The Long-term Implantation Effects Of Amniotic Mesenchymal Stem Cells Conditioned Culture With Amniotic Epithelial Cells In NOD/SCID Mice And Labeling Of Amniotic Mesenchymal Stem Cells Infection By Lentiviral Vector

Objective(s):Hematopoietic stem cell transplantation is one of the most effective treatments of blood diseases,for example blood system malignancy,severe aplastic anemia,severe immunodeficiency disease and some hereditary diseases.Mesenchymal stem cells are stem/progenitor cells of bone marrow stromal cells and are non-hematopoietic cells with multiple differentiation potential.It can not only differentiate into osteoblasts,chondrocytes and adipocytes in the mesoderm,but also cross-epithelial differentiation.MSC has hematopoietic support and immunomodulatory function,in vitro can support long-term hematopoietic start-up cells(LTC-IC)growth.in vivo,MSC aiso can promote hematopoietic implantation and reconstruction,and can prevent and treat GVHD,at the same time,it plays an important role in tissue damage repair.In the transplant,intravenous infusion method required a larger number of MSC,but the number of MSC implanted in the body is limited,so to explore the MSC to promote damage to the organization of the damaged method is very important.Many studies have confirmed that the SDF-1/CXCR4 axis plays a very important role in the migration and specific homing of the MSC.It’s also been shown that the co-culture with amniotic epithelial cells(AEC)resulted in up-regulation of cell surface CXCR4 of AMSC,and amniotic mesenchymal stem cells can maintain their good cell activity.It also increases the ability of AMSCs to migrate in vitro and the number of cells homing to the bone marrow shortly after implantation into the body,enabling faster hematopoietic recovery of the recipient.Our previous experiments have demonstrated that CXCR4 can be expressed in AMSCs.In a serum-free environment,the use of the AEC conditioned medium prepared by the autocrine characterization of AEC can up-regulate the expression of CXCR4 in AMSC,increase its ability to migrate in vitro and the number of cells homing to the bone marrow shortly after implantation into the body.It also can be faster to promote receptor hematopoietic recovery.The blocking effect of CXCR4 with neutralizing antibody significantly reduced the number of homing and the promotion of hematopoietic recovery,and confirmed that CXCR4 plays an important role in the homing process of AMSC.The first part of this study focuses on whether AMSCs can be homing to the bone marrow and long-term survival and implantation into NOD/SCID mice,and observe the effect of SDF-1/CXCR4 axis on homing and long-term implantation in AMSC-implanted mice.The second part of the study,to study the construction Method of a lentiviral vector containing Enhanced-green fluorescent protein gene and the infection ability of AMSC,in order to obtain a stable GFP expressing AMSC.Methods:In the first part,AMSC and AEC were isolated from human amniotic tissue by tissue block method and enzymatic digestion method respectively.The experiment was divided into five groups:AEC conditioned group,AEC conditioned + CXCR4 neutralizing antibody group(pre-incubated with anti-CXCR4 neutralizing monoclonal antibody lOug/pre-incubated cells),serum culture group,serum culture + CXCR4 neutralized Antibody group(pre-transplanted with anti-CXCR4 neutralizing monoclonal antibody lOug/pre-incubated cells),blank control group(injection of equal volume of saline).We injected the different treated AMSCs into the NOD/SCID mice,which were irradiated by sub lethal dose.After injection,After injection,observe the mental state of the mice.At 6 moths after transplantation,the mice were sacrificed,and take the mouse bone marrow for detection.A portion of the bone marrow was used to extract bone marrow genomic DNA,and into the drawn standard curve formula to obtain the sample in the sample of human cells and the proportion of mouse cells,detection of human cell implantation.Another part of the bone marrow with flow cytometry to detect the expression of HLA-I,CD45,CD34,CD10,CD33 and CD38 antibody.The remaining bone marrow was cultured and immobilized on Dexter bone marrow stromal cells for a long time.Detection of Human Cell Implantation by Human HLA-classⅠ Monoclonal Antibody,and cell differentiation was detected by human-specific endothelial cell marker vWF and human-specific fibroblast monoclonal antibody 5B5.In the second part,AMSC were isolated from human amniotic tissue by tissue block method.AMSCs were infected with lentivirus carrying GFP at different multiplicity of infection(MOI,respectively,set to 5、10、15、20、25、30、35、40、45and50).GFP expression efficiency was observed using the fluorescence microscope.Results:1.The results showed that human DNA was not detected in the bone marrow of serum in the serum culture group after 6 months of transplantation.And the human DNA in AEC conditioned group was about 3.4%of the total DNA content,and theimplantation rate of CXCR4 neutralizing antibody pretreatment group was significantly decreased.2.The results of flow cytometry analysis showed that HLA-I +human cells could be detected in AEC conditioned medium,and the HLA-I + human cells in CXCR4 neutralizing antibody group were significantly decreased,and CD45,CD34,CD10,CD33,CD38 test results were negative.3.Immunohistochemical staining showed that the positive rate of 5B5 antibody and vWF + staining were positive in the AEC conditioned group.CXCR4 neutralizing antibody group,the positive rate of two antibody staining was significantly reduced,serum group antibody staining results were negative;4.The lentiviral vector system carrying the green fluorescent protein(GFP)gene was able to infect AMSC,and the transfection was the best when the virus was infected with MOI = 30.Conclusion(s):AEC conditioned medium can significantly enhances the ability of AMSCs to be implanted in recipient mice;Stromal cell-derived factor-1/CXCR4 axis plays an important role in homogenization of AMSCs into bone marrow and long-term implantation;AMSC implanted bone marrow and will not differentiate into hematopoietic cells to participate in the recipient’s hematopoietic reconstitution;we constructed the green fluorescent protein(GFP)transfection system by lentiviral vector,and this transfect system could labeled the amniotic mesenchymal stem cells successful in vitro.