| In long time, the injury and regeneration of central nervous system is a problem forhuman health, which scientists devote themselves to investigate. From the century of19th to 20th, people found gradually that central and peripheral nerve could regenerate after injury in vertebrate and amphibian animals. But in mammalian animals, they confirmed CNS could not regenerate and peripheral nervous system could regenerate after injury. And this standpoint prevented much from therapy of nervous system disease.Johnasson had proved that there has a lot of neural stem cells in central nervous system .This prove made a new area in neurobiology research. Neural stem cells is a cell that has proliferation and differentiation potential in all it's life, it could make symmetry or asymmetry cleavage. It generate three cells : neurol, astrocyte and oligodendrocyte.The first mechanism of cranilcerebral injury was diffuse cell death, then was thesecond cell damage, It was cell apoptosis, at last astrocyte became cicatricial. At present, we could use few method to therapy central nervous system damage: Use nerve growth factor to accelerate regeneration of damage constitution, accelerate regeneration of axonal, plerosis damage myelin sheath, recover conductance of nervous to reinforcement the compliance of central nervous system. In clinical the main method to therpy brain injury was the application of the rehabilitation training and the use of neurotrophic factor, but the effect was not sure. Because it was very hard to repair damage neuron. The investigation of neural stem cell proved a new method to therpy brain injury.Our investigation made neural stem cells inject to rat's damage brain to observe the recovery of nervous function and the action of rising of neuron quantity after cell transplantation. It proved themry background for using of neural stem cells in clinical.Materials and methodsl.Main reagent: DMEM, DMEM/F12 (1:1), r-h EGF, r-h bFGF, B27, Nestin antibody, et al.2. Primary culture and subculturing of NSCs: the structure of subependymal region and hippocampus are taken from embryo 14 to 16 days of rats, and is digested into simple cell by 0.25% trypsin and 0.02% EDTA (1:1), then is evaluated by Nestin immunocytochemistry.3. Fabrication the model of rats' severe traumatic brain injury : use improved Feeney method, the rat was anesthesiaed, then put on stereotactic apparatus to make rats' severe traumatic brain injury model.4. Remove the neural stem cells: Using stereotactic apparatus inject neural stem cells to rat's brain one day after damage.5. Judgement praxiology after damage: Using Bederson neurologic defect score to detect functional recover of rat.6. Immunohistochemistry staining: Fifteen days after damage,we put all rats to death to make Immunohistochemistry staining.7. Statistics treatment: SPSS11.0 statistical computer programs were used, if P<0.05, it means that difference between two groups is significant.Results1. There is less NSCs in primary culture technology. We observe that many cells stick to the floor of plate at the first day, and a few of them stretch out processus. At the 5th day, the adhesive cells disrupt, and take on death phenomena, however, those non-adhesive cells suspend in culture medium, which become more and more and form some small cell colonies. Those passaged cells suspend in culture medium in regularly spherical shape and have non-prominence in form. At the 7th day, they grow bigger and bigger and become a great clone containing numerous cells, which named neurosphere.Neurosphere is a Nestin positive cells clone.2. The score of rats in experiment bundle was lower than rats in contrast bundle, there had significant difference.3. The number of neuron in experiment bundle was higher than neuron in contrast bundle, there had significant difference.Conclusions1. We have cultured NSCs successfully in primary and passaged culture technique. And we can investigate the proliferation and differentiation of NSCs in future. It provides a stable bases in cell transplantation in neurosurger...
|
PDF Full Text Request