Experimental Research Of Local Injection Of Merkel Cells Combined With External Application Of MEBO On Nerve Regeneration After Plantar Wound In Rats

Objective:Through extraction,isolation,culture and effective identification of MCs in SD rats,and observe the effect of local injection of Merkel cells combined with external application of MEBO on neural regeneration after plantar wound in rats.Methods:1)The foot pads,beard pads and back skins from neonatal SD rats(1~3d)were collected and the MCs were isolated and extracted by enzyme digestion.Then MCs were inoculated on poly lysine(PLL)coated culture flask.The expression of cytokeratin CK20 was determined by immunohistochemical method.And the expression of Piezo2 m RNA in MCs was detected by qPCR.ELISA was used to detect the secretion levels of CGRP on day 2,4,6,8 and 10.2)SD rat model of plantar wound was established and all rats were divided into five groups according to means of intervention: Group A(MCs injection +MEBO);group B(MCs local injection);group C(external application of MEBO);group D(negative control group);group E(positive control group with mouse nerve growth factor injection).Rats in MCs groups were injected MCs at the wound central and at 3,6,9,12 o’clock direction of the wound edge respectively with the MCs concentration of 1 x 106/mL.Additional one injection was given 24 hours later at each site with 25 μL per time.MEBO groups were treated with external application of MEBO according to the corresponding group once a day until the wound healing.Positive control group and blank control group were given equal volume of nerve growth factor,and PBS injection.The time of wound healing was observed and the wound healing rate of each time point in each group was calculated.Moreover,the recovery of plantar sensation was observed by swab test.Six rats were randomly selected in each group on day 7,14 and 21 after intervention and the tissues were obtained to make paraffin sections.Immunofluorescence staining was used to observe thegrowth of new skin nerve fibers.3)All the rats underwent the detection of paw withdrawal reflex of plantar by swab in each group 7,14 and 21 days after modeling,and records and statistics of the frequency was carried out to calculate the percentage.Results:1)By neutral protease and trypsin digest skin tissue of SD rats can get more Merkel cells,antibody CK20 staining showed that MCs positive reaction was detected by immunocytochemical.2)Using neutral protease and trypsin digestion method can be obtained MCs in the experiment,Ham,s F-12 medium was used as basic medium,add bFGF(20ng/ml)can make Merkel cells stable proliferation.The qPCR results showed that the expression level of Piezo2 mRNA in Merkel cells was 0.0270±0.0042.Enzyme linked immunosorbent assay(ELISA)method was used to detect the concentration of calcitonin gene related peptide(CGRP)in the supernatant of Merkel cells,the concentration increased following the extension of incubation time(2,4,6,8 and 10 days),and faster in 6D to 8D,it reacts Merkel cell proliferation indirectly.3)There was no significant difference in the degree of swelling and inflammatory reaction.The healing time of the two groups of MEBO was earlier than that of other experimental groups,from group A to group E of healing time is respectively 14.33±0.69 d,16.44± 0.86 d,14.83± 0.71 d,17.44± 0.98 d and 16.27± 0.67 d.At the same time,the wound healing rate in group A and group C was better than the other control group(P <0.05),all wounds healed on 21 d.On the comparison of foot reflex,the A group had lower reflex threshold and more sensitive to stimulation(P < 0.05)except group E,compared with the other three groups.The results suggested that Merkel cells combined with MEBO can promote the recovery of tactile sensation in the wound of rat planta pedis.4)Immunofluorescence staining showed that the number and diameter of nerve fibers in group A were better than those in negative control group or single treatment groups,which was similar to positive control group(group E).Conclusion: 1)Neutral protease and trypsin enzyme digestion method is an effectivemethod to obtain the rat Merkel cells.In vitro culture system,including Ham,s F-12 medium and bFGF,MCs can proliferate.2)Local injection of Merkel cells combined with external application of MEBO can accelerate wound healing and promote nerve regeneration of skin.