Construction Of Vector Carrying TGF-β1 Gene And Its Expression In Mouse Bone Marrow Mesenchymal Stem Cells

Chapter I The isolation and identification of mouse bone marrow mesenchymal stem cells (BMSCs) in vitroObjective Establish the method of isolation, purification and identification of the mouse bone marrow mesenchymal stem cells (BMSCs) in vitro.Method Cultured mouse BMSCs with the simple and available method of adherent cells separation, and observed cell morphology and growth; detected mouse BMSCs surface antigen CD34, CD45, CD105 and CD106 expression and purity with flow cytometry.BMSCs from mice was plated into tissue culture flasks. Adhered cells were allowed to grow to about 75% 80% confluency and then trypsinized and reseeded. Microscope were performed to observe the changes of cell morphology during this procedure. Cells were analyzed with a FACS can for CD34, CD45, CD105 and CD106 expression.Results After several discarding supernatant (24h-72h), the percent of adhered cells grew more, and there were colony formation were seen.the cell appeared long spindle. At the first 2 6th days, the cells growed very slow, however, cells growed faster in 7 10 days, which were "whirlpool-like". Cultured for 10-12d, cells grew to about 75% 80% confluency, BMSCs extracted from the BM formed a homogeneous population of cells by the second passage. Cell viability is 92.6 1.8%. The analysis of cell surface phenotype indicated that the BMSC population was positive for CD105, CD106, whereas negative for CD34 and CD45, with 90% (or more) uniformity.Conclusion The method was very simple, convenient and useful, by which mouse BMSCs were successfully isolated and identified. Chaper CONSTRUCTION OF VECTOR CARRYING TGF-AND ITS EXPRESSION IN MOUSE BONE MARROW MESENCHYMAL STEM CELLSObjective To construct a vector that carried TGF-β1 gene and express the fusion protein TGF-β1 in mouse marrow mesenchymal stem cell (BMSCs) by transfecting the recombinant plasmid into BMSCs.Methods The full-length coding sequence of TGF-β1 was amplified from the cDNA of mouse lung sample by PCR, and was directionally cloned into the Xba I/Bst BI-digested pCDH1-MCS1-EF1-copGFP to generate pCDH1-TGF-β1-EF1-copGFP. The recombinant plasmid was transfected into BMSCs and was observed by laser confocal microscopy and real time PCR.Results After PCR and sequence analysis, the recombinant plasmid pCDH1-TGF1-EF1-copGFP was constructed successfully. What s more, the TGF-β1 can expressed in BMSCs.Conclusion The recombinant plasmid pCDH1-TGF-β1-EF1-copGFP was constructed successfully and it can express the protein TGF-β1 in BMSCs, which provided a better understanding of the speci city of the biological behavior of BMSCs regulated by TGF-β1.