Effect Of The Microenvironment On The Biological Effects Of HUC-MSCs-Exos During The Plastic Stage Of Wound Healing

Objective: To investigate whether the human umbilical cord mesenchymal stem cell exosomes that stimulated by mouse scar skin homogenate has biological effects on fibroblasts the effector cells in the process of wound healing and regulates the process of scar remodeling.Methods:(1)Extraction and identification of exosomes of human umbilical cord mesenchymal stem cells stimulated by microenvironment during scar molding stage: the scar tissue of mice was epithelialized and the scar tissue homogenate(STH)was obtained by physical grinding and high-speed centrifugation.Human umbilical cord mesenchymal stem cells(hUC-MSCs)were stimulated by STH.The exosomes of human umbilical cord mesenchymal stem cells stimulated by scar tissue homogenate(STH-hUC-MSCs-Exos)were isolated and extracted by Total Exosome Isolation kit and identified by Western-blot,Nanosight and electron microscope.(2)Effect of STHhUC-MSCs-Exos on proliferation,migration and secretion of wound healing related factors of skin fibroblasts: scar tissue homogenate was set up with high(100?g/m L),middle(50?g/m L)and low(30?g/m L)concentration groups to stimulate hUC-MSCs,with traditional hUC-MSCs-Exos as positive control.CCK8 method and scratch test were used to detect the effects of hUC-MSCs-Exos stimulated by different concentrations of scar tissue homogenate on the proliferation and migration of skin fibroblasts.q PCR technique was used to detect the effect of STH-hUC-MSCs-Exos on the ability of fibroblasts to secrete factors related to wound healing.(3)Study on the effects of STH-hUC-MSCs-Exos on the process of scar remodeling: C57/BL6 mice were used to establish skin wound healing models,and model groups,hUC-MSCs groups,and traditional hUC-MSCs-Exos groups(Tra-Exos group),human umbilical cord mesenchymal stem cell exosomes stimulated with scar tissue homogenate(STHhUC-MSCs-Exos group),on the 14 th day of wound healing,that is,after the wound epithelialization is completed,the corresponding treatments are performed according to the grouping.Intervention.On the 3rd and 5th day after intervention,the sampleswere taken for HE staining,Masson staining and CD31 immunofluorescence staining,and the distribution of collagen and microangiogenesis were observed.Results:(1)Western-blot results showed that STH-hUC-MSCs-Exos expressed CD9,CD63 and HSP-70 exocrine labeled proteins,Nanosight results showed that STH-hUCMSCs-Exos was about 130 nm in diameter,which met the requirement of exosome size,and STH-hUC-MSCs-Exos was a cup-shaped bilayer lipid membrane structure under transmission electron microscope.(2)Cell proliferation test: STH-hUC-MSCs-Exos could promote the proliferation of fibroblasts,and the promoting ability increased with the decrease of scar homogenate concentration,and the promoting effect of low homogenate exocrine group was significantly higher than that of Tra-Exos group(P<0.01).(3)Scratch test: STH-hUC-MSCs-Exos had the ability to promote the migration of fibroblasts,and the promoting effect was gradually enhanced with the decrease of scar homogenate concentration,and the promoting effect of low homogenate exocrine was statistically significant compared with Tra-Exos group(P<0.001).(4)q PCR results showed that STH-hUC-MSCs-Exos could down-regulate the expression of pro-fibrosis factors TGF-?1.(5)HE staining: on the 3rd day,compared with other groups,the epidermis of STH-hUC-MSCs-Exos group was thinner and the skin structure was clear,and the skin accessory structure increased and arranged neatly;on the 5th day,the skin structure of all groups became more orderly than that of the third day,and the skin appendages of STH-hUC-MSCs-Exos group produced the most and arranged most neatly.(6)Masson staining: Masson staining: on the 3rd day,the collagen fibers in the STH-hUC-MSCs-Exos group were the most symmetrical and neatly arranged;compared with the model group,the collagen volume fraction of the mouse scar model decreased after intervention in each exocrine group or stem cell group.On the 5th day,compared with the 3rd day,the collagen fibers in each group were arranged more neatly,and the collagen volume fraction in STH-hUCMSCs-Exos group was the lowest,which was statistically significant compared with other groups.(7)CD31 immunofluorescence staining: each exocrine group and stem cell group can inhibit the formation of microvessels.On the 3rd day,the inhibitory effect of STH-hUC-MSCs-Exos on microangiogenesis was the strongest,and it was statistically significant compared with hUC-MSCs group(P <0.01),and on the 5th day,STH-hUC-MSCs-Exos still showed an effective inhibitory effect on microangiogenesis compared with hUC-MSCs group(P <0.05).Conclusion: The microenvironment of scar molding stage can optimize the biological effects of hUC-MSCs-Exos and enhance the ability of hUC-MSCs-Exos to promote the proliferation and migration of fibroblasts.STH-hUC-MSCs-Exos can down-regulate the expression of fibrosis-promoting factor TGF-?1,reduce collagen deposition and inhibit microangiogenesis to alleviate scars.