| [Objective]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 (SO 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 to the hemopoietic system ,however its function on the hemopoietic system has been unknown yet. MBP also shows the autoimmungenic characteristics , and experimental allergic encephalomyelitis(EAE) as a good animal model for the human autoimmune demyelinating disease multiple sclerosis (MS) can be caused when animals immunized with MBPs and Freund' s complete adjuvant (FCA). Oral tolerance administration of MBP is considered an effective method to treat MS and other demyelinating diseases. 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 suppresse 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 cellproliferation 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 CNS diseases. On the base of the previous construction of minigenes in our laboratory , we utilized pSCEP-MBP-CAT mini gene , containing the regulatory elements of human 01(1) collagen(COLIA1) and MBP cDNA , to transfect into human fibroblast(FB), smooth muscle cell (SMC) and Yunnan tin miner lung cancer ( YTMLC-90 ) with LipofectAMINE? meanwhile pSVCEP-NGF-CAT and pSVCEP-BDNF-CAT , and empty-carrier pSVCEP-CAT-transfected FB, SMC and YTMLC-90 as positive and negative controls respectively. The ectopic expression of foreign MBP, BDNF or NGF gene in these non-neural cells driven by COLIA1 was indivadually detected using ELISA, immune slit blot and Western transfer blot. And the effects of foreign genes on cell proliferation and apoptosis in these non-neural cells were explored by using a series of technology such as 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)-mediated dUTP-fluorescein nick end labeling (TUNEL). [methods] 1. Establishments of MBP, BDNF and NGF minigene-tranfected FB,SMC and YTMLC-90: Recombint DNA of pSCEP-MBP-CAT, pSVCEP-NGF-CAT, pSVCEP-BDNF-CAT and pSVCEP-CAT was separately extracted with 'Lysis by alkali' methods, and transfected into FB, SMC and YTMLC-90 by LipofectAMINE?. MBP , BDNF or NGF minigene-tranfected FB, SMC and YTMLC-90 were selected with G418.2. Expression evaluation of MBP, BDNF or NGF minigene in FB, SMC and YTMLC-90: 1 X 106 cells of each group was digested with 0.25 % trypsin and collected the cells into a tube. The total protein of cells was extracted, and qualitatively and quantitatively assayed the ectopic expression of MBP, BDNF or NGF in FB, SMC and YTMLC-90 with Immuno slit blot, Western transfer blot and ELISA. Finally , expression product was localized in FB, SMC and YTMLC-90 with immunocytochemistry methods.3. Identifiction of cell proliferation and apoptosis in MBP, BDNF and NGF minigene-tranfected FB , SM...
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