| PART1The differentiation and proliferation of rhesus monkey embryonic stem cells and allotransplantationObjective: To investigate the characteristics of proliferation and differentiation of the neural stem cells from rhesus monkey embryonic stem cells through establishing a stable and high-efficient neural differentiation system in vitro and observe the impact of nervous system function and apoptosis after neural stem cells (NSCs) transplantation in rats brain of cerebral contusion and explore the molecular mechanisms, promote the clinical application of neural stem cells.Methods:The differentiation and proliferation of the GFP marked rhesus monkey embryonic stem cells with bFGF/EGF into rosettes neural stem cells, were studied in the present project; through the radiostereostatics,transplanted5ul (1X105/ul) neural stem cell to the hippocampus of rhesus monkey, made brain tissue frozen section after two months, identified the survival and differentiation of neural stem cells transplanted in the body with immunohistochemical and confocal laser.Results:1) A stable and high-efficient neural differentiation system was established.2) The rosettes neural stem cells differentiated from the rhesus monkey embryonic stem cells could maintain the rosettes-shape (The true sense almighty neural progenitor cell) after proliferation with bFGF/EGF.3) After neural stem cell transplanted into two rhesus monkey brain two months, found that cell can survive, and differentiated into neurons and glial cells. Conclusion:The rosettes neural stem cells differentiated from rhesus monkey embryonic stem cells could maintain its characteristics after proliferation with bFGF/EGF and it could survive and differentiate into neurons after transplanted into the rhesus monkey brain, which will be giving solid support to the clinical application of neural stem cells in the future. PART2The influence of neurological function, apoptosis and gene expression after neural stem cells transplanted in rats with brain injuryObjective: To observe the impact of nervous system function and apoptosis after neural stem cells (NSCs) transplantation in rats brain of cerebral contusion and explore the molecular mechanisms.Methods:24SD rats were randomly divided into three groups:sham group, the surgical group and NSCs transplantation group. Rats brain injury model of cortical motor area induced by freefalling, NSCs transplanted into in four points of rats injury brain, and observed recovery of neurological function and survival of transplanted NSCs in the brain tissue, migration and apoptosis, apoptotic gene expression.Results:NSS score of the NSCs transplantation group significantly declined after7days; NSCs can survive and migrate in the host brain tissue; the number of apoptotic cells significantly reduced;BCL-XL expression was significantly increased, compared with the simply surgery group (not transplanted NSCs),the differences were statistically significant.Conclusion:NSCs can improve the nerve function of brain contusion rats; NSCs can survive in vivo, reduced apoptosis and increased apoptotic gene BCL-XL expression. PART3Glaucoma optic nerve injury model applied neural stem cell transplantation researchObjective: Investigate an appropriate method of modeling glaucoma for stem cells transplantation study and promote the clinical application of neural stem cells.Methods:Dexamethasone was injected unilaterally into the conjunctiva of New Zealand rabbit at the dose of2.5mg (5mg/ml),3times a week (every other day in work days), had last for8weeks. Then neural stem cells were injected into the vitreous body of the model eye4mm behind the cornea rim, after5months, gathered the eyes, made pathological, and observed neural stem cell growth survival through the confocal microscopy.Results:Eye ground photography showed that compared to the control eyes, the optic nerve head was expanded, the blood vessel was geniculate, while the ocular media kept transparent; hematoxylin-eosin (HE) stain of RNFL sections showed the optic neuron death of the model eyes. Heidelberg Retina Tomography (HRT) results showed consistent changes of the model eyes with pathophysiology of Glaucoma including, decrease1.10±0.88mm2in Rim area, increase0.17±0.13in Cup/disk ratio and decrease0.44±0.31mm in Mean retinal nerve fiber layer (RNFL) thickness, was different (p<0.001); And observed neural stem cells were survived in vitreous.Conclusion:This chronic glaucoma mode made by this experiment is a simple and reliable, repeatability strong, and applies neural stem cell transplantation research; the intraocular pressure fluctuations with glaucoma optic nerve injury was minor and will not affect the growth of cells transplanted, this experiment keep eyes clear refractive, suitable for observation neural stem cells after transplantation PART4The research of neural stem cells transplants in rabbit eyes with glaucoma optic nerve injuryObjective: To explore the specific feasibility about transplantation into eyes with glaucoma optic nerve injury.Methods: After Animal models of glaucoma optic nerve injury were established, with different methods transplantating rhesus monkey neural stem cells into eyes (model eye and control eyes), after five months, and gathered the eye balls, using different methods of specimen, fixed, slice, through the confocal microscopy, observed growth, survival, the migration, integration and response of neural stem cell in the eye.Results:The live neural stem cells were visible in the vitreous body of rabbit eyes (model eyes)with glaucoma optic nerve injury, did not observed the migration and integration of the neural stem cells, there was not the neural stem cells in control eyes; The results were related to transplantation methods, specimen production methods and the immune response.Conclusion:The rhesus monkey neural stem cells can better survived in the vitreous body of rabbit eyes with glaucoma optic nerve injury, were the foundation for the follow-up studies, further proof that the third part of the experiment was suitable for neural stem cell transplantation research; the rhesus monkey neural stem cells transplanted in vitreous did not migrate and integrate into the retina, transplantation method should be further improved; the live growth of neural stem cells were only observed in the model eyes (injection dexamethasone), did not observe in control eyes, and found response phenomenon in eyes, this give us new think about how to control the immune rejection response for the next step study. PART5Pathological changes of extraocular muscule and proprioceptors in extraocular muscles of the patients with monocular glaucoma damageObjective: To observe the ultrastructure of extraocular muscle (EOM) and proprioceptors in extraocular muscles (EMP) in intermittent exotropia (sensory strabismus) with monocular glaucoma damage and discuss it’s etiology and pathothogenesis. Promote the further understanding of glaucoma nerve disease, and provides tissue morphological basis for follow-up study of neural stem cell transplantation.Methods: The samples were obstained from medial rectus of10patients who were suffering from intermittent exotropia with monocular glaucoma damage, were about4-5mm, width of10mm of eye muscles as the experimental group (10cases, light microscopy, transmission electron microscopy,10cases),10corneal graft donors were as the normal control group (5cases by light microscopy,5cases by transmission electron microscopy), after being stained pathological specimens,the samples were examined under light microscopy(LM) and transmission electron microscopy(TEM)to observe the structure of extraocular muscle and EMP of two groups.Results:Compared with normal control group, the muscle fibers of medial rectus of the patients with intermittent exotropia and monocular glaucoma damage arranged disorderly, showed atrophied and edema, mitochondrial degeneration, the proprioceptors in extraocular muscle from intermittent exotropia showed ultrastructual change, inner and outer membranes were incomplete, intraspindle muscle fibers arranged disorderly,the formation of residues were found in myelinated nerve axoplasmic,lack of mitochondria, the thick dense collagen fiber hyperplasia appeared between Schwann cells and inner membrane, microfilaments and microtubules of the nerve fibers decreased, and nerve demyelination in severe cases.Conclusion:The ultrastructure of medial rectus of patients with intermittent exotropia was pathological changed compared with that of the controls. The motor pathways of the monocular glaucoma damage showed ultrastructual change. |
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