Proteins Expression Of Neural Stem Cells And Correlation Analysis Between The Expression Of Proteins And Human Glioma

BACKGROUNDNeural stem cells are those multipotential cells in the central nervous system (CNS), which are capable of self-renewing and giving rise to fully differentiated brain cells (neuron, astrocyte and oligodendrocyte). Till now, neural stem cells can be isolated and purified from embryo and adult rodents or human brain. Neural stem cells not only contribute to the regeneration of neurons and the repair of brain tisse, but also are promising for the gene therapy in the nervous system diseases through gene modification. Those neural stem cells express ectogenic neurotransmitters, neuro-nutrition factors and metabolic enzymes, thus provide a new therapy way for many refractory diseases in nervous system. Although those cells persist during the whole adult life, their abilities to self-repair the damadged nervous tissue are relatively minor due to the limitation of absolute quantity and local microenviroment. Therefore, the ectogenous transplantation with neural stem cells is considered as one of the most prospective therapies in nervous system diseases.METHODSDue to their properties of self-renewal and proliferation, the neural stem cells are isolated from the cerebral cortex, subependymal region and hippocampus of rat embryos and cultured in serum-free medium supplemented with growth factors, bFGF and EGF, which stimulate the growth of nueral stem cells.RESULTSNeural stem cells have been successfully isolated and cultured from brain of neonatal rats, and could be induced to differentiate into the neuron, astrocyte and oligodendroglia. Moreover, after three, ten, twenty passages the cells are still the same as the primary neural stem cells, and all of the fully differentiated cells of three types could be detected after inducement of differentiation.CONCLUSIONNeural stem cells are a kind of neural progenitor cells with the properties of self-renewal and multipotential. They can differentiate into many neural cell phenotypes, such as the neuron, astrocyte and oligodendroglia. The successful isolation and culture of NSCs in vitro is valuable to cell transplantation in the diseases of central nervous system. BACKGROUNDPeroxisome proliferators-activated receptor (PPAR) is one of the nucleo-hormone receptor superfamily, with three subtypes: PPARα, PPARγand PPARβ/δ. Although they are encoded by different genes, they share the similar amino acid sequence, especially the amino acid sequence of DNA binding domain and ligand binding domain. PPARγis a ligand-activated nulear factor, belonging to type II nuclear receptor superfamily. PPARγand its relative pathways play important roles in the insulin sensitivity, the tissue homeostasis and the regulation of cell functions.P21 gene, also called CIP I (cyclin dependent kinase interacting protein 1) , WAF1 (wild-type p53-activated fragment 1)or SDI1 (senescent cell-derived inhibitor 1)gene, is a downstream gene of P53 and an important member of cyclin dependent kinase inhibitors involved in regulation of cell cycle. P21WAF1/CIP1 gene is a key factor to suppress growth of tumor cells as well as a regulator of cell cycle and senescence.P16 gene is the gene encoding cyclin dependent kinase 4 inhibitor, also called multiple tumor suppressor gene (MTS1) or CDKN2. Under normal condition, P16 expression level peaks at late G1 stage, which might relate with the functional status of Rb protein. The possible way through which P16 protein regulates the cell cycle is that cyclin D1 increases at late G1 and binds with CDK4 or CDK6 to activate them, which promotes the phosphorylation of Rb protein, thus E2F is separated from Rb protein and finally cell proliferation is induced. Like P21WAF1/CIP1, P16 is also a member of cyclin dependent kinase inhivitors (CKIs), which negatively regulate the cell cycle.METHODSCell immunofluorescence, western blot and RT-PCR were employed to detect the expression levels of PPARγ, P21WAF1/CIP1 and P16 protein or mRNA repectively at different stages of NSCs.RESULTS1. The cell immunofluorescence showed that PPARγ, P21WAF1/CIP1 and P16 protein were stained as green in NSCs. Flourescence located mostly in the cell nucleus and also some in the cytoplasm, which indicated the location of these three proteins is mainly in the cell nucleus.2. Western blot showed positive bands of PPARγ, P21WAF1/CIP1 or P16 after incubation with their antibodies. With the time of differentiation, gradually weakened bands of PPARγsuggested the decrease of its expression in NSCs, while gradually strengthened bands of P21WAF1/CIP1 and P16 indictated the increase of their expression.3. RT-PCR showed that cDNA of PPARγ, P21WAF1/CIP1 and P16 were detected in both Undifferentiated and differentiated NSCs. According with western blot results, PPARγsignificantly reduced since the differentiation began. In contrast, the cDNA of P21WAF1/CIP1 and P16 increased gradually during the differentiation process of NSCs, especially that of P21WAF1/CIP1.CONCLUSIONWith the time of NSCs differentiation, mRNA and protein of PPARγdropped off, while those of P21WAF1/CIP1 and P16 ascended. PPARγ, P21WAF1/CIP1 and P16 might be involved in the regulation of NSCs proliferation and differentiation. PPARγinduced NSCs self-renewal and reduced the differentiating NSCs; whereas, P21WAF1/CIP1 and P16 may prefer to promote NSCs differentiation rather than proliferation. BACKGROUNDGliomas include the most common primary brain tumors and the vast majority of them are astrocytomas or oligodendrogliomas. A considerable number of advances on therapeutics of malignant gliomas have been achieved in the last decade. But unfortunately, surgical cure, as the basic treatment of malignant gliomas, is practically impossible to resect effectively beyond the limits of macroscopically visible tumor without drastically compromising neurologic function. As the result, gliomas portend a poor prognosis. In order to resolve this problem, many investigations have been performed and recent discoveries have shed light on the molecular events which deregulate the cell cycle in brain tumors. Mutational inactivation of the tumor suppressor genes is a frequent feature of many human neoplasms. However, the molecular mechanisms by which the tumor suppressor protein may contribute to deregulation of cellular growth and malignant progression are not clearly understood yet. P21WAF1/CIP1 and P16 proteins are two known tumor suppressor proteins. Analyses of the expression of the molecules of P21WAF1/CIP1 and P16 in glioma may be useful in predicting behavior of tumors in order to identify which kind of gliomas is most amenable to therapy. Therefore, the aim of this study is to investigate the correlation between the expression levels of P21WAF1/ CIP1, P16 proteins and the grading of glioma.METHODSCell immunofluorescence, western blot and RT-PCR were employed to investigate not only the expression of P21WAF1/ CIP1 and P16 proteins in glioma and normal brain tissue as well as in T98G and HA cell lines, but also the correlation between their expression levels and the grading of glioma.RESULTS1. Expression of P21WAF1/CIP1 and P16 in Human Glioma Cell Line (T98G)P21WAF1/CIP1 and P16 were expressed in Human Glioma Cell Line (T98G) as well as in normal human astrocyte cell line (HA). However, strongly positive reactions appeared in nucleus of most normal cells, shown by diffuse dark brown-yellow reaction products, whereas dramatic decrease of positive cells was shown in T98G cells because of lower expression of P21WAF1/CIP1 and P16 in them.2. Expression of P21WAF1/CIP1 and P16 in Human Glioma tissue The incidence of positive cases of P21WAF1/CIP1 and P16 waslower in carcinomas with grades III-IV than in those with grades I-II (p=0.005). Moreover, the coordinate expression levels of the two genes in glioma tissues were significantly decreased following the higher grading of carcinomas.3. Semi-Quantitative RT-PCR Analysis for the Changes in Expression Levels of P21WAF1/CIP1 and P16The expression levels of two genes in normal brain tissues were significantly higher than those in human glioma tissues. Moreover, the band intensity in normal brain tissues was found 1.28 (P21WAF1/CIP1) and 1.07 (P16) fold stronger than that in grade I human brain glioma tissues and the ratio of the expression levels of P21WAF1/CIP1 and P16 gene in normal brain tissues to human brain glioma tissues dramatically increased following the tumor's grading.CONCLUSIONIn conclusion, our results suggested that the expression levels of P21WAF1/CIP1 and P16 are inversely correlated with the clinical grading of gliomas significantly and the two genes are coordinate expressed in tumor tissues. It indicates that P21WAF1/CIP1 and P16 may take part in not only gliomas carcinogenesis, but also tumor progression of patients.