Experimental Study Of Glycosylated Adipose-derived Stem Cells Promoting Nerve Regeneration Of Single Perforator Flap In SD Rats

Objective:To investigate the effects and possible mechanisms of glycosylated adipose-derived stem cells on nerve regeneration of single perforator flaps in SD rats,and to provide a preliminary research basis for promoting sensory recovery of clinical flaps.Methods:1.After the patient's informed consent and the approval of the ethics committee of Zunyi Medical University,subcutaneous adipose tissue was obtained from liposuction of normal people in burn and plastic surgery of Zunyi Medical University,and centrifuged with type?collagenase digestion machinery to extract the subcutaneous fat from normal humans.Extraction of adipose-derived stem cells?ASCs?,low-dose DMEM?1g/L?medium containing 10%fetal bovine serum?FBS?+1%double antibody,culture in a 37?,5%CO₂ incubator,and use 0.25%trypsin digestion.2.P3 nASCs were identified for identification,and cell surface markers CD105,CD90,CD73,CD45,CD44,CD34,CD11b,CD19,and HLA-DR were detected by flow cytometry.Adipogenic,osteogenic,and cartilage-induced differentiation experiments were performed,and oil red"O",alizarin red,and alixin blue staining were used to identify the effects of adipogenic,osteogenic,and cartilage-induced differentiation,respectively.3.Take P3 nASCs and use a high-glucose DMEM?4.5g/L?glycosylation induction medium containing 0.2%advanced glycation end products?AGES?,15%FBS+1%double antibody,at 37°C,The cells were cultured in a 5%CO2 constant temperature incubator.The medium was changed once every 2-3 days,and glycosylated adipose-derived stem cells?gASCs?were obtained after 21 consecutive days of induction.4.Supernatants of nASCs and gASCs were collected,and the expression of human brain-derived neurotrophic factor?BDNF?,nerve growth factor?NGF?,and human neurotrophic factor 3?NT-3?were detected by ELISA.5.Take 72 8-week-old SD female rats,and make an in situ replanted denervated single perforation flap in the abdominal wall of each rat.According to the random number table method,divide them into normal human ASCs intervention group,sugar Basic ASCs intervention group and phosphate buffered saline solution intervention group?NC group?,24 rats in each group,the first two groups of rat skin flaps were injected with 0.2mL nASCs and gASCs suspension respectively,the number of cells was 1x10⁶,and the NC group was injected with 0.2mL PBS.6.Immediately after modeling,on the 10th day after the injection and on the 20th day after the injection,the skin tissue of the localized cell transplantation site was taken for histological examination,and the skin flap was evaluated using S100immunohistochemical staining and PGP9.5 immunofluorescence staining.Peripheral nerve regeneration,Image J software was used to calculate the percentage of positive expression under 5 high power microscopes.7.Statistical analysis:The experimental results were expressed as mean±standard deviation?±s?,and the data were processed using SPSS 20.0 statistical software.The LSD method was used to test for homogeneity of variance in multiple comparisons in the group,and the Dunnett's T3 method was used to test for variances.One-Way ANOVA was used for comparison between groups.Two independent samples were compared using independent sample t-test.P<0.05 were considered statistically significant.Results:1.A small number of primary cells adhere to each other after about 12 hours,and the growth rate of primary cells is slower,long spindle-shaped or polygonal.After48 hours,the growth rate of the cells is accelerated,and the fusion rate can reach 80%after 8-10 days.After passage,the cell growth rate is significantly faster,and it can be passaged again after 5-7 days.The results of cell surface markers extracted by flow cytometry showed that CD44?98.5%?,CD73?99.96%?,CD90?97.09%?,CD105?98.00%?were positively expressed,and CD45/CD34/CD11b/CD19/HLA-DR?1.56%?were negatively expressed;after adipogenic,osteogenic,and cartilage induced differentiation,the extracted cells showed adipogenic,osteogenic,and The differentiation potential of chondrocytes suggests that the extracted and cultured cells are adipose-derived stem cells.2.Take P3 nASCs to start glycosylation induction culture.As the induction time prolongs,gASCs will slow down the proliferation rate compared to nASCs.It will take 7-9 days to further passaging,while nASCs still remain for 5-7 days and can be passaged again.Morphically gASCs There was no significant difference between morphology and nASCs,and both of them grew and arranged in a radial vortex.3.Take the P3 generation of nASCs and gASCs to collect the two groups of cell culture supernatants,and perform ELISA to detect the expression levels of BDNF,NGF,NT-3.BDNF results:gASCs secretion is?1301.12±7.0?pg/mL,nASCs?1218.06?±5.1)pg/mL;NGF results:gASCs secretion was?432.37±1.2?pg/mL,nASCs?399.59±0.7?pg/mL;NT-3 results:gASCs secretion was?659.85±3.8?pg/mL,NACSs?638.45±1.6?pg/mL.The quantitative levels of the three neurotrophic factors in the gASCs group were higher than those in the nASCs group,and the differences were statistically significant?P<0.05?.4.S100 immunohistochemical results showed that immediately after modeling,there was no statistical difference in the positive percentage of newborn SC cells between the groups?P>0.05?;the gASCs group and nASCs group were higher than NC on the10th and 20th days after the intervention.Group,the difference was statistically significant?P<0.05?,of which the gASCs group was higher than the nASCs group,and the difference was statistically significant?P<0.05?.5.Quantitative analysis of PGP9.5 immunofluorescence showed that there was no statistical difference in the positive percentage of neonatal nerve fibers between the groups immediately after modeling?P>0.05?.The gASCs group and nASCs group were all on the 10th and 20th days after intervention.Compared with the NC group,the difference was statistically significant?P<0.05?.The gASCs group was higher than the nASCs group,and the difference was statistically significant?P<0.05?.Conclusion:1.Local transplantation of two different sources of ASCs can promote percutaneous nerve regeneration in SD rats.The mechanism may be related to increased Schwann cells and nerve fiber regeneration.2.Compared with nASCs,gASCs can promote nerve regeneration of perforator flaps in SD rats.The mechanism may be related to the secretion of more BDNF?NGF?NT-3.