The Detection Of Protein Secretion And Osteogenic Activity Of Adipose Tissue Derived Stromal Cells Transfected By VEGF Gene

ObjectiveThis study is to isolate adipose tissue-derived stromal cells from human adipose tissue, investigate the biological characteristics, and contruct recombinant plasmids of pSELECT-GFPzeo-VEGF connected by human vascular endothelial growth factor that was extracted from human vascular tissue and bicistronic expression vector with green fluorescent protein. The purpose aimed to combine the different generations of ADSCs and pSELECT-GFPzeo-VEGF by gene transfection and idenfy the expression of VEGF mRNA, the protein level variation of VEGF and the osteogenic activity of the transfected ADSCs.MethodsThe ADSCs were isolated from human adipose tissue digested by collagenase. Then the cells were cultured and passaged in the nutritive medium. The surface phenotype of the cultured ADSCs were identified by flow cytometry. The biological appearance and characteristics of the ADSCs were investigated and the cell growing line was draw. The oil red "O" staining was carried out after adipogenic induction, von kossa and ALP staining after osteogenic induction and toluidine blue staining after chondrogenic induction in condition medium in order to identify the success of multi-indirectional differentiation. Firstly, the total RNA of the human vessel tissue were extracted by Trizol reagent and composed to cDNA by reverse transcription. The gene orders of VEGF mature peptide were amplificated from cDNA by nested PCR. The acquired objective genes were combined with the linear vector of pGEM-Easy. After identified and sequenced, the recombinat plasmid vectors were recovered which were used to transform the competence Escherichia coli DH5α. Secondly, after digested by zymase, the recombinant cloning vector pTA2-VEGF and green bicistronic expression vector with green fluorescent protein were connected to the recombinant plasmids of pSELECT-GFP zeo-VEGF. After identified and sequenced one more time, the pSELECT-GFP zeo-VEGF were recovered in a great quantity. Finally, the2nd,3rd,4th and5th generations of ADSCs were transfected respectively by the recombinant plasmids of pSELECT-GFP zeo-VEGF after mediated by liposome. After transfection, the immunofluoresecnce identify were perfomed and the expression volumn level of VEGF mRNA were detected by RT-PCR. The supernatant liquid of the ADSCs groups transfected by pSELECT-GFP zeo-VEGF and transfected by empty vectors was collected in6h、12h、48h and72h respectively after transfection to detect the secretory volume of VEGF by ELISA which was analyzed by statistics. In addition, the ADSCs of2nd generation transfected by pSELECT-GFP zeo-VEGF, transfected by empty vectors and the untransfected ADSCs were cultured respectively in the osteogenic condition to detect the secretory volumn level of ALP, osteocalcin and laminin in the supernatant liquid.ResultsThe surface phenotype of the cultured cells, which were isolated by collagenase digestion and density gradient centrifμgation and differential adherent culture method, is positive. Therefor, the cells were identified to be the ADSCs with characteristics of stem cells and have multi-indirectional differentiation to be adipose cells, osteoblasts and chondrocytes by staining after cultured in different condition medium. Recombinant plasmids, pSELECT-GFPzeo-VEGF, can be successfully constructed by VEGF gene combined with bicistronic expression vector carrying green fluorescent protein. The objective gene of VEGF mediated by lipidosome can be transfected to the ADSCs in vitro successfully and identified under fluorescent microscope. The expression level of VEGF mRNA in the supernatant liquid increased significantly in the ADSCs group transfected by pSELECT-GFP zeo-VEGF, but unsignificantly in the group transfected by blank vectors. The difference that analyzed by statistics is significant (P<0.05). After ADSCs were cultured in osteogenic condintion medium, the secretory volume of ALP, osteocalcin and laminin in the ADSCs group transfected by pSELECT-GFP zeo-VEGF was much higher than the group transfected by blank vectors and the untransfected ADSCs group. The difference that analyzed by statistics is significant (P<0.05).ConclusionThe ADSCs with the capability of directional differentiation can be acquired from the adipose tissue by using collagenase digestion combined with density gradient centrifugation and differential adherent culture method. Gene segments with human VEGF can transfect into human ADSCs successfully with liposome mediated. The transfected ADSCs have the capability to express VEGF mRNA with biological activity continously in vitro and secrete VEGF protein. ALP, osteocalcin and laminin can be secreted abundantly from the transfected ADSCs which were cultured in the osteogenic condition medium. The angiogenic therapy was combined with stromal cells transplanted efficiently in the experiment. The ADSCs transfected by pSELECT-GFP zeo-VEGF have facilitated osteogenesis, vascularization and adhering the scaffolds to modify and construct tissue engineering bone and cartilage. The mothed provided a certain experimental foundation for the treatment of ununion, delayed union and bone defect in the clinic.