Investigation Regarding Tactility Recovery Effects Of Joint Transplantation Of Merkel Cells And Schwann Cell-like Cells On Plantar Wounds

Objective:To observe the tactility-recovery effects of joint transplantation of MCs and SCLCs on the plantar wounds,through an in-vitro separation,cultivation and evaluation of MCs,SCs,hAMSCs as well as SCLCs.Methods:1.Skin of plantar and beard area were obtained aseptically from 3-day-old newborn SD rat pups.A 2 step digestion of neutral protease and trypsin was utilized to isolate and extract MCs,which had been cultured for 6 days.The cultured MCs were then identified by cellular immunofluorescence,while Western Blot was used to detect the expression of protein CK20 and protein Piezo2.CGRP secretion was measured at different times by ELISA.2.Sciatic nerves were obtained aseptically from 3-day-old newborn SD rat pups.Trypsin plus type II collagenase digestion was applied here for isolation of SCs,the P3 generation of which were also identified by cellular immunofluorescence.3.The hAMSCs were isolated from the human amnions of full-term births,which were collected after Cesarean section,using digestion of trypsin and collagenase.Cellular immunofluorescence was applied to identify the P3 generation of hAMSCs.4.With the added inducible factors,P3 generation of hAMSCs were then induced into SCLCs.Performing cellular immunofluorescence for cell identification and Western Blot for detecting protein expression of S-100,P75 and GFAP.5.Transplanted cells were divided into four groups(10 rats for each)to establish a full plantar skin defects model of SD rat pups,including sham-operated group,model group(normal saline),control(SCs and MCs)and operated group(SCLCs and MCs),among which status of healing wounds were observed.Contraction force threshold of wounds plantar was measured using Vonfren fibre on day 0,7,10 and 14 respectively whereas on day 0,7 and 14,swab-stimulated plantar contraction was performed Western Blot had been used for detecting expression for both protein Piezo2 and MBP upon the collected healed woundsResults:1.The primary MCs could be obtained by enzymatic digestion,of which CK20 and Piezo 2 showed a hyper chromogenic reaction in cellular immunofluorescence.Results of Western Blot and qPCR indicated that expression level of the operated group was significantly higher than the control group with a p-value smaller than 0.05.Secretion of CGRP increased with time(p<0.05)2.Enzymatic digestion was considered to be able to isolate the primary SCs,the P3 generation of which was identified through cellular immunofluorescence,where hyper chromogenic reactions were observed on S-100,P75 and GFAP3.hAMSCs were successfully extracted via enzymatic digestion and cultivated until its P3 generation,which were then identified by immunofluorescence.The hAMSCs group was identified by cell immunofluorescence,vimentin was highly expressed,CK19 was not expressed4.Cellular immunofluorescence identification was performed onto the induced SCLCs,showing hyper chromogenic reactions on S-100,P75 as well as GFAP while no chromogenic reactions were obtained on hAMSCs group.A significantly higher expression level(p<0.05)of SCLCs group was detected compared to hAMSCs group according to the results of Western Blot5.The sham-operated group showed a fastest wounds healing speed,followed respectively by the control group and operated group,whereas the model group turned out to be with a slowest healing speed.A opposite result as healing speed had been obtained regarding to both the level of swab-stimulated contraction and measured thresholds for the plantar-contraction,which was considered to be statistically different(p<0.05).Protein expressions of both Piezo2 and MBP of healed wounds were detected through Western Blot,it was found that the expression of these two proteins in the model group was significantly lower than that in the experimental group and the control group,meanwhile,the statistical was obviously differenceConclusion:SCLCs combined with MCs can promote tactile recovery of skin wounds.