【作者】 王顺祥； 高峰； 宋西进； 吴晓慧； 彭利； 唐瑞峰； 张萌； 肖燕； 张风瑞；

【Author】 Wang Shun-Xiang Gao Feng Song Xi-Jin Department of Hepatobiliary Surgery, The Fourth Hospital of Hebei medical university & Hebei provincial tumor hospital, Shijiazhuang, 050011, China

【机构】 河北医科大学第四医院暨河北省肿瘤医院肝胆外科；

【摘要】 目的:探讨肿瘤坏死因子-α(TNF-α)及白细胞介素-1 β(IL-1 β)对人肝癌细胞系乙酰肝素酶(Hpa)表达的影响。方法:肝癌细胞株SMMC-7721分对照组和试验组。对照组细胞常规培养,试验组细胞分别施加TNF—α:1U／ mL、10 U／mL、102U／mL、103U／mL、104U／mL,IL— 1 β:0．1 ng／ml、0．5 ng／ml、1．0 ng／ml、5．0ng／ml、 10ng／ml,分别培养12h、24h、48h后收获细胞。在相差显微镜下观察细胞形态变化,四甲基偶氮唑蓝(MTT)比色法测定细胞增殖活性,RT—PCR半定量法测定细胞Hpa mRNA 的表达,Western-blot法测定细胞Hpa蛋白的表达,以?- actin作为内参照标准,将Hpa与?-actin比值作为Hpa表达水平的参数,对Hpa产物相对定量。结果:1)与正常对照组相比,不同浓度的TNF-α、IL-1 β作用后,SMMC-7721 细胞形态无明显变化。细胞增殖活性比较,当药物浓度增加至TNF-α为103U／ml、IL-1 β为5．0ng／ml时,其吸光度值(A值)较对照组明显降低,差异有显著性意义(P<0．05),随各浓度TNF-α、IL-1β作用时间延长,SMMC-7721细胞形态亦无明显变化,但细胞增殖活性逐渐降低(P<0．05),2)在同一时间内,TNF-α在10U／mL、102U／mL、103U／mL浓度组,SMMC-7721细胞Hpa mRNA表达较对照组均明显升高,且随TNF-α浓度的增加,Hpa mRNA表达增加(P <0．05),在各浓度组,随时间的延长,SMMC-7721细胞Hpa mRNA表达差异无统计学意义(P>0．05)。IL-1 β在0．5 ng／ ml、1．0 ng／ml、5．0ng／ml浓度组,SMMC-7721细胞Hpa mRNA表达在同一时间内较对照组均明显升高,且随浓度的增加而增加(P<0．05),但不存在时间依赖性(P>0．05)。3)在同一时间内,TNF—α在10U／mL、102U／mL、103U／mL浓度组,SMMC-7721细胞Hpa蛋白表达量较对照组均明显升高,且随TNF-α浓度的增加,Hpa蛋白表达增加(P<0．05), 在各浓度组,随时间的延长,SMMC-7721细胞Hpa蛋白表达量增加,差异有显著性(P>0．05)。IL-1 β在0．5 ng／ml、1． 0 ng／ml、5．0ng／ml浓度组,SMMC-7721细胞Hpa mRNA 表达在同一时间内较对照组均明显升高,且随浓度的增加 Hpa蛋白表达量增加(P<0．05),而且随时间的延长而增加(P <0．05)。结论:TNF-α及IL-1β能够在核酸和蛋白水平上促进肝癌细胞合成并分泌Hpa,从而间接促进肿瘤的侵袭和转移。更多还原

【Abstract】 Objective: To investigate the effect of Tumor Necrosis Factor- α (TNF- α) and Interlukin-1 β (IL-1 β) on heparanase(Hpa) Expression in Hepatic Cancer Cell Line SMMC-7721. Methods: SMMC-7721 cells were divided as control group and experiment group.The former were cultured with normal condition, the later were treated by TNF- α or IL-1 β with various concentration and time. All of the cells were collected on time. The morphological changes of SMMC-7721 cells were studied microscopically and the proliferation of SMMC-7721 were detected by methyl thiazoly tetrazolium(MTT). The ex- pression of Hpa mRNA level in cultured SMMC-7721 was evaluated by relative quantitative reverse transcription polymerase chain reaction(RT-PCR). The Hpa protein level in cytoplasm was evaluated by western blot Results: Compared with concrol group,there was a little morphological changes in SMMC-7721 with TNF- α or IL-1 β treatment, and no chang of cell proliferation in corresponding time until the TNF- α concentration up to 103U/ml or the IL-1 β concentration up to 5.0ng/ml. But the cell proliferation in corresponding concentration were obviously different with time extension. The expression of Hpa mRNA was enhanced gradually with the TNF- α or IL-1 β concentration increasing. There was a dependence on the concentration when the concentration of TNF-α was lower than or equal to 103U/ml, or the concentration of IL-1 β was lower than or equal to 5.0ng/ml. Furthermore, the effect of promotion was close to peak when the TNF-α concentration up to 103U/ml or the IL-1 β concentration up to 5.0ng/ml,but no time-effect pattern observed. The Hpa protein level in cytoplasm was elevated gradually with the concentration increasing, too. Both the concentration effect pattern and the time-effect pattern were obvious. Conclusion: TNF- α and IL-1 β can increase the expression of Hpa at level of mRNA and protein in hepatic cancer cell line.更多还原