Zhang Xia, Ouyang Xue-Nong, Li Xiao-Dong, Zhao Jian, Guo Ya-Jun
Nanjing Command, Fuzhou General Hospital, Fuzhou 350025, China.
Zhonghua Gan Zang Bing Za Zhi. 2005 Jan;13(1):38-41.
To introduce the newly found gene TIP30/CC3 into a hepatoma cell line PLC/PRF/5 and select the stable expression clones. The growth and cell cycles were studied with the clones stably expressing TIP30/CC3 or anti-TIP30/CC3, and the effects of TIP30/CC3 gene on hepatoma cells were analyzed.
The internal expression of TIP30/CC3 protein was detected with Western blot, then TIP30/CC3 or anti-TIP30/CC3 cDNA was subcloned into a constitutive vector pcDNA3 followed by transfection into PLC/PRF/5. Stable expression clones were selected. The cell growth curve was made and cell cycles detected using flow cytometry. To confirm the results in vitro, stable-expressing cells were implanted subcutaneously into nude mice and time of tumor formation recorded and tumor volume measured.
PLC-anti-TIP30 grew faster than the others. Three days after transfection, live cells of PLC-anti-TIP30 were 14.010(4), in comparison with the control PLC-DNA3 and PLC/PRF/5, the differences were statistically significant. Live cells of PLC-TIP30 were 4.910(4), significantly less than the two control groups. Six days after transfection, live cells of PLC-anti-TIP30 were 25.010(4), significantly more than the controls PLC-DNA3 and PLC/PRF/5. Live cells of PLC-TIP30 were 12.410(4), significantly less than the two control groups. Cell cycle analysis showed that PLC-anti-TIP30 proliferated faster, 22.4% cells were in G0/G1 (gap) phases and 58.6% cells in S (DNA synthesis) phase. The growth of the PLC-anti-TIP30 cell was retarded and many cells were arrested from G1 to S phases. Cells in G0/G1 and S phase were 44.2% and 33.3% respectively. Furthermore, the average time of tumor formation was shorter in anti-TIP30 group and longer in TIP30/CC3 group, and times were 6.0 d (with control groups) and 15.6 d (with control groups) respectively. Tumors in the nude mice grew faster in PLC-anti-TIP30 group and slower in PLC-TIP30 group.
Tumor suppressor gene TIP30/CC3 can inhibit the proliferation of tumor cells and interfere in its cell cycles. It can be used as a valuable tool for hepatoma biotherapy including gene therapy.
将新发现的基因TIP30/CC3导入肝癌细胞系PLC/PRF/5中，筛选稳定表达克隆。对稳定表达TIP30/CC3或抗TIP30/CC3的克隆进行生长及细胞周期研究，分析TIP30/CC3基因对肝癌细胞的作用。
采用蛋白质印迹法检测TIP30/CC3蛋白的内源性表达，然后将TIP30/CC3或抗TIP30/CC3的互补DNA（cDNA）亚克隆至组成型载体pcDNA3中，随后转染至PLC/PRF/5细胞。筛选稳定表达克隆。绘制细胞生长曲线，采用流式细胞术检测细胞周期。为在体外验证结果，将稳定表达细胞皮下接种于裸鼠，记录肿瘤形成时间并测量肿瘤体积。
PLC-抗TIP30生长速度比其他细胞快。转染3天后，PLC-抗TIP30的活细胞数为14.0×10⁴，与对照PLC-DNA3和PLC/PRF/5相比，差异具有统计学意义。PLC-TIP30的活细胞数为4.9×10⁴，显著少于两个对照组。转染6天后，PLC-抗TIP30的活细胞数为25.0×10⁴，显著多于对照PLC-DNA3和PLC/PRF/5。PLC-TIP30的活细胞数为12.4×10⁴，显著少于两个对照组。细胞周期分析显示，PLC-抗TIP30增殖更快，22.4%的细胞处于G0/G1期（间隙期），58.6%的细胞处于S期（DNA合成期）。PLC-抗TIP30细胞生长受阻，许多细胞在从G1期到S期被阻滞。处于G0/G1期和S期的细胞分别为44.2%和33.3%。此外，抗TIP30组肿瘤形成的平均时间较短，TIP30/CC3组较长，分别为6.0天（与对照组相比）和15.6天（与对照组相比）。裸鼠体内肿瘤在PLC-抗TIP30组生长较快，在PLC-TIP30组生长较慢。
抑癌基因TIP30/CC3可抑制肿瘤细胞增殖并干扰其细胞周期。它可作为包括基因治疗在内的肝癌生物治疗的有价值工具。
