| It had long been thought that there were no neural stem cells (NSCs) in the central nervous system of adult human and mammal just a decade before. Once the neurons in adult human brain and spinal cord were injured and died, they could not be regenerated. This dogma was surmounted until 1992, when Reynolds firstly reported that multi-potency neural stem cells could be separated and cultured from adult mammal striatum. Recently, embryonic stem cells (ES) and NSCs were utilized successfully in the treatment of many degenerative diseases of central nervous system. NSCs have been widely studied for neural regeneration, restoration, and neural function rebuilding. But for ethical and logistical problems, or source reason, NSCs can't be acquired easily. Moreover, most NSCs lineages built at present came from murine and have significant difference with that from mankind. Therefore, many researchers focus on searching for an ideal kind of stem cells which can replace NSCs now. Bone marrow mesenchymal stem cells(BMSCs)have been proved to have multiple differentiation potentials and can differentiate into osteoblasts, chondrocytes, myocyte, adipocytes, cardiac myocyte, vessel endothelial cells, hepatocyte, ligament, corium even neural cells in appropriate conditions. Recently, researchers found brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) could induce BMSCs differentiate into neurons and neuroglial cells, but the differentiation ratio of neuron was rather low. So how to raise the ratio of differentiation into neurons become the focal theme in neuroscience field. Receptor activator of NF-Kappa B ligand (RANKL) is a new found cytokine, and can up-regulate two receptors for biogenic monoamines which canmodulate neurite growth: the receptors for histamine (H2R) and the α1-adrenergic catecholamine receptor (ADRA1A). RANKL may play an important role in the regulation of cell growth through activating NF-κB. However, it has never been reported whether RANKL play a role in the neural differentiation of BMSCs. Here we investigate the effects of RANKL on the differentiation of adult human bone marrow mesenchymal stem cells (hBMSCs) into neural cells. Our experiment includes four aspects: (1) RANKL induces hBMSCs differentiate into neural cells in vitro; (2) the influence of hBMSCs intraspinal cord transplantation on the neurological functional outcome after spinal cord injury in rats; (3) the effect of RANKL on neural differentiation and neurological functional outcome following hBMSCs transplantation into injured spinal cord in adult rats; (4) the role of RANKL in hBMSCs intracranial transplantation following focal cerebral ischemia in adult rats. In this study, we object to explore if RANKL play an important role in orientating differentiation into neuron of hBMSCs, and provide a theoretical foundation for the use of RANKL and hBMSCs in the neuroscience field.1．RANKL induces hBMSCs differentiate into neural cells in vitro The purpose of this part is to identify the feasibility of RANKL inducing hBMSCs differentiate into neual cells in vitro and the effect of RANKL on the differentiation. Human bone marrow was obtained from healthy adult human ribs resected for chest surgery approach using aseptic technique. The mononuclear cells were separated and plated into plastic culture flask containing DMEM, cultured in a CO2 incubator at 37℃. When the adherent cells paved the flask fully, they were digested and passaged, regarded as the first passage of hBMSCs. Cells harvested from second passage were resuspended into culture dishes at a concentration of 1×104/ml. The medium of control group was replaced by DF12 medium containing insuline,transferrin, progesterone and all-trans-retinoic acid. The medium of RANKL, BDNF and RANKL+BDNF group was added sRANKL 20ng/ml, BDNF 100ng/ml and sRANKL 20ng/ml + BDNF 100ng/ml respectively. The cells growth was observed successively using an invert microscope. Immunocytochemical analysis for the expression of neuron marker neuron-sp...
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