| Background:Brain lesion results in dysfunction of brain due to the destruct of blood-brain barrier, abnormal metabolism of neural presenter, accumulation of inflammatory factor, and obstruction of microcirculation in brain. The traditional therapy mainly aimed at inhibiting the progression of disease by one or several strategies. Recently, embryological researches indicate the establishment and function remaining of neurocyte clusters depends on the mutual reactions among some particular signals like: growth factors, nutrition factors, and the synaptic connection between neurocytes. Therefore, new researches such as: ameliorating the brain microcirculation, increasing cell protection and enhancing repairing factors, become the therapy focus. And then therapy medical injection, usage of embryonic tissue and gene modified cell transplantation to the brain lesion region have been tried and gained some kind of success. Whereas, the development in this area is very slowbecause of the lack of embryonic tissue and the ethical inhibition. The discover of NSCs (neural stem cells) and their special biocharactor give lights to solution of this problem, which can be used as the source of cell transplant, replace and substitute ruptured cells in lesion region and activate intrinsic NSCs, reestablish neural circuit. They can also be used in transgenetic technique, in which gene segment coding neural nutrition factor is transmitted into NSCs, the NSCs afterward are transplanted to the lesion region as gene therapy carrier and make up the brain dysfunction. The methods above bring forth a bright application foreground for brain lesion therapy. At present, the research in this area is still at the early stage. There are problems need to be considered as:1) The pathophysiological process of neural cell lost, axon demyelination and colloid cicatrice formation.2) The characteristic of NSCs3) The source of NSCs4) The migration and directional differentiation of NSCs5) The gene controlling the differentiation of NSCs, and their modulation and inactivationIn our research, we testing on Sprague-Daeley (SD) rats aimed at discussing the problems above. Methods and ResultsEstablishing an separation in vitro, culture, identification, cryopreservation and thawing NSCs technique, observation the growth, proliferation and differentiation characteristic of neural stem cell: laparotomies were applied to SD pregnant rats, then the cerebral cortex andsubcortex tissue were separated by trypsin and mechanical aspiration. Cells were then suspended and cultured in modified neural stem cell culture medium. The clone cluster of NSCs were harvested and purified by limited dilution to get the daughter cell clone. Immunocytochemistry was used to identify the character and differentiation capacity of NSCs and to observe the activity and biological characteristic of the NSCs after cryopreservation and thawing. It is found that there is NSCs in both the cerebral cortex and subcortex tissue. These NSCs formed neural clone spheres after primary culture and subculture, which have the stem cell characteristic as well as the proliferation capacity. The immunocytochemistric assay show that the cells obtained express positively stem cell marker, nestin. The neural cloning spheres could differentiate into nerve cells, astrocytes and glial cells. For cryopreservation, cells were suspended in N2 complete medium complemented with 20% fetus bovine serum (FBS) and 10% DMSO and kept in -180°C. After thawing, cells were firstly suspended in medium contained 10% FBS for 3hours and then in normal medium. The post-cryopreservation and postthaw cells haven't difference in activity, configuration, differentiation and antigen expression with cells without cryopreservation. The cryopreservation period has no effect on survival ratio to our cells.NSCs were transduced with AdVec-EGFP plasma, an replication deficient adenoviral vectors(AdVec) mediated the enhanced green fluorescent protein (EGFP) vector. And the transduction efficiency and the expression of EGFP have been analyzed. Afterwards, the virus suspension was used to transfect neural cells directly. Fluorescent microscope was used to determinate transfection ratio and EGFP expression. After 6 hourspost-transfection, EGFP start to expressing, and there is a peak at 48 hours post-transfection. EGFP kept expressing till one month later.Survival, migration and differentiation of neural stem cells transfected by EGFP expressing vector after being transplanted to cerebral lesion region: Cerebral lesion model was made in motor area of SD rat. Neural stem cells expressing EGFP were transplanted to cerebral lesion region, then cell survival, migration and differentiation in different time points were observed by confocal microscope. It was detected that the NSCs transplanted survived in lesion region and dispersed outside. The expression ratio of stem cell marker, Nestin, increased after NSCs transplantation.Observation of transplanted NSCs affection to cerebral lesion focus and to apoptosis of neighbor cell: Cerebral lesion model of SD rat in motor area, region frl and fr3, which induced paralysis on rat left crura. Exterior original NSCs were transplanted to lesion area and at different time points. Cell apoptosis were detected by flow cytometry and in situ DNA terminal fragment label (TUNEL). The result indicated transplantation of NSCs shortened the functional loss period and significantly reduced the appearance of apoptosis.The affection to lesion focus and extracellular matrix after NSCs transplantation: Lesion model of SD rat in motor area, region frl and fr3, which induced paralysis on rat left crura. Exterior original NSCs were transplanted to lesion area and at different time points. Laminin expression in this area was determinated by immunohistochemistry, which is propitious to migration of transplanted cells.ConclusionThe NSCs isolated and cultivated in modified medium have proliferating capability and potential differential ability. They express positively nestin show that they are neural stem cells. Cryopreservation and thawing has no affection to the activity and their biological characteristic. The NSCs transfection induced by adenovirus is idea method for transfection, which has high efficiency and facility as well as low cell mortality. Transplanted NSCs can survive and migrate in cerebral lesion area. They change cerebral local microenvironment, inhibit cell apoptosis,increase the level of repairing factors and promote the recovering of function loss. |
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