CORELATION BETWEEN HEPATIC SINUSOIDAL ENDOTHELIAL CELLS AND HUMAN COLORECTAL CARCINOMA CELLS IN LIVER METASTASIS

肝窦内皮细胞与人结直肠癌细胞肝转移的相关性

• 批准号: 09671218
• 负责人: SUGIHARA Kenichi
• 金额: $ 1.98万
• 依托单位: Tokyo Medical and Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671218/
• 关键词: LIVER; METASTASIS; CARCINOMA; 大腸癌; 肝転移; 肝類洞壁内皮細胞

The liver is the major site for metastasis by colorectal carcinoma (CRC) and may have different mechanisms that inhibit CRC growth. We have used a tumor cell-sinusoidal endothelial cell (SEC) coculture system to evaluate whether SEC are cytotoxic to weakly or highly metastatic CRC cells. SEC were isolated from the livers of normal Swiss mice by a portal vein enzymatic infusion method and established as monolayers in 96 well microtiter plates. Confluent SEC monolayers contained 93% endothelial cells (by low density lipoprotein (LDL) receptor staining) and 7% Kupffer cells. When CRC cells were prelabeled with the vital fluorescent dyes rhodamine-dextran and calcein AM and then cocultured with confluent SEC monolayers to assess viability, the % metabolic activity of Clone A cells, a weakly metastatic CRC, was significantly lower than that of CX-1 cells, a highly metastatic CRC, after 4 hrs of coculture (p<0.05). Pretreatment of SEC gadolinium chloride (GaClィイD23ィエD2), an inhibitor of Kupffer cell function, did not block the effect of SEC on Clone A cell. After 24 hrs of coculture with Clone A, SEC produced 114±5.0 nM nitrite vs 123±5.0 and 8±2.0 for SEC and Clone A cells cultured alone, respectively. Furthermore, when 10ィイD1-6ィエD1 to 1 mM NィイD1GィエD1-methyl-L-arginine (NMMA) was added to SEC and Clone A cocultures, toxicity to Clone A cells was blocked as nitrite production was inhibited by dose greater than 10ィイD1-2ィエD1 mM. Unstimulated murine SEC are more toxic to the weakly metastatic Clone A cells than highly metastatic CX-1 cells. Thus, hepatic SEC may be a major host effector cell population that eliminates weakly metastatic CRC through the production of nitric oxide.

肝脏是结直肠癌（CRC）转移的主要部位，可能有不同的机制抑制结直肠癌的生长。我们使用肿瘤细胞-窦状内皮细胞（SEC）共培养系统来评估SEC是否对弱或高转移性CRC细胞具有细胞毒性。采用门静脉酶灌注法从正常瑞士小鼠肝脏中分离出SEC，并在96孔微滴板上形成单层。合流SEC单层含有93%的内皮细胞（低密度脂蛋白（LDL）受体染色）和7%的库普弗细胞。用重要荧光染料罗丹明-葡聚糖和钙黄蛋白AM对CRC细胞进行预标记，然后与SEC单层共培养，共培养4小时后，弱转移性CRC克隆A细胞的%代谢活性显著低于高转移性CRC CX-1细胞（p<0.05）。SEC氯化钆的预处理(GaClィイc15ィエD2),枯氏细胞功能的抑制剂,并没有阻止秒克隆细胞的影响。与克隆A共培养24 h后，SEC产生的亚硝酸盐为114±5.0 nM，而SEC和克隆A单独培养的亚硝酸盐分别为123±5.0 nM和8±2.0 nM。此外，当SEC和克隆A共培养物中添加10 μ l- D1-6 μ l- D1至1 mM N μ l- D1-甲基精氨酸（NMMA）时，亚硝酸盐的产生被10 μ l- D1-2 μ l- D1 mM的剂量抑制，对克隆A细胞的毒性被阻断。未受刺激的小鼠SEC对弱转移的克隆A细胞的毒性比高转移的CX-1细胞更大。因此，肝SEC可能是通过产生一氧化氮来消除弱转移性结直肠癌的主要宿主效应细胞群。