不同转移潜能的小鼠肝癌淋巴道转移和淋巴管生成的研究

目的探讨小鼠淋巴道高、低转移潜能的肝癌细胞的体内淋巴道转移状况和淋巴管生成对淋巴道转移的影响。方法将淋巴道高、低转移潜能的肝癌细胞接种于Balb／C小鼠，观察成瘤及转移情况，对肿瘤组织进行淋巴管染色，观察淋巴管生成情况。另取高、低转移潜能的细胞株进行体外淋巴管生成实验并进行小鼠肿瘤转移基因芯片检测，对血管内皮细胞生长因子C、D（VEGF—C、D）进行半定量逆转录聚合酶链反应及实时定量聚合酶链反应分析。结果高，低转移细胞在小鼠髂总动脉旁、肾门淋巴结的转移差异有统计学意义（P=0．0l8）。高转移潜能组诱导淋巴管生成的数量大于低转移组和对照组（P=0．032）。高转移组的CD44、E-cadherin．HER2／neu、H—Ras．VEGF—C的表达均高于低转移组，nm23A．nm23-E4、pl6ink4a、CD61等均低于低转移组。半定量逆转录聚合酶链反应表明，高转移组vEGF—C高于低转移组，VEGF—D低于低转移组。实时定量聚合酶链反应分析高转移组的VEGF—D分泌显著小于低转移组，vEGF—C／VEGF—D在高转移组明显高于低转移组。结论肝癌的淋巴道转移与淋巴管生成有关，VEGF—C、D相关基因表达的改变影响淋巴管生成。VEGF—C／VEGF—D比值可能是有效判断并影响肝癌淋巴道转移潜能的指标之一。

The relationship between lymphangiogenesis and lymphatic metastasis in murine hepatic carcinoma of high and low metastatic potentialities

OBJECTIVES: To study the relationship between lymphangiogenesis and lymphatic metastasis in mice bearing hepatic carcinoma and analyze the mechanism of the lymphatic metastasis. METHODS: Hepatic carcinoma cell lines of high and low potentialities of lymphatic metastasis were injected into the footpads of Balb/c mice. Their metastases to lymph nodes were examined. The tumor tissues of each group were stained with 5'-nucleotidase-ALP to observe the lymphoangiogenesis. The total RNA of high and low metastatic potential cell lines were extracted for metastasis gene DNA array. The vascular endothelial cell growth factor C (VEGF-C) and VEGF-D of each cell line were detected using semi-quantitative RT-PCR and were further quantatively analyzed using real time PCR. RESULTS: The para-common iliac a. and renal hilar lymph nodes metastases of the high metastatic potential cells were significantly higher than in the controls (P>0.05). The quantity of lymphatic vessels in the high metastasis group was significantly larger than that of the control group (P<0.05). The expressions of CD44, E-cadherin, HER2/neu, H-Ras and VEGF-C in the high metastasis group were higher than those in the low metastasis group shown by the cDNA micro array experiment but the expressions of nm23A, nm23-E4, p16ink4a, CD61 were lower. The VEGF-C expression was higher and the VEGF-D was lower in the high metastasis group compared to those of the low metastasis group shown by semi-quantitative RT-PCR. The secretion of VEGF-D was significantly lower and the ratio of VEGF-C/VEGF-D was significantly higher in the high metastasis group than the low metastasis group (P<0.05). CONCLUSIONS: The lymphatic metastasis of hepatic carcinoma is related to lymphoangiogenesis. The changes of VEGF-C and VEGF-D expressions might be a cause influencing the lymphoangiogenesis. VEGF-C/VEGF-D might be an effective parameter in affecting lymphatic metastases.