Research Of The Differentiation Of Rat Bone Marrow-derived Mesenchymal Stem Cells Into Neuron-like Cells Induced By Xinmailong Injection

ObjectiveIn recent years, isolation and culturing of desired target cells by cell engineering techniques, has become a research hotspot of neurodegenerative diseases’cell replacement therapy. Bone marrow-derived mesenchymal stem cells with abundant source materials have advantages of easy separation, purification and culturing in vitro, they also have multilineage differentiation potential advantages. So bone marrow-derived mesenchymal stem cells are good target cells for alternative treatment and are widely used in the field of neural regeneration. Xinmailong injection is made of dried Periplaneta americana after impregnation, decompression concentration, separation and dissolution. This study used rat bone marrow-derived mesenchymal stem cells as the object, Xinmailong injection as the inducing agent, to explore the feasibility of Xinmailong injection inducing rat bone marrow-derived mesenchymal stem cells (BMSCs) to differentiate into neuron-like cells.Methods1.The bone marrow-derived mesenchymal stem cells of rat were separated and purified by passage culture. After successive isolation, purification, subculture and proliferation, the morphology was observed with phase contrast microscope.2.Following passage, cell surface markers CD29, CD90, CD34and CD45were measured using flow cytometry. 3.Xinmailong injection was used to induce the third generation BMSCs into neuron-like cells. The morphologic changes at different induction time points were observed.4.At4h,12h,36h after induction, the expressions of Nestin, Neuron specific enolase(NSE), Microtubule associated protein-2(MAP2), Glial fibrillary acidic protein(GFAP) were detected by immunofluorescence assay.5.At36h after induction, NSE, GFAP were determined by RT-PCR.Results1.The primary culture cells of BMSCs were scattered with other cells, had strong refractivity. at1d, some of the mononuclear round cells adhered to the wall, and at3d, most of the the mononuclear round cells adhered to the wall, and congregated together. At8-10d, the cells showed clusters growth, each set of clusters was radially or spiral, cells covered the dish bottom and began to fusion. After subculturing, cell proliferation speeded, and the third to five generations had been purified from the spindle cells, a fish like arrangement, after six generations, cell growth slowed down, became broad and flat.2.Flow cytometry showed great expression of CD29(99.06%) and CD90(99.61%) and low expression of CD34(2.03%) and CD45(2.00%).3.2h after induction, the cells’morphology was beginning to change, part of the long fusiform cells became small, into the irregular shape or circular shape.36h after induction, most of the rat BMSCs incubated with Xinmailong injection differentiated into neuron-like cells, revealing cytodendrite, showing typical nerve cells’forms with two polar, multipolar and taper.4. At4h,12h after induction, the positive rates of Nestin were (81.0±1.6)%and (22.5±1.9)%, and at36h after induction, Nestin was negative. At4h after induction, NSE and MAP2were negative. At12h,36h after induction, the positive rates of NSE were (73.5±2.2)%and (94.3±1.8)%and those of MAP2were (80.0±2.2)%and (96.4±2.8)%. MAP2was negative all the time.5.By using RT-PCR,36h after induction, the expression of NSE was positive and GFAP was negative in the experimental group. Conclusions1.Total marrow culture was a simple new method which was used for the isolation, purification and cultivation in vitro of bone marrow-derived mesenchymal stem cells.2.Xinmailong injection could rapidly and efficiently induce rat BMSCs to differentiate into neuron-like cells.3.Before rat BMSCs differentiated into neuron-like cells induced by Xinmailong injection, they passed through a neural precursor cell stage.