Construct And Identify The Way Of Lentivirus Transduced Connexin43 Gene Expression In Murine Bone Marrow-derived Mesenchymal Stem Cell In Vitro

BackgroundGraft-versus-host disease?GVHD?is a major cause of overall survival and the quality of life after hemopoietic stem cell transplantation.Therapy for acute and/or chronic GVHD relies primarily on administration of corticosteroids.however,50% of the patients do not respond to the corticosteroids and need second-line drug treatment,but because of the lack of uniform standards of second-line treatments,lead to the therapeutic effect of uncertainty and a wide range of adverse effects.In addition,some patients of chronic GVHD are steroid-dependent.and the qualities of life of patients are influenced by the side effects,such as infection,hyperglycaemia,hypertension,osteoporosis and dyspepsia,which are caused by long-term systemic steroid use.Bone marrow mesenchymal stem cells?BMSC?have attracted much attention with the manifestations of low immunogenicity,multi-directional differentiation potential,immune adjustment feature and promoting tissue repair by migrating to the damaged tissue.Researches indicated that the infusion of BMSC is effective for steroid resistant GVHD without adverse reactions related infusion.Steady state of bone marrow hematopoietic microenvironment depends on the frequency of signal transduction between bone marrow cells,and gap junction intercellular communication?GJIC?contributed by connexin 43?CX43?is common way of direct communication between bone marrow stromal cells.The CX43 expression defects of BMSC affect the implantation of hematopoietic stem cells and hematopoietic recovery after chemotherapy.And we suppose that the down-regulation or absence of Cx43 expression and the miopragia of GJIC probably induce the abnormal hematopoietic regulation of leukemic bone marrow stromal cells.Based on the background above,by constructing the CX43 gene modified BMSC,we try to provide theoretical and experimental evidence for the possible outcome of lentivirus transduced CX43 gene in BMSC increasing information communications between BMSC and BMSC,or BMSC and other cells from the bone marrow.ObjectiveTo establish the culture method for murine bone marrow-derived mesenchymal stem cells?BMSC?in vitro.Lentiviral vectors carrying the connexin43?CX43?gene are constructed and transfect BMSC.The aim to build foundations for investigating the effect of BMSC transfected with pHBLV^TM-CX43-GFP in treatment of graft versus host disease.Methods1.BMSC by whole bone marrow adherence method was adopted,the specific surface markers was defected in flow cytometry,osteogenic,adipogenic induction and endothelial cells differentiated were performed on BMSC.Testing the manifestations of alizarin red staining,oil red staining as well as immunofluorescence and the quality of tube formed after the cells induced.2.Plasmids containing the CX43 gene and lentiviral vectors were digested using Eco RI/XbaI restriction enzymes,and the target gene fragments were cloned into the lentiviral vectors to result in CX43 recombinant lentiviral vectors.CX43 recombinant lentiviral vectors were identified by agarose gel electrophoresis,and sequencing was carried on only for correct vectors after identification.The successfully-constructed CX43 recombinant lentiviral vectors and packaging plasmids were mixed and co-transfected into 293 FT cell for packaging and producing virus.Then the virus was collecting,concentrated and titered in 293 FT cells.Finally,The expression of CX43 gene was assessed by Real Time PCR.Then,used pHBLV^TM-CX43-GFP and pHBLV^TM –GFP to infect the murine BMSC,respectively,and detected the change of Cx43 expression in these BMSC by Immunofluorescence,immunohistochemistry and Western blot methods.Result1.Flow cytometry showed that BMSC were positive for CD73,CD44,CD29,CD90,and negative for CD34,CD105.After osteogenic induction,alizarin red staining showed that alizarin red was positive in cells.lipid droplets of cells was obviously discovered and oil red staining positive after adipogenic induction.The result of BMSC differentiation into endothelial cells showed that CD31 and CD34 expressions increased significantly by immunofluorescence after 21 days of induction.And the cells could form capillaries tube-like structure on Matrigel.2.The results from agarose gel electrophoresis and sequencing proved that CX43 recombinant lentiviral vectors were constructed correctly.Lentiviral concentrated virus suspension titering1*108/mL.The expression of CX43 protein was significantly increasing by Real Time PCR in 293 FT cells.3.The green fluorescence could be observed in BMSC about 24 h after the recombinant lentiviral p HBLVTM-CX43-GFP and pHBLV^TM–GFP were transfected into BMSC.By Immunofluorescence,immunohistochemistry and Western blot methods,the Cx43 expression of BMSC transfected with pHBLV^TM-CX43-GFP was confirmed to be significantly higher than the control groups?P<0.05?.Conclusion1.we have Successfully established a method of murine BMSC isolated culture.2.Succefully constructed the recombinant lentiviral vector pHBLV^TM-CX43-GFP which highly expressed the CX43 gene.The means of Lentivirus transduced connexin43 gene expression were constructed in bone marrow-derived mesenchymal stem cell in vitro.