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目的 观察外源性N端截短的视网膜母细胞瘤 (Rb)基因对骨肉瘤细胞株OS732的生长和细胞间缝隙连接信息通讯能力的影响。方法 构建N端截短的长度为 1 6 5kbRb基因的真核表达质粒 ,并用DOTAP将其导入骨肉瘤细胞株OS732。应用逆转录 聚合酶链反应 (RT PCR)和Northernblot检测Rb基因的表达 ;用细胞计数、流式细胞仪分析和软琼脂克隆形成试验观察OS732细胞的生长状况 ;用半定量RT PCR法和罗氏黄荧光传输法检测细胞间缝隙连接信息通讯的能力。结果 转染N端截短的Rb基因后 ,OS732细胞内可检测到内源性和外源性Rb基因的mRNA表达 ;OS732细胞的形态发生改变 ,其生长速度减慢 ,软琼脂形成集落能力降低 ,细胞周期阻滞于G0 G1期 ;缝隙连接蛋白基因Connexin4 3的表达和细胞间信息通讯能力增强。结论 N端截短的Rb基因可抑制骨肉瘤细胞株OS732的恶性生长表型以及增强细胞间信息通讯能力
Objective To investigate the effect of exogenous N-terminal truncated retinoblastoma (Rb) gene on the growth and cell-cell gap junctional information communication of osteosarcoma cell line OS732. Methods The eukaryotic expression plasmid of N-terminal truncated length of 165kb was constructed and introduced into Osteosarcoma cell line OS732 by DOTAP. The expression of Rb gene was detected by reverse transcription polymerase chain reaction (RT PCR) and Northern blotting. The growth of OS732 cells was observed by cell counting, flow cytometry analysis and soft agar colony formation assay. Semi-quantitative RT-PCR and Roche’s yellow Fluorescence transmission method to detect the ability of cell gap to connect information and communication. Results The mRNA expression of endogenous and exogenous Rb gene was detected in OS732 cells transfected with N-terminal truncated Rb gene. The morphology of OS732 cells was changed, the growth rate was slowed, and the soft agar-forming ability was reduced , Cell cycle arrest in G0 G1 phase; connexin gene connexin43 expression and intercellular communication enhanced. Conclusion The N-terminal truncated Rb gene inhibits the malignant phenotype of osteosarcoma cell line OS732 and enhances the ability of information communication among cells