The Role Of Schwann Cell-like Cells Derived From Human Amniotic Mesenchymal Stem Cells In The Healing Of Denervated Wound In Rats

Objective:To investigate the role of Schwann cell-like cells derived from human amniotic mesenchymal stem cells in the process of denervation wound healing,and to provide new ideas for the clinical treatment of refractory denervation wounds such as diabetic foot and pressure ulcer.Methods:1.Extraction,culture and identification of hA-MSC:intact amniotic membrane of healthy pregnant women of childbearing age was taken from the operating room,ha MSc was isolated by trypsin combined with type Ⅱ collagenase digestion method and subcultured.Cell morphology was observed under inverted phase contrast microscope and photographed.CK19 and vimentin were detected by cell immunofluorescence,and cell surface markers CD90,CD44,CD73 and CD 105 were detected by flow cytometry CD34,CD11b,CD19,CD45 and HLA-DR2.SCLC and SCLC identification:when the density of the third generation of hA-MSC reached 80%-90%in 6-well plate,induction medium 1(DMEM/F12 medium containing 1mmol/L β-mercaptoethanol),induction medium 2(DMEM/F12 medium containing 35ng/ml ATRA and 10%FBS)and induction medium 3(DMEM/F12 medium containing 10%FBS,5mmol/L forskolin and 10NG/ml forskolin)were added in sequence BFGF,5ng/ml PDGF-AA and 200ng/ml Heregulin β-1)were cultured for2weeks.Cell morphology was observed by inverted phase contrast microscope,and the expressions of S-100,p75 and GFAP were detected by Western blot.3.Establish the denervated wound model in rats:after abdominal anesthesia,shaved the back,dissected the sciatic nerves on both sides by surgical means,removed the 1 cm nerve bundle at the distal end of one side(experimental group),and left the other side untreated(control group).Two days later,a 5 mm diameter round full-thickness skin was removed from the sole of both feet,and the wound healing was observed on the 3rd,7th,10th and 14th days The healing speed of the control group was faster than that of the experimental group,which was verified by histology.4.Local transplantation of SCLCs and NC into denervated wound of rats:the sciatic nerves on both sides were dissected surgically and the nerve bundles 1cm distal to both sides were excised,and the wound treatment was the same as above;two days later,a 5mm diameter round full-thickness skin was excised from the plantar of both sides,In the experimental group,200 ul of suspension of the third generation SCLCs(1 × 106)was injected subcutaneously around the wound,while in the control group,200 ul of normal saline was injected subcutaneously around the wound.5.Observe the wound healing after transplantation:take photos on the 3rd,7th,10th and 14th day,and calculate the wound size.He staining was used to observe the skin structure and wound contraction.Masson staining was used to observe the number and arrangement of subcutaneous collagen fibers.Five fields were randomly selected under the microscope,and Image J was used to calculate the relative collagen expression;CD31 immunofluorescence was used to observe neovascularization,Myofibroblasts were observed by α-SMA immunofluorescence,and the relative intensity of immunofluorescence of α-SMA and CD31 were calculated by image J.6.Statistical analysis:the experimental results were expressed as mean ± standard deviation(x±s)and analyzed by SPSS 19.0 statistical software.After the normality test of the measurement data,the comparison of the two groups at the same time point was performed by Paired sample t test;P<0.05 is statistically significant.Results:1.Human amniotic mesenchymal stem cells(hA-MSCs)were successfully isolated from human amniotic membrane.The primary culture of hA-MSCs grew slowly and mixed with impurities.After 24 hours,a small number of cells adhered to the wall,and single long fusiform and polygonal cells adhered to the wall were observed.After 48 hours of culture,the morphology of hA-MSC showed diversity,the number of cells increased significantly,and the morphology gradually changed into long fusiform.After passage,the number of cells increased The cell density could reach 80%-90%within 4-6 days,and the cell morphology was long fusiform and whirlpool.2.Flow cytometry showed that hA-MSC were positive for CD90(98.52%),CD44(98.56%),CD73(99.57%)and CD105(82.56%),but negative for CD45,CD34,CDllb,CD 19 and HLA-DR(0.37%).Immunofluorescence results:vimentin was highly expressed,but CK19 was not.3.hA-MSC was successfully induced to differentiate into SCLC in vitro:some cells died and fell off 24 hours after the induction solution was added for 1 hour.72 hours after adding inducer 2,a large number of cells were observed to proliferate,and most of the cells still grew in spindle and vortex shape.The results showed that the processes around the cells were slender,some nuclei were enlarged,stereoscopic sense was enhanced,and they were arranged in fusiform and whirlpool shape.western blot was used to detect S-100,p75 and GFAP in SCLC and hA-MSC.4.The denervation wound model of rats was successfully established.The difference of plantar wound area between the two sides appeared from the 7th day.The healing speed of the control group(wound area 4.744 ± 1.055mm2)was faster than that of the experimental group(wound area 7.184 ± 1.211mm2)(P<0.05);by the 14th day,the control group had completely healed,while the experimental group had not completely healed.He staining showed that the width of the wound in the experimental group was larger than that in the control group,and autophagy and small ulcer appeared in some hind limbs of the experimental group,which proved that the denervation wound model was successfully established.5.Local transplantation of SCLC and NS into denervated wound in rats:On the 3rd day after operation,the wound size of SCLC group was(7.043 ± 0.649mm2)and NS group was(8.341 ± 0.339mm2).On the 7th day after operation,the wound size of SCLC group was(3.565 ± 0.537mm2)and NS group was(5.695 ± 0.545mm2).On the 10th day after operation,the wound size of SCLC group was(1.758 ± 0.357mm2)and NS group was(2.322±0.398mm2)Incomplete healing.HE staining showed that the width of wound in SCLC group was smaller than that in control group on the 7th,10th and 14th day,and the subcutaneous structure recovered faster.Masson staining:the number of collagen in SCLC group was more than that in NS group on the 7th,10th and 14th day(P<0.05).CD31 immunofluorescence results showed that compared with NS group,SCLC group had more neovascularization on the 7th,10th and 14th day(P<0.05).The results of α-SMA immunofluorescence showed that on the 10th and 14th day,compared with the NS group,the SCLC group was significantly better than the NS group The expression area of a-SMA was significantly more(P<0.05).Conclusion:Schwann like cells derived from human amniotic mesenchymal stem cells can accelerate wound healing by accelerating wound contraction,increasing collagen deposition,promoting angiogenesis and myofibroblast formation.