Construction Of Eukaryotic Expression Plasmid PcDNA3-NGF And Its Expression In Rat Bone Marrow Stromal Cells

Objective: By building the bioactive eukaryotic expression plasmidpcDNA3-NGF and transfecting to rat bone marrow stromal stem cells(BMSCs)，the expression of plasmid is observed to provide a theoretical support for furthergene transplant in treatment fracture animal experiments.Methods: Taking about50mg of the brain tissue from rats, and extract total RNAby TRNzol-A+method. Generate NGF full-length cDNA fragments using reversetranscirption PCR (RT-PCR) rfom the total RNA amplification. Identify acquiredcDNA by agarose gel electrophoresis, and connect with pcDNA3to the eukaryoticexpression plasmid pcDNA3-NGF with T4DNA ligase. Inoculating positiveclones e.coli with a recombinant plasmid in AMP LB medium to37°C shakingincubator and culture for24hours to amplify. Extract recombinant plasmid fromamplified bacteira according to extraction kit instructions and conifrm with doubleenzyme digestion of Hindlll and BamHI. Store in-20°C for later use. Recover rat BMSCs, and divide transfected cells into4groups: A with transfected plasmid inliposome2000; B with transfected plasmid in liposomes; C with liposomes withempty plasmid and D with DMEM (Low glucose) containing10%fet al bovineserum as a negative control. Using the liposome Lipofectamine2000transfectionmethod, recombinant plasmid pcDNA3-NGF was transfected into BMSCs.Thencompare the number of cells in each group under the inverted light microscope.And test the expression of NGF in the collected supernatant fluid, using RT-PCRmethod.Results:1. The construction of eukaryotic expression plasmid pcDNA3-NGF: theNGF gene connected with plasmid pcDNA3is identified by double enzymedigestion identification, and confirmed to be of the same sequence with NGF.Atfer being digestion by double enzyme of Hind III and BamHI, the length ofenzyme rfagment rfom recombinant plasmid pcDNA3-NGF is consistent withinserted gene fragment of504bp;2. The recombinant plasmid is transfected byliposome Lipofectamine2000according to its operation manual. And the cellgrowth was observed with inverted optical microscope. Cells displayed differentgrowth patterns: the expeirment group cells presented better growth condition thanthe liposome transfection group and the empty plasmid. A large number of cellswere fragmented in the nucleus, and are highly irregular in morphology withundefined cell membrane while blank control group, had no significant change.3.Atfer recombinant plasmid pcDNA3-NGF transfecting to rat BMSCs72hourslater by liposome Lipofectamine2000, using RT-PCR test, agarose gel electrophoresis shows a positive stripe in the place of about504bp.Conclusions:1. The NGF gene and plasmid pcDNA3can be connected and isconsistent with the NGF gene sequences. It can be used to support a theoretical forNGF gene transfection studies in treatment of fractures.2. Using the liposomeLipofectamine2000transfection method, NGF recombinant plasmid can besuccessfully transfected to marrow stromal stem cells. Rat BMSCs undertransfection with pcDNA3-NGF presented the ability of proliferation,differentiation, and showed NGF expression. These results provided a theory basisfor gene transplant therapy studies in the treatment of fractures.