小鼠肝癌细胞淋巴结转移的基质金属蛋白酶谱

Matrix Metalloproteinases Map of Lymphogenous Metastasis of Hepatocarcinoma Cell in Mice

背景与目的:肿瘤转移是恶性肿瘤致死的主要因素之一,淋巴结转移是癌早期最常见的转移方式,且淋巴结转移灶可以成为进一步血道转移的桥头堡,但癌淋巴道转移的分子机制尚不清楚。本实验旨在探讨小鼠肝癌细胞分泌基质金属蛋白酶(MMPs)及表达Fas-L与其淋巴结转移的关系。方法:采用酶谱法检测具有不同转移能力的小鼠肝癌细胞Hca-F(高转移)和Hca-P(低转移)分别在淋巴结匀浆、肝组织匀浆或脾组织匀浆存在的情况下分泌的基质金属蛋白酶谱;以流式细胞仪检测荷瘤鼠淋巴细胞的生长分数;以TUNEL检测淋巴结内淋巴细胞的凋亡情况;以免疫组化法检测Hca-F和Hca-P细胞表达Fas-L的情况。结果:在淋巴结匀浆存在下Hca-F和Hca-P细胞分泌MMP-9显著增多(P<0.01):RPMI-1640培养基中,Hca-F和Hca-P细胞分泌MMP-9分别为1256±157、2642±385;加入淋巴结匀浆后,则分别为12403±894、9086±686,同时分泌大量的MMP-2(Hca-F为7364±2001,Hca-P为2997±1990)和MMP-9的活性型(Hca-F为7297±1657,Hca-P为3914±1253),且Hca-F细胞分泌量多于Hca-P(P<0.05)。而在肝组织或脾组织匀浆存在下,Hca-F和Hca-P细胞均不分泌基质金属蛋白酶。Hca-F组荷瘤鼠引流淋巴结内的淋巴细胞的增殖高峰出现于荷瘤后第14日,并迅速下降。

Background & Objective: Metastasis is the leading cause of tumor related death, in which lymphogenous metastasis is the most common pattern and also a bridgehead of further metastasis. However, the molecular mechanism of metastasis is uncertain. This study was designed to investigate the correlation between the metastasis of hepatocarcinoma cell(HCC) to lymphnode and matrix metalloproteinases (MMPs) activity and the expression of Fas ligand of tumor cells in lymphnodes. Methods: The maps of MMPs in supernatants of mouse HCCs with different metastatic potential Hca F(high potential) and Hca P cells(low potential) cultured with extract of lymph node, liver or spleen were examined by zymographic analysis. Growth fraction of lymphocytes in lymph nodes of tumor burden mice was detected by flow cytometry. The apoptosis signals of macrophages in lymph nodes were observed with TUNEL staining. The expressions of Fas ligand of Hca F and Hca P cells in the mice were examined by immunohistochemistry. Results: With the presence of the extraction of lymph note, the quantity of MMP 9 significantly increased (P< 0.01): in RPMI1640 medium, the quantity of MMP 9 produced by Hca F and Hca P were 1256±157 and 2642±385, respectively. After the addition of extraction of lymph node , the quantity of MMP 9 increased to 12403±894 and 9086±686,respectively, together with the secretion of a large amount of MMP 2 (Hca F:7364±2001,Hca P:2997±1990) and active MMP 9 (Hca F:7297±1657,Hca P:3914±1253), and the amount of which was also more in Hca F than in Hca P cells(P< 0.05). There was no MMPs detected when the extraction of liver and spleen is present. The growth fraction of lymphocytes was as follow: in the Hca F cells, the proliferating peak of lymphocytes appeared on the 14th day post inoculation and then decreased rapidly, while in the Hca P cells , the peak appeared on the 7th day post inoculation and then kept at a high level. TUNEL staining showed that the positive signals of macrophages were detected around Hca F cells. The expression of Fas ligand of Hca F cells was stronger than that of Hca P cells (P< 0.01). Conclusion: The MMPs secretion of Hca F and Hca P cell depend on the environment of lymph nodes. The increased expression of Fas ligand protein in Hca F tumor cells with high lymphogenous metastatic potential in lymph nodes may help tumor cells escape from being killed by host lymphocytes.