Liao Y, Tang Z, Sun F
Liver Cancer Institute, Shanghai Medical University.
Zhonghua Yi Xue Za Zhi. 1996 Sep;76(9):650-3.
To investigate if treatment with antisense H-ras oligodeoxynucleotides (ODNs) modulates tumor growth, apoptosis and metastasis of a high metastatic tumor model of human hepatocellular carcinoma (HCC) in nude mice LCI-D20, in which over-expression of H-ras has been identified.
LCI-D20 cells in primary culture were treated with 10 mumol/L antisense ODNs in vitro. 1.5 x 10(6) LCI-D20 cells with or without pretreatment were inoculated into each elevated subcutaneous (s.c) flap in fourteen nude mice, 6 animals for antisense H-ras ODN treated cells, 4 for H-ras non-specific antisense ODN treated cells, the rest 4 for cells without pretreatment.
In in vitro cell culture study, 5-day continuous suppression of H-ras expression by antisense H-ras ODNs resulted in significant inhibition of the proliferation of LCI-D20 cells (t = 31.529, P < 0.01). Cell cycle analysis showed a significant decrease in S phase (36.0 +/- 1.4) and a remarkable increase in G1/G0 fraction (56.7 +/- 1.1) after exposure to antisense H-ras ODNs in comparison with the cells without any treatment (58.5 +/- 0.9, t = 13.519, P < 0.01, 37.4 +/- 0.7, t = 14.802, P < 0.01). In situ end-labeling (ISEL) detection showed that apoptotic cell death was significantly increased in cells with 5-day treatment of antisense H-ras ODNs (34.0% +/- 4.5%) in comparing with cells without treatment (2.5% +/- 1.2%, t = 13. 434, P < 0.01) or treated with non-specific antisense ODNs (4.8% +/- 1.4%, t = 12.453, P < 0.01) at the corresponding time. In in vivo experiment, after six-week observation, tumor growth in antisense H-ras treated animals was significantly retarded in comparison with that of the untreated (t = 3.509, P < 0.01) or nonspecific antisense ODN treated animals (t = 3.452, P < 0.01).
Specific inhibition of H-ras expression by antisense H-ras ODNs could not only induce apoptotic cell death, inhibit the growth rate of LCI-D20 cells in vitro and in vivo, but also alter in vivo tumorigenesity and metastatic potential of LCI-D20 cells.
研究反义H-ras寡脱氧核苷酸（ODNs）治疗是否能调节人肝细胞癌（HCC）高转移肿瘤模型裸鼠LCI-D20的肿瘤生长、凋亡和转移，该模型中已发现H-ras过表达。
原代培养的LCI-D20细胞在体外用10 μmol/L反义ODNs处理。将1.5×10⁶个经或未经预处理的LCI-D20细胞接种到14只裸鼠的每个皮下（s.c）皮瓣隆起处，6只动物接种反义H-ras ODN处理的细胞，4只接种H-ras非特异性反义ODN处理的细胞，其余4只接种未经预处理的细胞。
在体外细胞培养研究中，反义H-ras ODNs连续5天抑制H-ras表达导致LCI-D20细胞增殖受到显著抑制（t = 31.529，P < 0.01）。细胞周期分析显示，与未处理的细胞相比，暴露于反义H-ras ODNs后，S期显著减少（36.0 ± 1.4），G1/G0期显著增加（56.7 ± 1.1）（未处理细胞分别为58.5 ± 0.9，t = 13.519，P < 0.01；37.4 ± 0.7，t = 14.802，P < 0.01）。原位末端标记（ISEL）检测显示，与未处理的细胞（2.5% ± 1.2%，t = 13.434，P < 0.01）或用非特异性反义ODNs处理的细胞（4.8% ± 1.4%，t = 12.453，P < 0.01）相比，经反义H-ras ODNs处理5天的细胞凋亡性细胞死亡显著增加。在体内实验中，经过六周观察，与未处理的动物（t = 3.509，P < 0.01）或用非特异性反义ODN处理的动物相比，反义H-ras处理的动物肿瘤生长明显迟缓（t = 3.452，P < 0.01）。
反义H-ras ODNs特异性抑制H-ras表达不仅能诱导凋亡性细胞死亡，在体外和体内抑制LCI-D20细胞的生长速度，还能改变LCI-D20细胞的体内致瘤性和转移潜能。
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