Effects Of Biological Function After NGFβ/HIF-1α Gene Transfection On SD Rat Bone Marrow Mesenchymal Stem Cells And Safety Evaluation

ObjectiveThis experiment aimed at transfecting the gene of NGFβ/HIF-1α on SD ratbone marrow mesenchymal stem cells.Study the effects of biological functionafter NGF β/HIF-1α gene transfection on SD rat bone marrow mesenchymalstem cells and safety evaluation.To lay the foundation for the application oftissue engineering method for the treatment of spinal cord injury.MethodsFirst of all, recovery the frozen BMSCs cells of SD rats, the adherentcultured BMSCs cells of SD rats, cell fusion rate of more than85%BMSCs cellsfor experimental study. Then, lentivirus expression vector carrying the geneencoding NGF β/HIF-1α were constructed, setting-out the MOI, using lentiviralvector carrying the target gene that with MOI to transfect BMSCs cells, Westernblot testing the expression level of NGF β and HIF-1α. The subjects weredivided into five groups: group A, control group; group B, no-load lentivirusgroup; group C, NGF β gene transfection group; group D, HIF-1α genetransfection group; group E, NGF β/HIF-1α gene transfected group. Finally,theultrastructure was observed by transmission electron microscope, the cellsvalue-added was detected by MTT, Cell cycle was analyzed by flow cytometry,In vitro colony formation in soft agar assaied cell tumorigenicity. Data usingSPSS19.0statistical software, the differences between two groups were analysis of variance.Results1. Observation under inverted microscope, BMSCs adherent growth,cultured3-5days, BMSCs proliferated rapidly, the cell morphology of fibroblastlike into fusiform, part of a broad flat polygon, whirlpool, cells fusion ratereached80%-90%.2. After transfection of lentivirus, no fluorescence expression in group A,group B showed green fluorescence, group C showed red fluorescence, groupD showed green fluorescence, group E showed yellow fluorescence. After72h,MOI=100, fluorescence was distributed in whole cell, transfection rate mayreach above85%.3. Western blot detected in A group and B group had no obvious NGF β/HIF-1α expression, only expression NGF β protein in the group C, onlyexpression HIF-1protein in group D, NGF β/HIF-1α in group E, two proteinswere expressed.4. Cellular ultrastructure were observed under transmission electronmicroscope, in group B and group D, abundant organelles, normal morphology,no significant difference compared with in group A; in group C and group E,abundant organelles, normal morphology, no significant difference comparedwith in group A,but in the cytoplasm of some cells with nerve filament formationcan be seen.5. Cells value-added were detected by MTT results show that in fifth daysthe proliferative capacity of BMSC tends to peak, the experimental group,negative control group, positive control group had no significant difference.6. Flow cytometry cell cycle results show that the experimental group,negative control group, positive control group had no significant difference.7. In soft agar colony forming experiment results show no cell colonyformation. Conclusions1. NGF β/HIF-1α gene transfected BMSCs sustainable and efficientexpression HIF-1α and NGF β.2. NGF β/HIF-1α gene transfected BMSCs with biological functions ofgood and safe.