Lentiviral Vector Infection On Induction And Differentiation Of Mesenchymal Stem Cells(rMSCs)

Part â…  isolation culture and identification of rat Mesenchymal stem cells(rMSCs)Experiment 1 isolation and culture of rat Mesenchymal stem cells(rMSCs)Objective: to isolate and culture rat Mesenchymal stem cells (rMSCs). To grasp the techniques of rMSCs culture and investigate the biological characters of rMSCs. Materials and Methods: 4- month-old SD rats were used, follow the methods of Friedenstein, because of the characters of rMSCs adhereing to tissue culture flask, (as rMSCs is adherent to tissue petri dish,) monocytes were isolated by gradient centrifugation first; then pure adherent rMSCs were obtained by the property that rMSCs is adherent to plastic petri dish. Results: rMSCs are rapidly adherent, grow in clone, the morphology of cell are homogeneous fusiform, within 24hs fibrous like cells were seen sporadic adherent, some cell clony were seen in 2-3 days, and they increased rapidly in 7～10 days , gradually grow into mixture. As the cell clony grown, they became monolayer at last, after passage, rMSCs no longer growed in clony but in homogeneous distribution. rMSCs number of primary culture is about 10⁵, and passage 10 is about 10¹².Conclusions: isolation and culture of rMSCs are feasible, and have powerful ability of cell proliferation .Experiment 2 identification of rat bone marrow Mesenchymal stem cells (rMSCs)Objective: surface antigen was used to identify the property of cultured cells, to prepare the seed cells of next step, methods and materials: to detect the surface antigen of rat bone marrow MSCs with flow cytometry,include CD29, CD44, CD90, CD105, CD34, CD45, CD11b. results: in contrast to the control group, the surface antigen CD29, CD44, CD90, CD105 of rat bone marrow rMSCs were positive; surface antigen CD34, CD45, CD11b were negative, and major MHO â… ,â…¡(major histocompatibility complex-â… ,â…¡) were negative too.Conclusions: rat bone marrow rMSCs surface markers are multiplicity, which express the surface markers of interstitial cells, endotheliocytes and epidermic cells, generally CD29(the member of integrin family), adhesion molecule CD44, CD105 and CD166, surface marker CD90 of T lymphocytes in thoracic gland and periphery circulation and so on were important markers of bone marrow rMSCs.Part â…¡ The comparison between adenoviral and lentiviral vector infection on bone marrow rMSCsObjective: lentiviral vector system with green fluorescent protein (GFP) gene and adenovirus vector system infect bone marrow rMSCs, to observe expression and persistence time after infection and to evaluate two vector system. Materials and methods: recombinant plasmid pHIV-CS-CDF-CG-PRE (including GFP gene) and packaging plasmid pRSV-Rev, pMDLg/pPRE, pMD.G infects 293 T cells and collect virosome; adenoviral vector(including GFP gene) were bought, to infect rat rMSCs respectively, to observe the expression of GFP; to detect fluorescent protein expression with flow cytometry. Results: A week after infection, both infection efficiency of lentiviral and adenoviral vector system on rMSCs are high, fluorescent protein express well, GFP expression of lentiviral system is 90%, adenoviral system 71%; GFP expression of rMSCs infected by lentiviral system is good, about 83.2%; however expression of adenoviral system infected decreased obviously 3 weeks later. after 5 weeks, expression of lentiviral system still remain good , about 79% , in contrastadenovirus system only remain 0.3%.Conclusion: the infection efficiency and persistence time of lentiviral vector system infection to rat RMSCs are significant superior than adenovirus vector system.part â…¢ Lentivirus vector construction of human bone morphogenetic protein 2 geneObjective: Human bone morphogenetic protein 2 was cloned to lentiviral vector, to prepare for the next study of gene therapy. Materials and methods: total RNA are isolated from liver; to design primer of two enzyme digestion site of Nhe â… ,Age â… , to amplify the total sequence of BMP-2 gene, cloned to lentiviral recombinant plasmid pHIV-CS-CDF-CG-PRE, polymerase chain reaction(PCR) amplification, enzyme digestion, sequencing etc were used to screen and identify recon. Results: successfully amplified total sequence of BMP-2 gene about 1.2kb, sequence match analysis with human BMP-2 in genebank, open-reading frame is 100% coincident.part â…£ package and expression in vitro of lentiviral vector with BMP-2 geneObjectives: packaging lentiviral vector, to determine the virus titer and detect the protein expression. Materials and methods: recombinant plasmidpHIV-CS-CDF-CG-PRE(including GFP gene) and packaging plasmid pRSV-Rev, pMDLg/pPRE, pMD. G infects 293 cells and collect virosome; using ELISA to detect virus titer of lentivirus which were recombined with hBMP-2 and packaged in 293 cells. hBMP-2 gene expression were detected by indirect immunofluorescence analysis and western blotting detection in vitro. Results: lentiviral vector system package successfully, the virus titer is 2.92 × 10⁹, indirect immunofluorescence and Western Blotting reveal that expression of hBMP-2 gene is good invitro.part â…¤ directional differentiation of rMSCs infected by lentiviral vector with hBMP-2Objectives: to observe the change of rMSCs induced by lentivral vector, to detect if rMSCs transform to osteoblast as expectation. Materials and methods: after lentiviral vector induced rMSCs, in the 0,3,6,9,12,15,18 and 21 day, alkaline phosphatase (ALP)activity is evaluated; to determin calcium ion concentration with quantitative assay; immunohistochemistry is used to detect the expression of type I collagen. Results: as the induced time prolonging, in rMSCs after induction, alkaline phosphatase (ALP)activity is gradually increasing, but ALP activity of rMSCs that were not induced had no obvious change after 21 days culture. After induced, Calcium ion concentration is continuous increasing, reached climax after 15 days, then get in stationary phase. Calcium deposition had no marked change in the group that were not induced; type I collagen expression increased obviously in induced rMSCs and staining is positive, in contrast, which had no change in the control. Conclusions: with immunohistochemistry and histochemistry methods we revealed that inducedrMSCs can produce type I collagen, ALP and had the ability of mineralization, which confirmed rMSCs can be induced to differentiate into osteoblast by lentivral vector.