Clarification of the lymph node metastasis mechanism by orthotopic implantation models and application to inhibition of the metastasis

为了研究癌细胞转移的分子机制，我们建立了肝转移模型。采用细胞悬浮技术将人胃癌细胞株AZ521 （5 × 10^6/0.1ml）原位移植至裸鼠胃内。选取6-7周龄的雌性BALB/c nu/nu小鼠，体重18-22g。6 ~ 8周后，切除AZ521细胞的肝脏和局部淋巴结，建立AZH1G和AZL5G。用AZH1G和AZL1G重复同样的步骤，在第5轮逐步筛选时建立AZH5G和AZL5G。我们还通过腹腔和原位逐步选择方法建立了AZ3P3G。1) AZL5G小鼠淋巴结转移率为85.0%，MKNL3G小鼠淋巴结转移率为60.0%。2)各转移细胞在体内的生长均显著大于亲本AZ521。3)细胞运动试验表明，各转移细胞的迁移能力明显高于亲本细胞系。4) FACS分析表明，α_1和α_2整合素在AZL5G细胞中的表达率显著高于AZ521。MKNL3G细胞α_2整合素表达上调。此外，综合分析高转移细胞系和低转移同源细胞系的生物学行为，证实了黏附活性和VEGF产生的差异。AZL5G上调的基因为内皮素a受体和tgf - β II R α。综上所述，我们的实验模型将有助于研究人胃癌的转移机制。MKNL3G上调基因和抑制方法目前正在研究中。

To investigate the molecular mechanisms by which carcinoma cells metastasize, we have established liver metastatic models. Human gastric carcinoma cell line AZ521 (5 × 10^6/0.1ml) was transplanted orthotopically into the stomach of nude mice using cell suspension technique. Athymic female BALB/c nu/nu mice, 6-7 weeks old, weighing 18-22g were used. After from 6 to 8 weeks, the liver and regional lymph nodes with a few metastatic foci of AZ521 cells were dissected, we established AZH1G and AZL5G. The same procedure was repeated using AZH1G and AZL1G, and AZH5G and AZL5G was established upon the fifth cycle of stepwise selection. We have also established AZ3P3G using intraperitoneally and orthotopically stepwise selection methods. 1) AZL5G produced lymph node mestastasis in 85.0%, and MKNL3G produced lymph node mestastasis in 60.0% of mice. 2) Each metastatic cell grew significantly larger than parental AZ521 in vivo. 3) A cell motility assay demonstrated that the migration ability of each metastatic cell was clearly higher than that of parental cell lines. 4) The expression of several cell adhesion molecules by FACS analysis showed that the incidence of α_1 and α_2 integrins expression in AZL5G cells was significantly higher than in AZ521. Then, MKNL3G cells expressed suggestive up-regulation of α_2 integrin. Moreover, Comprehensive analysis of the biological behavior of highly metastatic cell lines and the less metastatic same parental lines were demonstrated the differences in adhesive activity and VEGF production. Up-regulated genes were endothelin-A receptor and TGF-beta II R alpha in AZL5G. Above all, our experimental models should be useful for investigation of the mechanisms of metastasis in human gastric carcinoma. Up-regulated genes in MKNL3G and inhibitory methods are currently underway.