the model control group.The results showed that the wound healing of h AMSCs group,IL-10-h AMSCs group and empty plasmid group was better than that of model control group,IL-10-h AMSCs group was better than h AMSCs and empty plasmid group.The difference was statistically significant(P < 0.05).h AMSCs using CM-Dil staining,IL-10-h AMSCs and empty plasmid-h AMSCs expression of GFP itself,cut frozen sections after modeling injection 14 days,scattered fluorescence were observed by fluorescence microscopy,indicated that h AMSCs,IL-10-h AMSCs and empty plasmid-h AMSCs in colonization and survival of the organization.5?The results of HE showed that the epithelium of normal mice were intact,accompanied by hair follicles.1d,there were amounts of inflammatory cells infiltration inthe wound of h AMSCs group,IL-10-h AMSCs group,empty plasmid group,the upper parts of the wound were necrotic and crusted,inflammatory cells infiltration in IL-10-h AMSCs was lower than the others.3d,the inflammatory cells infiltration in h AMSCs group,IL-10-h AMSCs group,empty plasmid group were obviously lower than the model control group,the morphology of collagen in hAMSCs group,IL-10-hAMSCs group,empty plasmid group were uniform and the gap were moderate.the morphology of collagen in model group was vary in size.the arrangement was loose and disordered.IL-10-h AMSCs group was the most;7d,model group was still a large number of inflammatory cell infiltration,fibroblasts are large in number,the collagen is dense.h AMSCs group,IL-10-h AMSCs group,empty plasmid group inflammatory cell infiltration was lower than before,and were less than the model control group,the group of IL-10-h AMSCs,inflammatory infiltration was the weakest,the amount of fibroblasts were least and the collagen were moderate;14d model group has a small amount of inflammatory cell infiltration,h AMSCs group,IL-10-h AMSCs group,empty plasmid group wound inflammatory cells were significantly reduced,IL-10-h AMSCs group had little inflammatory cells.collagen in h AMSCs group,IL-10-h AMSCs group,empty plasmid group were vary moderate.The amount of fibroblasts in model group was largest.To observe the normal mouse skin tissue under the microscope,Masson staining results showed that collagen stained blue,the structure was network,the arrangment was regular.1day,a large number of necrotic tissue and inflammatory cell infiltrationi in every groups.3day,in model control group,the number of collagen was small and the gap was wide.the arrangement of the collagen in the control group was disordered,the size was different,and a large number of inflammatory cells were infiltrated.The collagen in h AMSCs group,IL-10-h AMSCs group,empty plasmid group were arranged in a relatively orderly manner.The morphology of IL-10-h AMSCs group was uniform,the cross-linking was close,and the volume fraction was better than the other three groups,the difference was statistically significant(P<0.05).7day,h AMSCs group,IL-10-h AMSCs group,empty plasmid group,compared with the model group,collagen arranged in a network structure,the collagen gap was moderate,and the number of collagen was slightly reduced.In the model control group,the collagen fibers were arranged in a whirlpool shape,disordered and disorderly,with large and large collagen and dense collagen space.The difference in volume fraction between the IL-10-h AMSCs group and the other three groups was statistically significant(P<0.05).14 day,the collagen of model control group were vary in size,the amount was big and the arrangment was disorder.empty plasmid group,h AMSCs group and IL-10-h AMSCs group were arranged in net work,the space of collagen was moderate.The structure and space of collagen in IL-10-h AMSCs group were best,the amount of collagen was less than h AMSCs and empty plasmid group.The results of immunohistochemistry showed that the expression of TGF-beta 1,MMP-1 and TIMP-1 were positive but little in the blank group,and the positive results were all cytoplasm brown and nuclear blue.Results of TGF-beta 1:1day?3day after modeling,TGF-beta 1 positive rate gradually increased in the model group,IL-10-h AMSCs group increased obviously(P < 0.05);7days ? 14 days after modeling,compared with the model group,h AMSCs group,the positive rate of IL-10-h AMSCs group,empty plasmid group were significantly decreased,and the IL-10-h AMSCs group decreased most(P<0.05).MMP-1 results: 1day-3day,the positive rate of MMP-1 was gradually increased in each group,and there was a significant difference compared with the blank group(P < 0.05).Except the blank group,there was no difference among the other groups(P > 0.05).After 7days,compared with the model group,the positive rate of h AMSCs group,IL-10-h AMSCs group,empty plasmid group were significantly higher,and the IL-10-h AMSCs group was the most(P<0.05),14 day,the positive rate in each group was decreased,the model group was significantly decreased.The difference was significant compared with IL-10-h AMSCs group.(P < 0.05).TIMP-1 results: before 7days,h AMSCs?IL-10-h AMSCs and empty plasmid group compared with the model group,the positive rate of TIMP-1 were higher,the difference compared to the model group and the blank group was significant(P < 0.05).IL-10-h AMSCs compared with empty plasmid group and h AMSCs group,the difference were significant(P<0.05),14 d,model group content increased rapidly,the three groups increased not that significantly,the difference was statistically significant(P < 0.05).6?The results of ELISA method and qPCR detection of mice back wounds and wound tissue showed the relative expression of TGF-beta 1 mRNA content: the blank group TGF-beta1 is low,the model control group TGF-beta1 content gradually increased,maximum was in day 7,then began to decline gradually after the peak.1d?3d,TGF-beta1 of h AMSCs?IL-10-h AMSCs and empty plasmid group increased significantly than model control group,and IL-10-h AMSCs group increased more significantly.7 day,IL-10-h AMSCs group compared with the empty plasmid group,h AMSCs group,TGF-beta1 decreased significantly,and the three groups were less than the model control group,with significant difference(P < 0.05)above.It is suggested that IL-10-h AMSCs can promote the secretion of TGF-beta1 in wound healing process in 1-3days,and inhibit the secretion of TGF-beta 1 in the wound surface after 7days.The content of MMP-1 and the relative expression of MMP-1mRNA: the results showed that the content of MMP-1 in the blank group was lower,and the MMP-1 content of the other four groups in 1-3days increased gradually,and then began to decline after the peak of 7 days.h AMSCs and IL-10-h AMSCs,empty plasmid group compared to the model control group,the content of MMP-1 in 1-7days were lower,and IL-10-h AMSCs group was the lowest.7 days,four groups of MMP-1 were increased,and the IL-10-h AMSCs group MMP-1 was significantly more than empty plasmid group,h AMSCs group and model group,the difference was statistically significant(P < 0.05).The results showed that the expression of MMP-1 in normal skin was very low.IL-10-h AMSCs can increase the content of MMP-1 in wound.The TIMP-1 content and the relative expression of TIMP-1mRNA : the results showed the blank group with low content of TIMP-1,TIMP-1 in the other four groups gradually increased,before 7 days,TIMP-1 in IL-10-h AMSCsgroup?h AMSCs and empty plasmid group compared with model control group were higher.The difference was statistically significant(P < 0.05),14 days,the content of TIMP-1 in model group increased rapidly,the other three groups increased not that obvious,h AMSCs group,IL-10-h AMSCs group,empty plasmid group compared to the blank control group and model group,the difference was significant(P < 0.05).The results showed that the expression of TIMP-1 in normal skin was very low.IL-10-h AMSCs can increase the content of TIMP-1 in wound surface.The ratio of TIMP-1/MMP-1: 1D,7D,there was no difference between groups,3D,the IL-10-h AMSCs ratio is greater than the model control group,the difference was statistically significant(P < 0.05),14 D,the ratio of IL-10-h AMSCs group is less than the model control group,the difference was statistically significant(P < 0.05).Conclusion:1?After the wound formation,the local transplantation of IL-10-h AMSCs can affect the expression of TGF-beta 1 by promoting first and then decreasing in the wound,promote wound healing,control the inflammatory reaction of wound,and improve the quality of wound healing2?IL-10-hAMSCs can decrease the ratio of TIMP-1/MMP-1 in wound healing,improve wound healing quality and promote wound healing.