The Effects Of Human 21.5kDa MBP Gene Transfection On Neural And Correlative Cell Proliferation And Apoptosis

Human brain myelin basic protein (MBP) is a class of specific proteins in the nerve tissue, which is synthesized both by oligodendrocytes in the central nervous system (CNS) and Schwann cells (SC) in the peripheral nervous system(PNC). MBP plays an important role on the insulation and fast transmission of nerve fibers. MBP possesses specificities of species, tissues and cells, as well as developmental stages and multiple physiological effects on development of the brain, differentiation of neural cells , myelinization and myelinogenesis. MBP distributes not only to the nervous system but also the hemopoietic system with its multiple functions. MBP is still a hot point in neuroscience though MBP and its gene have been studied and progressed. It remains unclear how MBP plays the role on growth development of the brain. Does MBP accelerate or suppress differentiation of neural cells through the apoptotic mechanism? There are few and discrepant reports dealing with this subject. It is very important to identify the exact effects of MBP on cell proliferation and apoptosis for not only the basic research on development of human brain, differentiation of neural cells, myelinization and myelinogenesis, but also clinical application to pathogenetic researches, diagnoses and treatment of the CNSdiseases. On the base of the previous study of our laboratory, we utilized pSCEP-MBP-CAT gene, containing the regulatory elements of human a 1 (l)collagen(COLIAl) and MBP cDNA, to transfect into rat cerebral neuron cell ,bone marrow stromal cells (BMSCs) and U-251 cell with LipofectAMINE? meanwhile pSVCEP-NGF-CAT and pSVCEP-BDNF-CAT, and empty-vectorpSVCEP-CAT-transfected cell as positive and negative controls respectively. The expression of foreign MBP, BDNF or NGF gene in these neural and correlative cells driven by COLIA1 was indivadually detected using ELISA, immuno-dot blot and Western transfer blot. And the effects of foreign genes on cell proliferation and apoptosis in these cells were explored by morphologic and ultra-structural changes observed by microscopy and transmmison electro-microscopy (TEM) , cell proliferation curve and apoptotic rate assayed with 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide(MTT) and flow cytometer(FCM), apoptotic DNA ladder analyzed through agarose gel electrophoresis , the characteristic appearence of individual apoptotic cells determined by immunocytochemistry assay and terminal deoxynucleotidyl transferase(TdT)-mediateddUTP-fluorescein nick end labeling (TUNEL). Results proved pSCEP-MBP-CAT, pSVCEP-NGF-CAT, pSVCEP-BDNF-CAT andpSVCEP-CAT plasmid DNA were successfully expressed in neural and correlative cells in vitro. The specific antigen activities of MBP , NGF and BDNF in the lysates were identified by ELISA and Western transfer blot and dot blot. The results of MTT method indicated that 21.5Kda MBP, BDNF and NGF had significantly promotive effects on the cellproliferation of neural and correlative cells. In contrast, H2O2 remarkably suppressed cell growth and induced apoptosis with dose-dependent and time-dependent effects. Compared with pSVCEP-CAT-transfected cells , 21.5kDa MBP, BDNF and NGF gene-transfected cells showed obviously anti-apoptotic effects. HE staining results revealed that 21.5kDa MBP, BDNF and NGF gene-transfected cells with same treatment of H2O2 were not found remarkably cell morphological changes compared to negative controls. Electron microscope observation results revealed that after treated by H2O2 the cells were changed such as size decreased, vacuoles increased, cytoplasm and nucleus shrunk and deformed, the chromatin condensed into small masses distributed along the inner surface of nuclear membrane. In contrast, 21.5kDa MBP, BDNF and NGF gene-transfected cells with the same treatment of H2O2 were not observed obviously apoptotic appearances as above. The DNA Ladder of apoptotic characteristics was observed in the maps of agarose gel electrophoresis for the empty vector transfected cells, but did not appeared in the maps o...