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目的:体外合成GADD45β基因全序列表达质粒后,研究GADD45β基因表达诱导对呈不同p53状态的肝癌细胞的作用及可能机制。方法:采用RT-PCR法获得GADD45β基因全序列,插入pDrive穿梭克隆载体和pIRES2-EGFP荧光表达载体后大量扩增获得DNA,结合p53全基因表达质粒pp53-EGFP转染HepG2、Hep3B细胞后,以[3H]胸腺嘧啶脱氧核苷掺入法(3H-Tdr)和细胞克隆形成法分析DNA合成变化及细胞生长能力;以双抗体夹心ELISA法测定TGF-β1表达变化。结果:成功合成GADD45β基因全序列和表达质粒,通过流式细胞仪收集转染阳性的荧光表达细胞能显著提高转染效率;转染GADD45β后,具有野生型p53基因的HepG2细胞的细胞克隆形成能力和DNA合成能力明显受到抑制,细胞凋亡明显增加,TGF-β1的表达亦明显受抑。与之相反,缺失p53基因的Hep3B需要同时共转染p53基因后,方出现抑制效应。结论:GADD45β基因能够有效抑制肝癌细胞的生长,其功能需要完整p53基因的辅助和(或)调控。GADD45表达和(或)功能异常,导致p53介导的DNA损伤修复途径异常或阻断,是肝脏细胞恶性转化及形成肿瘤的可能机制。
OBJECTIVE: To study the effect and possible mechanism of GADD45β gene expression induction on hepatoma cells with different p53 status after in vitro synthesis of GADD45β gene full-length expression plasmid. Methods: The full-length GADD45β gene was amplified by RT-PCR and inserted into the pDrive shuttle vector and pIRES2-EGFP vector. The amplified DNA was amplified and transfected into HepG2 and Hep3B cells with pp53-EGFP. [3H] thymidine incorporation (3H-Tdr) and cell clone formation assay DNA synthesis changes and cell growth capacity; double antibody sandwich ELISA assay TGF-β1 expression. RESULTS: The complete sequence of GADD45β gene and expression plasmid were successfully synthesized. Transfection efficiency of transfected cells was significantly increased by flow cytometry. After transfection with GADD45β, the cell clonality of HepG2 cells with wild-type p53 gene And DNA synthesis ability was significantly inhibited, significantly increased apoptosis, TGF-β1 expression was significantly inhibited. In contrast, Hep3B lacking the p53 gene needs to be co-transfected with the p53 gene at the same time, and the inhibitory effect appears. Conclusion: GADD45β gene can effectively inhibit the growth of hepatocellular carcinoma cells and its function requires the help and / or regulation of the complete p53 gene. GADD45 expression and / or dysfunction, leading to p53-mediated DNA damage repair pathway abnormalities or blocked, is a possible mechanism of malignant transformation of liver cells and tumor formation.