| Part One Magnetic Resonance Imaging of Transplantated Neural Stem Cells in Intracerebral Hemorrhage Rat ModelObjective:To observe the growth and differentiation of neural stem cells (NSCs) cultured from rat embryoes in vitro,and to observe their self-renewal and multi-potential differentiation ability,to build experimental intracerebral hemorrhage(ICH) rat models,to observe NSCs migration in ICH rat brain.Methods:NSCs were isolated,cultured and purified from rat embryos hippocampus,and were induced to differentiate with differentiation culture medium.ICH models in rats were made by transplanting autologous artery blood to caudate nucleus.SPIO was transfered to NSCs via lipofectin.NSCs labeled with SPIO were transplanted to lateral ventricle of ICH rats and were observed by MRI.Results:NSCs were isolated,cultured and purified from rat embryos,which could self-renew and differentiate to neuron or astrocyte in vitro.The ICH rat model built by transplanting autologous artery blood was stable.NSCs were labeled successfully by SPIO via lipofectin,and the migration of NSCs labled with SPIO could be tracked by MRI.Conclusions:NSCs can be isolated,cultured and purified from rat embryos. ICH rat model built by transplanting autologous artery blood to caudate nucleus is suitable for ICH study.NSCs labeled with SPIO provide a new method to observe NSCs migration in ICH rats. Part Two Recovery of Neural Function by Transplantation of Neural Stem Cells in Intracerebral Hemorrhage Rat ModelObjective:To evaluate the survival,migration and differentiation of transplanted NSCs surrounding hematoma and the possible recovery function of NSCs,and to investigate the repairing effect of NSCs on damaged neurofunction in intracerebral hemorrhage model rats.Methods:The NSCs were isolated,cultured,and evaluated from hippocampus of day E14 fetal SD rats.Eighteen rats were randomly divided into control group,PBS group and NSC transplantation group.Intracerebral hemorrhage rat models were established via injection of autologous arterial blood in caudate nucleus.Thirty minutes after model establishment,5μL NSC suspension with the concentration of 2×1011 L-1 was transplanted at four points surrounding hematoma site in the NSC transplantation group.Control group rats were performed sham operation.Forelimb placing test and corner turn test were performed in order to evaluating motor function recovery of experimental rats.Results:①Neurofunction scores:There was no significant difference 5 days after model establishment(P＞0.05).However,the scores were significantly improved in the NSC transplantation group 14-28 days after model establishment(P＜0.05).②Immunofluorescent double labeling: Apoptosis cells around hemotoma in the NSCs transplantation group were less than those in the PBS group.BrdU and MAP-2 or GFAP-positive cells were observed in cerebral tissue sections,indicating that NSCs could survive, migrate and differentiate in host brain and differentiate into neurons or astrocytes.Conclusion:NSCs Transplantation contributes to the recovery of neurofunction in cerebral hemorrhage rats through differentiation into neurons or astrocytes. Part Three GFP and bFGF Genes are Transferred to Neural Stem CellsObjective:To establish the method of transfering GFP and bFGF genes to NSCs. To investigate the gene express of bFGF in RNA and protein levels in the gene modified NSCs.Methods:NSCs were isolated,cultured and purified from rat embryos hippocampus.GFP and bFGF genes were transferred to NSCs via lentivirus vector.RtPCR and Westent-Blot methods were used to test transfer quality.Results:GFP and bFGF genes modified NSCs could survive and grow well in NSCs culture medium,bFGF RNA and protein could be identified successfully in bFGF gene modified NSCs.Conclusion:GFP and bFGF genes can be transferred efficiently to NSCs via lentivirus vectors. Part Four Recovery of Neural Function by Transplantation of bFGF Gene Modified Neural Stem Cells in Intracerebral Hemorrhage Rat ModelObjective:To isolate,culture and purify rat adult NSCs,and to test the hypothesis that the administration of NSCs or bFGF gene-modified NSCs to lateral ventricle could diminish rat neurologic deficits caused by intracerebral hemorrhage.Methods:NSCs were isolated,cultured and purified from rat embryos hippocampus.SPIO was transfered to NSCs via lipofectin,and GFP or GFP and bFGF genes were transferred to NSCs via lentivirus vetors.Experimental intracerebral hemorrhage(ICH) rat models were established via transplantation of autologous whole blood.Experimental rats were divided into three groups:group A:PBS transplantation group,group B:GFP gene modified NSCs transplantation group,and group C:GFP and bFGF genes modified NSCs group.The donors were injected into right lateral ventricle of ICH rats. We evaluated motor function recovery of experimental rats by forelimb placing test and corner turn test,difference of bFGF expressing by Western-Blot, NSCs migration and differentiation by MRI and immunobistochemical examination.Results:GFP or GFP and bFGF genes were transferred successfully to NSCs via lentivirus vectors.The GFP and bFGF gene modified NSCs group showed more bFGF expressing,more intrinsic neuron regeneration,and better functional recovery compared with the other two groups.Conclusion:Transplantation of bFGF gene modified NSCs contribute to the recovery of neurological functions in ICH rats. |
