| Objective: To verify the biocompatibility and immunogenicity of Scx-hAMSCs-HAAM complex based on whether hAMSCs modified by Scleraxis are compounded with human acellular amniotic membrane(HAAM).Methods: 1)The amniotic membrane tissues of full term placenta were taken from women with Cesarean Section,Human acellular amniotic membrane(HAAM)were prepared by enzymatic digestion and freeze-thaw methods,respectively.The HAAM prepared by the two methods were dehydrated,paraffin-embedded and sections stained with HE and observed under inverted microscope.The morphological features of HAAM prepared by different methods were compared and the decellularizing effect of the two methods was evaluated.By immunofluorescence staining,HAAM was placed under fluorescence microscope to observe the removal of cells and the loss of extracellular matrix(ECM)on the surface of human amniotic membrane after decellularizing treatment.2)Human amniotic membrane tissues were obtained by the same method and digested by trypsin and collagenase to obtain hAMSCs.hAMSCs were purified,cultured and passaged.The growth and arrangement of hAMSCs were observed under inverted microscope.The phenotype of hAMSCs was identified by flow cytometry in the P3 generation,and the proliferation activity of hAMSCs was detected by CCK-8.Scx lentivirus vector was constructed,and the virus was amplified and titered by 293 T cells.Scx-hAMSCs were obtained by transfection of P3 generation hAMSCs with appropriate titer.The expression of Scx gene in cells was detected by PCR and Western Blot,and the cell viability was detected by CCK-8.3)Scx-hAMSCs were co-cultured with HAAM-Scx-hAMSCs cells were stained for to detect their growth and distribution on HAAM at days for culture,and their morphological features were observed under inverted phase contrast microscopy and scanning electron microscopy(SEM).Then,blank control group,negative control group and experimental group were set up.HAAM and Scx-hAMSCs were implanted under the back fascia of SD rats.After one week,SD rats were killed.The tissues of operation area were taken for HE staining,paraffin embedding and section,and immunological rejection reaction of each group was observed under the microscope.Results: 1)Human acellular amniotic membrane(HAAM)prepared by enzymatic digestion had better decellularizing effect than that by freeze-thaw method.After decellularizing by enzymatic digestion,the surface of HAAM was smooth and no cell structure could be seen by HE staining.After immunofluorescence staining,the expression of HAAM cells treated by enzymatic digestion was negative,and there was no significant decrease in collagen of human amniotic membrane(HAM)before decellularizing.4)HAMSCs with stable proliferation could be obtained by digestion of HAM with trypsinase and collagenase.After purification,culture and passage,the cell morphology presented spindleshaped,the cell arrangement became dense and showed spiral growth.After the identification of P3 generation hAMSCs by flow cytometry,the mesenchymal stem cell surface markers CD73(92.1%),CD105(84.8%),CD90(94.7%)and CD44(94.1%)were highly expressed,CD19 + CD34 + CD11 b + CD45 + HLA-DR(0.3%)were negative expression.PCR detection of digestion products showed the size of bands at 600 bp.Different gradients were set to transfect 293 T cells,and The 2×108 TU/μL viral stock was screened out and transfected into P3 generation of hAMSCs with 6 μL viral stock.A large number of fluorescent expression was observed under fluorescence microscope after 24 and 48 hours,respectively.The results indicated that the transfection effect was good.The expression of Scx gene in Scx-hAMSCs trans fection group was hi gher than t hat in non-t ransfection group b y PCR(* P < 0.05).3)Scx-hAMSCs prepared in the previous step were compounded with HAAM.Immunofluorescence staining was performed 14 days after culture.Scx-hAMSCs covered HAAM with rod or spindle shape under fluorescence microscope.HE staining showed that,compared with the negative control group,there was inflammatory reaction in the surgical area 1 week after local operation of scx-hamscs-haam(experimental group).Infiltration of plasma cells and neutrophils was observed,granulation tissue and new capillary growth were observed locally,necrosis and scar repair co-existed,and the inflammatory reaction was more severe than that in the negative control group.Postoperative 1 month and the negative control group and experimental group,presents the inflammation disappeared under the mirror,the experimental group of visible light pink osteoporosis was similar to the amniotic membrane after epithelial tissue surrounding the local tissue,and has been attached with cell growth,the surface cells are arranged closely linear,cell-complex stents can be thought of SD rats after low immunogenicity.Conclusion: The main results are as follows: 1)the human acellular amniotic membrane scaffolds(HAAM),)with abundant extracellular matrix and good porosity can be obtained by enzymatic digestion of human amniotic membranes,which can be used as biological scaffolds to provide conditions for the growth and reproduction of seed cells.2)ScxhAMSCs-HAAM and HAAM show low immunogenicity in animals in a short period of time,which is helpful to the construction of tissue engineering ligaments and provides a potential source of graft. |
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