Study On The Effect Of Nanofat Combined With Mouse Nerve Growth Factor On Nerve Regeneration Of Free Anterolateral Femoral Perforator Flap

To investigate the effect of nanofat combined with Mouse nerve growth factor on sensory recovery after free anterolateral femoral perforator flap and its potential therapeutic mechanism,so as to provide a safe and effective clinical treatment and theoretical basis for promoting postoperative sensory recovery of the flap.Methods:One.Clinical study1.Patient recruitment: the inclusion and exclusion criteria were established and 16 patients who had undergone free anterolateral femoral perforator flap 4 months after operation were recruited.2.Preoperative sensory evaluation and sampling of the skin flap: the sensation of pain,temperature and two-point discrimination of the flap and its surrounding area were evaluated before operation,and the skin tissue of the flap was obtained by using a 5mm perforator.3.Preparation of the mixture of nanofat and mouse nerve growth factor: the nanofat was obtained by referring to the method of Tonnard,and the nanofat was mixed with mouse nerve growth solution at 9:1.4.Experimental intervention: Mouse nerve growth factor injection group(N group),nanofat injection group(Na group),Mouse nerve growth factor combined with nanofat group(NN group),physiological saline group(NS group).5.Postoperative evaluation: the sensation of the skin flap was evaluated according to the preoperative method,and the perforator was used again at the site of skin extraction before operation.Immunohistochemistry: the neovascularization of skin flap was observed by CD31 immunohistochemical staining and the nerve regeneration of skin flap was observed by S100 immunohistochemical staining.The number of neovascularization and positive expression were counted by Image J software after 5 visual fields.The difference between sensory evaluation and immunohistochemical data before and after operation was calculated and the statistical results were obtained.Two.Basic research.6.Using clinically prepared nanofat,human adipose-derived stem cell(h ADSCs),was extracted by type II collagenase digestion and transferred to the third generation for this experiment.7.The expression of h ADSCs surface markers CD90,CD105,CD73,CD34,CD11 b,CD19,CD45 and HLADR was detected by flow cytometry.The cells were induced by three-line induction reagents,and the results were identified by oil red,alizarin blue and alizarin red staining.8.Cell proliferation experiment: to study the effects of different concentrations of mouse nerve growth factor(0.9?g/m L,0.09?g/m L,0.009?g/m L)on the proliferation of h ADSCs cultured in vitro;briefly: the third generation h ADSCs was digested,resuscitated and counted.Grouping: experimental group,negative control group,blank control group.Seed plate: the counted h ADSCs was inoculated into a 96-well plate and cultured in a incubator.Adding medicine: the culture medium containing different concentrations of mouse nerve growth factor was added to continue the culture.Adding CCK-8 reagent: after 24 hours and 48 hours of culture,the serum-free medium containing 10% CCK-8 reagent was added and cultured for 2 hours.The optical density(OD value)of h ADSCs 450 nm was detected by enzyme labeling instrument.9.Enzyme-linked immunosorbent assay: to study the effect of mouse nerve growth factor on paracrine of adipose-derived stem cells cultured in vitro.In brief,the supernatant of P3 h ADSCs with different concentrations of mouse nerve growth factor was taken from the control group,and the corresponding standard was added according to the instructions of ELISA kit,then the enzyme conjugate(except the blank control hole)was added,and then the antibody was added,mixed and sealed,and incubated at 37 ? for 1 hour.Discard the liquid,wash,shake dry.Add chromogenic agents A and B,avoid light at 37 ?,and add terminating solution.The optical density(OD)of each hole was measured by enzyme labeling instrument at 450 nm wavelength.The sample concentration is calculated.Three.Statistical analysis The experimental results showed that the mean ± standard deviation(±s)was used for statistical analysis by SPSS 20.0 statistical software.After the normality test,LSD method was used to test the variance homogeneity of multiple comparisons between groups,and Dunnett' s T3 method was used to test the variance when the variance was uneven.Results:1.The difference of skin flap temperature sensation,two-point discrimination and tactile pressure sensation before and after injection of nanofat and or mouse nerve growth factor was larger than that of normal saline group,and the difference of skin flap temperature sensation,two-point discrimination and tactile pressure sensation in the combined group was the largest,which was 6 cases,18.73 ±2.72 mm and 1.24 ±0.26(specification),respectively.2.The results of immunohistochemistry showed that the difference of CD31 and S100 expression in skin flap before and after injection of nanofat and or mouse nerve growth factor was larger than that in normal saline group,and the difference in combined group was the largest,which was 22.00 ±2.58,11.81 ±4.63,respectively.3.There were many impurities in h ADSCs,P0 cells extracted from human nanofat.After48 hours of culture,most of the cells adhered to the bottom of the culture bottle and were fusiform and polygonal.There are few impurities in P3 cells,with regular cell morphology,long fusiform,and cross-linked with each other.4.Flow cytometry showed that h ADSCs expressed CD73,CD90,CD105,and almost no expression of CD45,CD34,CD11 b,CD19 and HLADR.After the induction and differentiation of the three lines,h ADSCs showed the ability of adipogenesis,osteogenesis and chondrogenesis.5.Cell proliferation test showed that 24 h and 48 h after h ADSCs,the optical density(OD)of 0.9?g/m L mouse nerve growth factor was 1.17 ±0.03 times and 1.28 ±0.07 times higher than that of the control group,respectively.enzyme linked immunosorbent assay showed that after co-culture of 0.9?g/m L mouse nerve growth factor with h ADSCs,the amount of neurotrophic factors(NGF and NT-3)secreted by h ADSCs was 4.40 ±0.44 and 1.76 ±0.17 times higher than that of the control group,respectively.Conclusion:1.Mouse nerve growth factor combined with nanofat transplantation can promote the sensory recovery of free anterolateral thigh skin flap,and the mechanism may be related to increased angiogenesis and nerve regeneration.2.Mouse nerve growth factor can promote the proliferation and paracrine of h ADSCs cultured in vitro.