RESUMO
OBJECTIVE: To establish an in vitro model of natural degeneration of lumbar endplate chondrocytes and explore the role and the expression change of Sox9 gene, an important gene in the differentiation and maturation of chondrocyte, in the process of natural degeneration of endplate chondrocytes. METHODS: The lumbar vertebrae of 35 SD rats were taken out to obtain the endplates. Endplate chondrocytes were isolated by enzyme digestion and cultured so as to establish an in vitro natural degeneration model of chondrocytes. The morphological appearances and biological characteristics of the chondrocytes of different generations were observed by HE staining, immunocytochemical staining and toluidine blue et cetera; RT-PCR was used to detect the mRNA expression of Sox9 gene and type II collagen in differential generations. RESULTS: The lumbar cartilaginous endplate chondrocytes of rat expressed collagen II, and it's phenotype and biological characteristics were similar to those of articular cartilage cells. When the cells were passaged to the forth or fifth generations they were fusiform and their proliferative speed decreased. Compared with the primary generation chondrocytes, the expression of Sox9 mRNA in the forth and fifth generation chondrocytes was markedly decreased (P < 0.05). And the mRNA expression level of type II collagen, regulated by Sox9 gene, decreased too (P < 0.05). The mRNA expression of Sox9 was positively correlated with the mRNA expression of type II collagen (r = 0.912, P < 0.05). CONCLUSION: A model of natural degeneration of lumbar endplate chondrocytes has been established successfully, thus providing a good cytological basis for the study of degeneration of lumbar endplate. Sox9 gene may play a role in the process of natural degeneration of endplate chondrocytes.
Assuntos
Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Deslocamento do Disco Intervertebral/metabolismo , Fatores de Transcrição SOX9/genética , Animais , Colágeno Tipo II , Modelos Animais de Doenças , Expressão Gênica , Vértebras Lombares , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
AIM: To establish a cell line stably expressing EGFRvIIIex (epidermal growth factor receptor variant III extracellular domain) and evaluate its immunogenicity. METHODS: NIH3T3 cells stably expressing EGFRvIIIex was obtained by screening NIH3T3 cells transfected with pLNCX2-EGFRvIIIex, a plasmid encoding EGFRvIIIex. The expression level of EGFRvIIIex was examined by immunohistochemical staining and Western blot. The cell clone with the highest expression of EGFRvIIIex named as 3T3-vIIIex was used to immunize BALB/c mice. The titer and specificity of murine antiserum was evaluated by ELISA, Western blot and immunofluorescent staining. RESULTS: A NIN3T3 stable cell line with high EGFRvIIIex expression was obtained. The titer of the antiserum against human EGFRvIIIex was about 10(-5). Western blot and immunofluorescent staining analysis revealed the high specificity of the antiserum. CONCLUSION: High titer of antiserum against EGFRvIIIex can be obtained by NIH3T3 cell lines stably expressing EGFRvIIIex.
