The Study On The Biological Properties Of Neural Stem Cells, NSCs Transplantation After Traumatic Brain Injury Improving Functional Outcome In Adult Rats, And Gene Therapy Of Experimental Brain Tumors

A neural stem cell can be most simply defined as any cell which is capable of self renewal for extended periods of time, the progeny from which is capable of forming the components of nervous tissue, differentiating into neurons, astrocytes and oligodendrocytes. Asymmetric division allows NSCs to generate a progenitor cell, in addition to another NSC, which has a limited potential for self renewal and often spontaneously stops dividing and differentiates. NSCs have been isolated from several sites from mammalian animal or human CNS, such as SVZ, dentate gyrus of hipppocampus, olfactory bulb, granular layer of cerebellum.With the recognition that NSCs, propagated in culture, could be reimplanted into mammalian brain where they could reintegrate appropriately, stem cell therapy is the new strategy to treat a variety of diseases, through replacing, repairing the injured tissue or organ, then strengthening the function of them. NSCs have many properties, such as genetic manipulability, sustained foreign gene expression, potential for normal reintegration into host cytoarchitecture and circuitry, ability to migrate, one stem cell may carry multiple transgene. Theseproperties make them appealing vehicles for CNS gene therapy and repair.Up to date, there have been few reports in the study on the isolation and biological properties of NSCs from hippocampus. Using NSCs in brain injury therapy has rarely been reported. Gene therapy of experimental brain tumors using NSCs expressing IL-12 has not been reported. In the study, we investigated the biological properties of neural stem cells, neuroprotective effect of NSCs transplants after experimental traumatic brain injury, and gene therapy of experimental brain tumors using NSCs expressing IL-12.Part One: Study on the biological properties of Neural Stem CellsThe study of the proliferating property and ultrastructure of EGF and FGF-2 dependent neural stem cells (NSCs) derived from the hippocampi of embryonic rats or human.1. The proliferation of NSCs was studied using the MTT colorimetric assay for quantitating NSC activity, and bromodeoxuridine (BrdU) labeling was used to study NSC proliferation. NSCs were examined by transmission electron microscopy (TEM). The techniques of immunohistofiuorescence and immunohistochemistry were applied to define the NSCs' antigenic characterization and differentiation potential. There are multipotential NSCs in the hippocampi of embryonic rats, their cellular viability increasing steadily during the first week in vitro. Many cells were immunoreactive for BrdU. Visualized by TEM NSCs were relatively small, with a large ratio of nucleus to cytoplasm and a prominent nucleolus. Heterochromatin was less condensed and scattered, with little debris. The cytoplasm contained many free ribosomes; there were few mitochondria (MIT) and rough endoplastic reticulum (RER).Under appropriate conditions, EGF/ FGF-2 stimulated the differentiation of NSCs,producing neurons and glia in definite proportions.2. Nestin is an intermediate filament protein found in CNS progenitor cells. Using immunocytochemistry, fluorescence-activated cell sorting, RT-PCR and Western Blot, we examined the expression of nestin in the NSCs isolated from human hippocampus. Nestin was detected in all samples. Virtually (>90%) almost cells in the spheres were immunoreactive for nestin and were not immunoreactive for NSE and GFAP. The mRNA expression of nestin was detected by RT-PCR. Nestin had an molecular mass of approximately 240 KD.3. EOF and FGF-2 have trophic effects on NSCs. Proliferation of cells declined immediately upon removal of EGF or FGF-2 although cells remained viable in the absence of these growth factors. Cells maintained in the presence of FGF-2 produced significantly more neurons than parallel cultures maintained without FGF-2.The presence or absence of EGF did not appear to affect the differentiation of neurons or astrocytes.4. When transplanted into the adult rat brain, the in vitro propagated cells migrated along the routes...