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目的:探讨癌基因Src在体外培养骨肉瘤细胞侵袭伪足形成中的作用。方法:构建Src sh RNA慢病毒表达载体,在HEK293T细胞中包装慢病毒,感染HT-1080骨肉瘤细胞,经嘌呤霉素加压筛选,获得稳定沉默Src基因的骨肉瘤细胞系HT-1080-sh Src;实时定量PCR和Western Blot法检测基因沉默效率;采用原位明胶酶谱法检测侵袭伪足形成;采用侵袭小室实验检测下调Src基因表达对HT-1080细胞侵袭力的影响。结果:成功构建稳定沉默Src基因的骨肉瘤细胞系HT-1080-sh Src及对照细胞系HT-1080-shluc,经实时定量PCR和Western Blot检测,与对照细胞系相比,HT-1080-sh Src细胞中Src基因表达下调3倍以上;下调HT-1080细胞中Src基因表达能显著抑制HT-1080细胞侵袭伪足形成及其对细胞外基质的降解能力;下调Src基因表达能显著抑制骨肉瘤细胞侵袭力。结论:癌基因Src参与调节骨肉瘤细胞HT-1080侵袭伪足形成,促进肿瘤侵袭、转移。
Objective: To investigate the role of oncogene Src in the formation of invasion pseudopods in osteosarcoma cells in vitro. Methods: The lentiviral vector of Src sh RNA was constructed. The lentivirus was packaged in HEK293T cells and infected with HT-1080 osteosarcoma cells. The osteosarcoma cell line HT-1080-sh with stable silencing Src gene was screened by puromycin. Src. The gene silencing efficiency was detected by real-time PCR and Western Blot. In situ gelatin zymography was used to detect the formation of invasion pseudopod. The invasion chamber assay was used to detect the effect of Src down-regulation on invasiveness of HT-1080 cells. RESULTS: The osteosarcoma cell line HT-1080-sh Src with stable silencing Src gene was successfully constructed and the control cell line HT-1080-shluc was successfully constructed. Compared with the control cell line, real-time PCR and Western Blot showed that HT-1080-sh The Src gene expression in HT-1080 cells was down-regulated more than 3-fold. The down-regulation of Src gene expression in HT-1080 cells significantly inhibited the invasion of HT-1080 cells and the degradation of extracellular matrix. The down-regulation of Src gene expression significantly inhibited osteosarcoma Cell invasiveness. Conclusion: The oncogene Src is involved in the formation of invasion pseudopods in HT-1080 osteosarcoma cells and promotes tumor invasion and metastasis.