Transfection Of Human-βNGF Plasmid Vectors Into BMSCs Of GFP Transgenic Mouse

Objective:To study the biological features of the mouse bone marrow stromal stem cells (BMSCs) transfected by human-β-nerve growth factor (human-β-NGF), to provide experimental support for the next step of the animal experiment and the clinical treatment of presbycusis. Methods:Take Positive clones of Escherichia coli with recombinant plasmid inoculate to37℃LB medium. Shaking culture for the night at37℃for24hours and amplificating bacteria. According to specification of the plasmid kit extract recombinant plasmid. The recombinant plasmid was analyzed and identified by the Hindlll and Xhol restriction endonuclease after amplified, Preserved at-20℃refrigerator. To revive the mouse bone marrow stromal stem cells by identifying the surface CD antigens with floe cytometry. Then put the BMSCs of GFP transgenic mouse into four groups:A is the group of recombinant plasmid encapsulated in liposomes; B is the liposome transfection group; C is the group of liposome parcel empty plasmid transfection; D is negative control with10%fetal bovine serum of DMEM/F12nutrient solution. The NGF plasmid vectors were transferred into the cultured BMSCs by Lipofectamine TM2000.Compared the number of cells between each group and observe the outgrowth of processes in the inverted optical microscope, and collected supernatant liquid of each group cells. The expression of human-β NGF was detected with ELISA. Results:The length of the endonuclease digestion which the recombinant plasmid of pcDNA3-P-NGF was about725bp after double digestion of restriction enzyme HindⅢ and XhoⅠ. It was consistent with the fragment length of the insert gene; The plasmid vectors were transferred by Lipofectamine TM2000, compared the number of cells between each group and observe the outgrowth of processes in the inverted optical microscope:The test group growth was good; The group of the liposome transfection and liposome parcel empty plasmid transfection cells growth were poor, and a large number of cells in cell membrane was not apparent, nuclear fragmentation, cell morphology chaos; The blank control group was not obvious change. Collected supernatant liquid of each group cells after48hours, the expression of human-β-NGF was detected with ELISA:group A was174.91±4.71pg/ml; group B was5.94±0.91pg/ml; group C was5.71±0.50pg/ml; group D was5.79±0.80pg/ml. The NGF protein expression quantity of Recombinant plasmid group obvious higher than any other groups. Conclusion:The NGF plasmid vectors were successfully transferred into BMSCs of GFP transgenic mouse cells. The proliferation and differentiation of BMSCs of GFP transgenic mouse after transfecton with human-βNGF were unalter, keeping the ability to produce NGF. In addition, the expression NGF can effectively protect the BMSCs which were injured in transfection process. The success of these studies can provide support for the next experiment of cochlear implant in mices.