The Transfection With GFP Gene And The Differentiation Of Bone Marrow Stroma Cells Into Neuron-like In Vitro

Objective To study the isolation, purification, amplification and biological character of mesenchymal stem cells ( BMSC) form rat bone marrow. To study the possibility that transforming growth factor - β, nerve growth factor and the traumatized spinal cord extracts (SCE) induces adult rat bone marrow stromal cells ( BMSC) into neurons for further investigation of an alternative for neuron regeneration and the treatment of spine cord injury. To explore the action of GFP labeled bone marrow stromal cells differentiate into neuron - like cells, which induced by transforming growth factor - βand nerve growth factor.Method(1) BMSC were isolated from the thighbone, shankbone of SD rats of 6 - 8 weeks, and were cultured in IMDM containing 100ml/L fetal bovine serum. They were purified by passage control and adhering to the culture plastic. We observed the growth status of BMSC, drew the growth curve, and analyzed the phenotype by immunocytochemistry. The bone marrow stromal cells from adult rats were isolated and cultured for 5 passages. The BMSC were incubated with IMDM containing 10% FRS and TGF - β( 1μg/L ) for 2 hours. Then the media were replaced by IMDM media consisting of NGF(50ng/mL). The morphological changes of the cells were observed under phase contrast microscope, the cells were stained immunocytochemically with NF,NSE and GFAP antibodies respectively.(2)The bone marrow stromal cells from adult rats were isolated and cul-tured for 5 passages. The extracts of normal spinal cord and injuried spinal cords were added into the cultures. The morphological changes of the cells were observed under phase contrast microscope, the cells were stained immunocyto chemically with NSE antibodies respectively.(3) After BMSC were transfected with recombinant adenovirus carrying GFP gene, they were induced by transforming growth factor - βand nerve growth factor. Then their distributions and differentiation were observed by microscopy and immunocytochemistry.Result(1) BMSC of passage 1 were oval, short spindle - shaped, long spindle -shaped or polygonal. After purification and amplification, they were uniform long spindle - shaped morphology. The living behavior of BMSC from passage 2 to passage 6 was quite stable, exhibiting a large expansive potential. BMSCs were passaged every seven days, and adhered to culture plastic within 24 hours. BMSC of passage 3 uniformly expressed CD54 (ICAM - 1) and FN ( Fibronec-tin) by immunocytochemistry. After 2 hour of induction with neurotrophic factor, some cells showed morphological changes, 12 hours later,many BMSC became neuron like cells and were stained positively with NF, NSE antibody, but negatively with GFAP.(2) After 24 hour of induction with neurotrophic factor, some cells showed morphological changes,48 hours later,many BMSC became neuron like cells and were stained positively with NSE antibody by the induction of traumatized spinal cord extracts ( SCE).(3) After 8 day of . transfection with GFP gene, BMSC were demonstrated positive CD54XFN expression and negative CD34. The GFP labeled bone marrow stromal cells expressed neuron - specific marker, such as neuron - specific enolase,but did not express glia fiber acid protein.