Construction Of A Lentivirus-mediated Vector For Fas Gene Silencing And Its Stable Expression In Human UC-MSCs

Objective:1,To isolate and culture umbilical cord mesenchymal stem cells fromhealthy person umbilical cord in vitro, Identification the biological characteristics ofUC-MSCs.2, To construct a Lentivirus-Mediated Vector for RNA interference(RNAi) ofFas, and carry out packaging, purification,concentration,and titer determination of slowvirus-like articles; In order to lay a experiment foundation for the next step infectingUC-MSCs，to achieve effective silencing the FAS gene expression of UC-MSCs.3,using the lentiviral vector siRNA of Fas gene, establish a human UC-MSCs cell linewith Fas Gene interference;Detection the gene and protein expression，in order to lay aexperiment foundation for the next step using low Fas expression gene of umbilical cordmesenchymal stem cell in the therapy aplastic anemia. and looking for new way fortreatment of aplastic anemia.Methods:1,UC-MSCs were isolated and cultured by tissue block adhering wallmethod, obtained adherent cells after2weeks culture and were observed cellmorphology under inverted phase contrast microscope, Identified cell surface markersby flow cytometry; Detection adipogenic and osteogenic differentiation capacity invitro;2, According to Fas gene mRNA sequence, Design four groups of the genetargeting Fas shRNA sequence, Synthesis, anneal to form double-stranded DNAfragments, And after BamHI, EcoRI double enzyme cut LV3carrier connection intocompetent cell transformation, to sequence and comparison analysis for the positiveclones, and PCR identification for the positive cloning, By transfected293T cells onpacking and lentivirus titer determination.3，Recombinant lentiviral infected humanumbilical cord mesenchymal stem cells by different multiple of infection (MOI,0,10,30，50,80and100). By recombinant lentiviral infected UC-MSCs with reporter genegreen fluorescent protein expression, the best MOI was selected; Recombinant lentiviralvector infected UC-MSCs at the best MOI, Real time-PCR and Western blot was used todetect the expressions of Fas mRNA and protein in the transfected cells. Results:1,culturing UC-MSCs10d by tissue block adhering wall method in vitro,Fibroblast-like cells climbing from the tissue; cultured cells of10generations have nosignificant change in morphology and proliferation; cells positive high expression ofCD44,CD73and negative expression of CD34.Experiments confirmed that UC-MSCshave adipogenic and osteogenic differentiation capacty in vitro.2, Restriction digestionand DNA sequencing showed that lentiviral vector was successfully constructed,the titerof lentivirus vectors was3x108TU/ml.3,When the MOI was30and the infectionefficiency was up to80%. The results of real-time PCR and western blot showed the Fasgene expression was significantly suppressed in UC-MSCs.Conclusion:1,Human umbilical cord mesenchymal stem cells were successfullyisolated in vitro and had the biological characteristics of mesenchymal stem cells.2,Therecombinant lentiviral vector siRNA of Fas gene was successfully constructed andpackaging high titers of virus.3，Selecting the optimal MOI=30, carrying entiviral vectorsiRNA of Fas gene infected UC-MSCs,the levels of Fas gene and protein expressionwere significantly reduced compared with the control group. Objective:1, To isolate and culture umbilical cord mesenchymal stem cells fromhealthy person umbilical cord in vitro, Identification the biological characteristics ofUC-MSCs.2,Exosomes were isolated from the culture supernatant with serum-free inthe P3-P5generation UC-MSCs,And study its biological characteristics.3, peripheralblood lymphocytes were isolated by lymphocyte separation medium (Ficoll), To discussthe immune effects of UC-MSCs derived exosomes on peripheral blood lymphocytes.Methods:1, UC-MSCs were isolated and cultured by tissue block adhering wallmethod, obtained adherent cells after2weeks culture and were observed cellmorphology under inverted phase contrast microscope, Identified cell surface markersby flow cytometry; Detection adipogenic and osteogenic differentiation capacity in vitro;2, collection the serum-free culture supernatant from P3-P5-generation UC-MSCs,obtain the cell culture supernatant concentrates by High-speed centrifugation, and theexosomes were isolated and purified with sucrose density gradient ultracentrifugationand ultrafiltration. Electron microscope observed the morphology of exosomes; then theimmunophenotype of exosomes were detected through CD44and CD73by flowcytometry.3,peripheral blood lymphocytes were isolated by lymphocyte separationmedium (Ficoll), which followed by exosomes and peripheral blood lymphocytesco-cultured, and then MTT assay was used to detect the proliferation of lymphocyteinfluenced by exosomes; and ELISA was used to detect the effects of exosomes onlymphocytes secreting IL-4and INF-γ.Results:1, As in the first portion, culturing UC-MSCs10d by tissue blockadhering wall method in vitro, Fibroblast-like cells climbing from the tissue; culturedcells of10generations have no significant change in morphology and proliferation;cells positive high expression of CD44, CD73and negative expression of CD34.Experiments confirmed that UC-MSCs have adipogenic and osteogenic differentiationcapacty in vitro.2, Electron microscope had observed exosomes were30-100nm in diameter elliptical membrane vesicles, and they high expressed adhesion moleculesCD44(54.36%) and stem cell marker CD73(30.56%);3, which followed by exosomesand peripheral blood lymphocytes co-cultured, MTT and ELISA results showed thatdifferent concentrations of exosomes inhibited proliferation of peripheral bloodlymphocytes and had a dose-dependent, also inhibited the secretion of cytokines IL-4and INF-γ with a dose-dependent.Conclusion:1, Human umbilical cord mesenchymal stem cells were successfullyisolated in vitro and had the biological characteristics of mesenchymal stem cells.2,The UC-MSCs derived exosomes wre isolated Successfully, Peripheral bloodlymphocytes wre isolated by lymphocyte separation medium (Ficoll) Successfully.3，UC-MSC-derived exosomes inhibited lymphocyte proliferation and secretion ofcytokine IL-4and INF-γ with a dose-dependent trend.