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。采用细胞悬浮法将人胃癌细胞株AZ521(5×106/0.1ml)原位移植到裸鼠的胃内。选用6-7周龄、体重18-22g的雌性BALB/c-nu/nu裸鼠。6~8周后，解剖有少量AZ521细胞转移灶的肝脏和区域淋巴结，建立AZH1G和AZL5G。用AZH1G和AZL1G重复相同的步骤，在第五轮逐步选择时建立AZH5G和AZL5G。我们还利用腹膜内和原位逐步选择的方法建立了AZ3P3G。1)AZL5G和MKNL3G分别有85.0%和60.0%的小鼠发生了淋巴结转移。2)体内各转移细胞生长均明显大于亲本AZ521。3)细胞运动分析表明，各转移细胞的迁移能力明显高于亲本细胞系。4)流式细胞仪分析多种细胞黏附分子的表达情况，发现α_1和α_2整合素在AZL5G细胞中的表达明显高于AZ521细胞。然后，MKNL3G细胞表达α_2整合素上调。此外，对高转移细胞系和低转移亲本细胞系的生物学行为的综合分析表明，高转移细胞系和低转移亲本细胞系的黏附活性和血管内皮生长因子的产生存在差异。在AZL5G中上调的基因是内皮素-A受体和转化生长因子-βII受体α。最重要的是，我们的实验模型对于研究人胃癌的转移机制是有用的。在MKNL3G中上调基因和抑制方法目前正在进行中。

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.