Gypenosides On The Spinal Cord Derived Neural Stem Cells In Vitro

Spinal cord injury (spinal cord injury, SCI) is a kind of serious neurodegenerative disease, directly or indirectly caused by the injury or by external forces. After SCI the man always have persistent sensory and motor dysfunction, from the epidemiological point of view, it had a high rate of permanent disability. Spinal cord injury in the world has exceeded three million,many countries were making active effort for the treatment of this chronic illness, medical research. Chinese traditional medicine for spinal cord injury awareness of the disease had over two thousand years of history, is rich in diagnosis and treatment techniques.The damaged area of SCI appears loss of neurons, the majority of the formation of hollow (cavity), a demyelinating axons phenomenon. Also the formation of glial scar, and further hampered by patients with spinal cord neural stem cells growth and differentiation of neurons in the recovery of the nervous system, but also adversely affect cell transplantation therapy. SCI can be divided into two parts:First, primary injury is damage to an instant force in the direct damage to the spinal cord. Including compression, tearing, etc.; one secondary injury in spinal cord tissue after SCI is chemia, trigger, such as local inflammation, edema and electrolyte changes, cytotoxic substances (such as free radicals, etc.) caused by ischemia-reperfusion injury accumulation, cell outside the abnormal increase of excitatory amino acid transmitters in the nerve cell cytotoxicity, etc. These changes can lead to injury secondary to expansion of the scope, the more the death of neurons, nerve fiber degeneration and demyelination.Jiaogulan (Gynostemma pentaphyllum (Thunb) Makino.) is a perennial creeping herb, for the first time solved Gynostemma contains dammarane type (dammarane) saponin components. Gypenosides XLIII (30mg·kg-1) could significantly enhance the mice learning and memory access process, so that brain Glu levels were significantly increased, GABA levels were significantly lower, and Glu/GABA learning and memory control system consistent with the theory, the theory suggests that Glu positive effect on learning and memory regulation, GABA negative effect on learning and memory regulation.2ml(1mg/ml) of the Gyp can significantly increase the neural stem cells to differentiate into neurons ratio. When the Gyp concentration was 100μg/ml, its expression by increasing the amount of PCNA can be significantly enhanced in vitro of neural precursor cells (NPCs) proliferation. Gyp and the active ingredient can reduce the volume of cerebral infarction, cerebral ischemia can reduce free radicals and excitatory amino acids, in reducing ischemic brain edema, increased cerebral blood flow, improve the blood-brain barrier permeability, protection brain tissue, inhibition of apoptosis play an important role.This study aimed to establish a mouse spinal cord neurons in vitro model, on the basis of this technology gypenosides (Gyp) on neural stem cell culture in vitro differentiation, transplantation for treatment of the body attempts to spinal cord injury research. The results are as follows:(1) Select the new Kunming mice, from the cervical and lumbosacral spinal cord isolated spinal cord segments, in vitro by mechanical method. Daily use of inverted microscope morphological changes of nerve cells, and counting time, the growth curves drawn on the nerve cell self-renewal and the ability to add value to study. Establish a set of neural stem cells in mouse spinal cord culture system. (2) will be the 3rd generation of spinal cord neural stem cells as a source of follow-up experiments cells, were added in the culture medium with different concentrations of Gyp solution, based 4μg/ml,20μg/ml, 100μg/ml concentration gradient in the experimental group three and do not add Gyp the control group. It was found, and do not add gypenosides (Gyp) compared to medium supplemented with the appropriate amount of Gyp can significantly promote the spinal cord neural stem cells, the formation of colony numbers increased significantly, and the colony became larger, making the spinal cord neural stem cells training easier, on cell proliferation was significantly promoted. And reached a concentration of added 20μg/ml gypenosides Gyp the best when it is medically difficult to solve the spinal nerve cell cultures and experimental basis for reference. (3) detected by immunocytochemistry, neuron-specific enolase (neuron specific enolase, NSE) and glial fibrillary acidic protein (glial fibrillary acidic protein, GFAP) expression, which Gynostemma group and control group of spinal cord culture neural stem cell surface antigen expression of stem cell characteristics were identified. (4) Select Add Gynostemma cultured for 1 day,5 days and 10 days of spinal cord nerve cells, using RT-PCR method of spinal cord neural stem cells in various periods of Nestin and endogenous bHLH transcription factor family gene neruoD/ngn2 and Mash-1 mRNA expression in the training process of the different levels. In neural stem cells and differentiation, Nestin in the differentiation of 5 days after the continuous expression, bHLH gene family, NeuroD, Ngn2, and Mash-1 expression in the more intense the first day, the 5th day and its expression was decreased 10 days trend. This shows that the Gyp vitro differentiation of neural stem cells affect the function, can induce differentiation into neurons, may start the bHLH family of transcription factors, so that neural stem cells differentiate into neurons.This study established for the spinal cord neural stem cells in vitro culture system, and Gynostemma in gypenosides spinal cord neural stem cells of made a more detailed study results show that gypenosides can significantly promote the spinal cord stem cells in vitro proliferation and growth, promote stem cell colony formation, can protect the spinal cord stem cell proliferation and differentiation in vitro, as medicine can not be solved in the spinal cord neural stem cells in vitro to provide technical and laboratory reference. Has some theoretical and practical significance.