SV40LTAg基因永生化软骨细胞体外培养的生物学特性

Observation of the biological behavior of in vitro cultured immotalized chondrocytes induced by SV40LTAg gene transfection

目的探讨永生化关节软骨细胞体外培养的生物学特性。方法用SV40LTAg(类人猿40病毒大T抗原)基因转染原代培养的关节软骨细胞,构建永生化关节软骨细胞系(immortalized cartilage chondrocytes)并体外培养,以正常关节软骨细胞(normal cartilage chondrocytes)作对照。倒置显微镜下观察永生化软骨细胞的形态学变化和增殖情况;应用甲苯胺蓝、番红O及Ⅱ型胶原免疫组织化学染色,观察永生化软骨细胞在培养过程中酸性粘多糖(GAG)和胶原分泌情况。结果永生化关节软骨细胞体外培养呈贴壁单层生长,长期传代(50代)仍有很强的增殖能力,形态呈现多角形和三角形,GAG和胶原染色均呈阳性。结论构建的永生化关节软骨细胞在体外培养能长期维持软骨细胞的特征性表型。

Objective To investigate the biological characteristics of immortalized chondrocytes cultured in vitro. Methods Primary chondrocytes were immortalized with SV40LTAg gene transfection and grown in monolayer culture. The biologicalcharacteristics of the cells were defined in terms of cell morphology and proliferative capacity observed under inverted micro-scope. GAG synthesis was assessed with toluidine blue and safranine O staining and type-Ⅱcollagen levels by immunohisto-chemistry. Results After 50 passages, the immortalized chondrocytes appeared polygonal or triangular with still vigorous pro-liferative capacity in monolayer culture and positivity for GAG and collagen type II as characteristic of chondrocytes. Con-clusion The immortalized chondrocytes cultured in vitro can maintain their phenotype in a long term.