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目的研究HBx羧基末端40个氨基酸对肝癌细胞增殖的影响及作用机制。方法用HBx及去除羧基末端40个氨基酸HBx3′40转染含野生型(wt)p53的SMMC7721细胞,经稳定筛选后,通过细胞生长曲线绘制以及平板克隆形成实验来观察HBx对细胞增殖的影响,并通过裸鼠体内的成瘤实验观察各移植瘤的大小及重量、增殖细胞核抗原(PCNA)表达的变化。结果细胞生长曲线表明HBx及HBx3′40的细胞生长速度明显较空载体增快;平板克隆形成试验表明克隆形成率,HBx组(48.7±8.1)%及HBx3′40组(82.8±6.0)%较对照组(26.9±3.5)%明显增多(P<0.05),pcDNA3HBx3′40组又较pcDNA3HBx组增多(P<0.05)。裸鼠成瘤实验pcDNA3HBx3′40组肿瘤的大小及重量(1.476±0.232、0.987±0.279)%及pcDNA3HBx组(0.412±0.212、0.395±0.159)%较对照组(0.051±0.024、0.033±0.004)%明显增大(P<0.05),肿瘤细胞的PCNA表达率pcDNA3HBx3′40组(69.25±3.77)%及pcDNA3HBx组(59.00±2.58)%也较对照组(37.67±2.52)%明显增强(P<0.05),且以HBx3′40组的表现更为明显(P<0.05)。结论HBx的羧基末端40个氨基酸可刺激细胞增殖,其突变可能与肝癌细胞发生过程中促进癌细胞的恶性转化有关。
Objective To study the effect of 40 amino acids of the carboxyl terminus of HBx on the proliferation of hepatoma cells and its mechanism. Methods HBx3’40 was transfected into SMMC7721 cells with wild type (wt) p53 by HBx and HBx3’40. After stable screening, the effect of HBx on cell proliferation was observed by cell growth curve and plate clone formation assay. The size and weight of each xenograft and the expression of proliferating cell nuclear antigen (PCNA) were observed by tumorigenesis in nude mice. Results The cell growth curve showed that the cell growth rate of HBx and HBx3’40 was significantly faster than that of empty vector. The clonogenic assay showed that the colony formation rate was higher in HBx (48.7 ± 8.1)% and HBx3’40 (82.8 ± 6.0)% The control group (26.9 ± 3.5)% increased significantly (P <0.05), while the pcDNA3HBx3’40 group increased more than the pcDNA3HBx group (P <0.05). The tumor size and weight (1.476 ± 0.232,0.987 ± 0.279)% and pcDNA3HBx group (0.412 ± 0.212,0.395 ± 0.159)% of pcDNA3HBx3’40 tumor group in nude mice were (0.051 ± 0.024, 0.033 ± 0.004)% (P <0.05). The expression of PCNA in tumor cells was significantly higher than that in control group (69.25 ± 3.77)% and pcDNA3HBx group (59.00 ± 2.58)% (37.67 ± 2.52)% ), And the performance of HBx3’40 group was more obvious (P <0.05). Conclusion The 40 amino acids of the carboxyl terminus of HBx can stimulate cell proliferation. The mutation may be related to the promotion of malignant transformation of cancer cells during the development of hepatocellular carcinoma cells.