【作者】 张雪琛；

【导师】 高亚杰；

【作者基本信息】 大连医科大学 ， 肿瘤学， 2009， 硕士

【摘要】 目的:研究盐酸洛拉曲克对体外培养人肝癌细胞HepG-2的增殖抑制作用和对肝癌细胞HepG-2凋亡抑制基因Bcl-2表达的影响。方法:MTT比色法观察盐酸洛拉曲克对人肝癌细胞株HepG-2增殖的抑制作用;用免疫细胞化学染色法检测其对人肝癌细胞HepG-2凋亡抑制基因Bcl-2的表达影响。结果:1、实验前HepG-2细胞为贴壁的梭形细胞,细胞排列紧密,生长增殖速度快,每2～3天需传一代;在加入化疗药物盐酸洛拉曲克12h后镜下观察细胞形态变为圆形,体积变小,细胞密度增加和细胞固缩,细胞膜和核膜完整,线粒体发生超浓缩和染色质进行性固缩,并向核周“崩溃”形成一个或多个块状结构、“致密球体”或向外“发芽”形成葡萄串样小球体。24后观察镜下视野模糊,细胞膜损伤﹑肿胀﹑裂解。2、肿瘤细胞体外药物敏感性检测即MTT法,试验设立三组药物即(ADM组、DDP组、盐酸洛拉曲克组)每组药物设立四个浓度梯度,同时设立空白对照组,结果显示,盐酸洛拉曲克对人肝癌细胞HepG-2的生长具有显著的抑制作用,促进肿瘤细胞的凋亡,高浓度组的生长抑制率可达80.15%,具有良好的量效关系,其中高浓度给药组的抑瘤率最高,其次是中浓度组,对每个瘤株均重复实验3次,实验结果的重复性比较好。当各组药物浓度达到最大时盐酸洛拉曲克组与阳性对照组(ADM组和DDP组)比较其肿瘤细胞的抑制率分为80.15%、48.12%、51.69%,差异有统计学意义(P=0.013,x2检验)。3、免疫细胞化学染色结果显示,对不同浓度盐酸洛拉曲克作用的肝癌HepG-2细胞Bcl-2的表达进行检测,未用药物干预组(对照组)阳性细胞呈棕黄色,染色较深,颗粒分布密集,低浓度组(2μg/ml)与对照组相比未见明显区别,中浓度组(20μg/ml)与高浓度组(200μg/ml)较对照组阳性细胞呈淡染,其中高浓度组尤为明显,颗粒分布疏松。随着盐酸洛拉曲克浓度的递增,凋亡指数逐渐增加,即肿瘤细胞迅速减少,而Bcl-2阳性细胞表达率逐渐下降。当药物浓度达到最大时(200μg/ml),凋亡指数最大达48.95±3.32,而Bcl-2的阳性表达率最低为10.62±1.21,同时与对照组比较P<0.05,具有统计学意义。结论:1、盐酸洛拉曲克对人肝癌细胞HepG-2的生长具有显著地抑制作用,其单药组生长抑制率明显高于ADM组和DDP组,且高剂量组抑制率明显高于其它剂量组,具有良好的量效关系。2、盐酸洛拉曲克能下调凋亡抑制基因Bcl-2的表达,促进肿瘤细胞的凋亡。更多还原

【Abstract】 Objective: Study nolatrexed hydrochloride on cultured human hepatoma cells HepG-2 inhibition of proliferation of hepatoma cells and HepG-2 apoptosis suppressor gene Bcl-2 expression.Methods: MTT colorimetric observation nolatrexed hydrochloride on human hepatoma cell line HepG-2 inhibition of proliferation; by immune- ocytochemical staining of human hepatoma cells HepG-2 apoptosis suppressor gene Bcl-2 expression affected .Results: 1. Before the experiment for the HepG-2 cell adherent of the spindle cells, cells with close cell growth speed, every 2 to 3 days to be mass generation; in hydrochloric acid by adding chemotherapy after 12h nolatrexed cell morphology were observed to round, smaller size, cell density and cell shrinkage, cell membrane and nuclear membrane integrity, mitochondria occurred concentrate and ultra-sexual chromatin condensation, and nuclear Week "collapse" to form one or more massive structure, " dense sphere, "or outside" germination a strand of grape the formation of small spherical samples. Observed after 24 endoscopic vision blurred, the membrane swelling﹑cracking﹑damage.2. Tumor cells in vitro drug susceptibility testing that is, MTT, to experiment with three groups of drugs that (ADM group, DDP group, hydrochloric acid nolatrexed group) to set up four in each group of drug concentration gradient, while the establishment of the control group, results showed that hydrochloric acid nolatrexed of human liver cancer cell growth of HepG-2 significantly inhibited to promote apoptosis of tumor cells, high concentration of growth inhibition rate of 80.15%, with a good dose-effect relationship, in which a high concentration to inhibition rate of the drug group, followed by concentration group for each experimental tumor lines are repeated 3 times, the repeatability of results is good; when the group reaches the maximum when the drug concentration nolatrexed hydrochloride group and positive control Group (ADM group and DDP group) compared to the inhibitory rate of tumor cells is divided into 80.15 percent, 48.12 percent, 51.69 percent, the difference was statistically significant (P = 0.013, test)3.Experiment of cell-mediated immunity, in accordance with hydrochloric acid concentration nolatrexed incremental administration, a significant increase in apoptotic index, that is, the rapid reduction of tumor cells, and Bcl-2 positive rate decreased significantly. When the drug reaches the maximum when the concentration (200μg/ml), apoptotic index up to 48.95±3.32, while the Bcl-2 positive expression rate of a minimum of 10.62±1.21, compared with the control group P <0.01, with statistical significance. Hydrochloric acid showed that in vitro nolatrexed of HepG2 human hepatoma cell proliferation inhibition are strong, can promote tumor cell apoptosis, reduced apoptosis inhibitory gene Bcl-2 expression.Conclusion: 1.Nolatrexed hydrochloride on human hepatocellular carcinoma HepG-2 with the significant growth inhibition, and its single-drug group was significantly higher than the rate of growth inhibition ADM group and DDP group, and the inhibitory rate of high-dose group was significantly higher than other dose groups, has a good dose-effect relationship.2.Nolatrexed hydrochloride on the in vitro proliferation of HepG-2 hepatoma cells have a stronger inhibitory effect can be reduced inhibitor of apoptosis Bcl-2 gene expression and promote apoptosis of tumor cells.更多还原