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目的:研究DNA依赖蛋白激酶催化亚基(DNA dependent protein kinase catalytic subunit,DNA-PKcs)在小鼠不同发育阶段肝脏组织和肿瘤组织中的表达,明确其促细胞增殖和在肿瘤发生发展中的作用及机制。方法:采用免疫组织化学方法和蛋白印迹检测小鼠不同发育阶段肝脏组织和肿瘤组织中DNA-PKcs的蛋白表达。通过干扰RNA技术沉默肝肿瘤HepG2细胞中DNA-PKcs的表达,通过细胞增殖实验和裸鼠致瘤实验观察肿瘤细胞增殖和致瘤能力的变化。蛋白印迹法检测细胞增殖相关信号通路蛋白p-GSK3β和c-myc的表达水平。结果:在发育过程中,随着细胞增殖能力降低,肝组织中DNA-PKcs表达水平下降,在成年鼠肝组织中DNA-PKcs只有微弱表达。而在肿瘤组织细胞中DNA-PKcs表达显著增高(P<0.01)。细胞生长曲线结果显示,DNA-PKcs表达抑制后,HepG2细胞增殖能力受到明显抑制(P<0.01),致瘤能力也显著降低。信号通路蛋白分析发现DNA-PKcs抑制导致GSK3β磷酸化水平和c-myc蛋白水平降低(P<0.01)。结论:DNA-PKcs表达水平与肝脏细胞的增殖能力密切相关。DNA-PKcs的高表达可能通过调节细胞的增殖,参与肝脏肿瘤的发生和发展过程,作用机制和Wnt/GSK/c-myc信号通路相关。
OBJECTIVE: To study the expression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in liver and tumor tissues at different developmental stages in mice and to clarify its role in promoting cell proliferation and tumorigenesis And mechanism. Methods: Immunohistochemistry and Western blotting were used to detect the protein expression of DNA-PKcs in liver and tumor tissues at different developmental stages. The expression of DNA-PKcs in hepatic tumor HepG2 cells was silenced by interfering RNA technology. The proliferation and tumorigenicity of tumor cells were observed by cell proliferation assay and tumorigenicity test in nude mice. Western blotting was used to detect the expression levels of p-GSK3β and c-myc, which are cell proliferation related signaling pathways. Results: During the development, the expression of DNA-PKcs in liver tissue decreased with the decrease of cell proliferation ability, and the expression of DNA-PKcs was only weakly expressed in liver tissue of adult mice. However, the expression of DNA-PKcs in tumor cells was significantly increased (P <0.01). The results of cell growth curve showed that the inhibition of DNA-PKcs expression inhibited the proliferation of HepG2 cells significantly (P <0.01), and the ability of tumorigenesis was also significantly reduced. Signal pathway protein analysis found that DNA-PKcs inhibition resulted in decreased GSK3β phosphorylation and c-myc protein level (P <0.01). Conclusion: The expression level of DNA-PKcs is closely related to the proliferation of liver cells. The high expression of DNA-PKcs may be related to the Wnt / GSK / c-myc signaling pathway by regulating cell proliferation and participating in the development and progression of liver tumors.