| Objective and background:Neural stem cells (NSCs) have been isolated and cultured from central neural system (CNS) of human and various animals one after another since NSCs were discovered in 1990, A traditional idea was exploded that neuron could not regenerate because the defining characteristics of NSCs have the ability of self-renewal and proliferation. The finding of NSCs has grate value of therapy for nerve injury, nerve degenerative diseases and so on and of deeply researches on growth, development and differentiation of animals. NSCs provide a new therapic method for structural and functional reconstruction of CNS and have extensively applied perspective. Consequently, researches into NSCs have become one of the most hot spots of life science now. At present, isolation, culture in vitro of NSCs has achieved great success, and studies on their bionomics and NSCs transplantation in CNS diseases have gained some gratifying results. But regeneration of CNS is a extremely complicated topic which still includes considerable question studied deeply.Hypoxic-ischemic brain injuries are clinical common diseases and frequently occurring illness, such as cerebral thrombosis, cerebral infarction, and cerebral angio spasm and so on, and result in fearful disability rate and death rate. Furthermore, hypoxic-ischemic brain injuries usually happen to perinatal foetus infant and newborn infants. They cause not only death, but also cerebral palsy, epilepsy, intelligence hypoevolutism or mental retardation and so on. According to statistics, in my country, there are about seven hundred fifty thousand newborn infants happened asphyxia every year, two hundred fifty thousand of which are disability or oligophrenia children. The investigation of recent year indicated that ischemia-reperfusion caused releasing too much excitatory amino acid (EAA) and bringing out excitability toxic effect on neurocyte. The toxic effect of EAA may be produced in the following two channels: the characteristic of the first channel is the inflowing of Na+, and the overexcitation of AMPA-R and KA-R mediate the neurocyte acute osmosis swelling; The characteristic of the second channel is the inflowing of Ca2+, and the overexcitation of NMDA mediate the neurocyte tardive osmosis swelling; The overloading of Ca2+ may be the common patho-mechanism of the neurocyte acute and tardive osmosis swelling which induced by overexcitation of EAA receptor, it may result in some sequelae such as neurologic impairment, intelligence falling behind and posture abnormity.Therefore, the aim of this research is observing the effect of gastrodin in more EAA after brain ischemia, and studying its influence in the differentiating of NSCs; Detecting whether the gastrodin can induce the NSCs differentiating toward neuron and improve intelligence falling behind or limbs activity disorder which resulted from NSCs differentiating into more glial cells after brain suffering from hypoxic-ischemic. This is significant for inducing the NSCs differentiating toward neurons and has grate value of therapy for ischemic cerebrovascular disease, nerve degenerative diseases, epilepsy, nerve injury, and so on.Materials and Methodology:The experimental animal is the newborn Wistar rat in 24 hour, collected the n ewborn rats' brain tissue and cultured it in vitro, and did the single-cell cloning test. Treating the NSCs with KA and gastrodin in differentiating, the concentration of KA in different group are 100μmol/l,500μmol/l,1mmol/l,2mmol/l,5mmol/l; and the concentration of gastrodin is 0.5mg/l. Detect the percentage changing after given KA and gastrodin by immumofluorescence and immunohistochemistry method; stained with AO/EB mixed fluorescent dye to estimate the apoptosis of NSCs after given KA; Determine the differentiating velocity changing of NSCs after given KA by MTT assay.Results1. The NSCs can differentiate into neurons and glial cells, the percentage of neurons is 23.07%±1.51 %, and the percentage of glial cells is 76.93 %±1.51 %.2. After treated by KA, the percentage of neurons is 13.99%±2.07%, and the percentage of glial cells is 86.01 %±2.07%. The cell percentage difference among the different concentration KA group has no statistic significance (F=1.43,p>0.05) ; The cell percentage difference between the normal group and KA group have notable statistic significance (F=368.21,p<0.001). KA can induce the NSCs differentiating toward glial cells efficiently.3. After treated by KA and gastrodin, the percentage of neurons is 17.16%±2.14%, and the percentage of glial cells is 82.84%±2.14%, compared with the cell percentage of KA group, there is notable statistic significance(F=15.6, p<0.01). Gastrodin can diminish the excitatory influence of the kanic acid in some degree, and induce the neural stem cells differentiating toward neurons.4. MTT assay found: after added the KA and gastrodin, some NSCs died soon, but the survivor differentiated quicklier than before.Conclusions1. The cells we cultured in this experiment can differentiate toward neurons, oligodendrocyte and horizontal cells, so they are NSCs.2. KA can induce the NSCs differentiating toward glial cells efficiently.3. Gastrodin can diminish the excitatory influence of the kanic acid in some degree, and induce the neural stem cells differentiating toward neurons. |
