Establishment and Analysis of the in vivo Human Tumor Angiogenesis Model

[目的]建立并分析“体内人肿瘤血管生成模型”。【材料与方法】将人大肠癌细胞系CCL 188、Colo 320、MIP 101和克隆A原位接种于裸鼠盲肠。采用RT-PCR、ELISA和免疫组化方法检测结直肠癌细胞中血管生成因子和淋巴管生成因子的表达。通过CD 31免疫染色和活体荧光显微镜（IVFM）观察每个细胞系的稳定GFP转染的肿瘤血管生成。用抗VEGFR 3抗体检测肿瘤淋巴管生成。[结果] CCL 188和CX-1细胞接种裸鼠脾脏后，几乎100%的小鼠在肝脏形成肿瘤，而CCL 188和CX-1细胞很少形成肝肿瘤。当将人CRC细胞原位移植到裸鼠体内时，Matrigel悬浮细胞产生的局部肿瘤的效力显著高于PBS或Cellmatri悬浮细胞。 关于我们 X.基质胶还仅通过两种高转移性CRC细胞系增强向肝脏的自发转移。然而，原位植入CRC细胞与基质胶没有增加腹膜转移。4种大肠癌细胞株均有不同程度的VEGF释放，其中CCL 188和CX-1的VEGF释放量高于MTP 101和克隆A。抗小鼠CD 31免疫染色显示在CRC细胞植入后3天诱导肿瘤血管生成。该结果与使用GFP转染子在IVFM下观察到的肿瘤血管生成的结果非常一致。两个高转移细胞系诱导的微血管密度高于其他两个细胞系。结直肠癌细胞VEGF-C和VEGF-D的表达与淋巴管密度相关，但与淋巴结转移能力无关。[结论] VEGF可诱导大肠癌细胞内肿瘤血管生成，可能在大肠癌肝转移的发生中起作用。而VEGF-C和VEGF-D虽然促进肿瘤淋巴管生成，但并不决定肿瘤的淋巴结转移能力。少

[Purpose] The purpose of this project was to establish and analyze the "in vivo human tumor angiogenesis model".[Materials and Methods] Four human colorectal carcinoma (CRC) cell lines, CCL188, Colo320, MIP101 and Clone A, were orthotopically implantated in the cecum of nude. Expression of angiogenic and lymphangiogenic factors in the CRC cells were analyzed by RT-PCR, ELISA and immunohistochemistry. Tumor angiogenesis was observed by CD31 immune-staining and intravital fluorescence microscopy (IVFM) with stable GFP-transfectants of each cell line. Tumor lymphangiogenesis was also examined with anti-VEGFR3 antibody.[Results] When inoculated into the spleens of nude mice CCL188 and CX-1 produced tumors in the liver in almost 100% of mice whereas the other two cell lines rarely formed hepatic tumors. When human CRC cells were implanted orthotopically in nude mice Matrigel-suspended cells produced local tumors with significantly higher efficacy than the cells suspended in PBS or Cellmatri … More x. Matrigel also enhanced spontaneous metastasis to the liver only by the two highly metastatic CRC cell lines. However, orthotopic implantation of CRC cells with Matrigel did not increase peritoneal metastasis. All the four CRC cell lines released VEGF with different extents while CCL188 and CX-1 did greater amount than did by MTP101 and Clone A. Anti-mouse CD31 immno-staining showed induction of tumor angiogenesis 3 days after implantation of CRC cells. The results were well compatible with the findings of tumor angiogenesis observed under the IVFM with GFP transfectants. The two highly metastatic cell lines induced higher density of microvessels than the other two cell lines did. Expressions of VEGF-C and VEGF-D in CRC cells showed correlation to lymph vessel density but did not correlate to the metastatic ability to lymph nodes.[Conclusions] VEGF, that induces tumor angiogenesis in CRC cells, may have effects on the production of hepatic metastasis. In contrast, VEGF-C and VEGF-D, that enhance tumor lymphangiogenesis, may not decide the metastatic ability to lymph nodes. Less