【作者】 邵应举；

【导师】 郑玉玲；

【作者基本信息】 郑州大学 ， 肿瘤学， 2010， 硕士

【摘要】 胃癌是常见的恶性肿瘤,在世界范围内最常见的恶性肿瘤中占第4位,死亡率占所有恶性肿瘤的第2位。在我国胃癌发病率和死亡率分别占恶性肿瘤的第1位和第3位。针对胃癌的治疗药物也层出不穷,短期内显示出良好的临床疗效。然而大规模的随访研究结果表明,肿瘤患者的远期生存率并没有因为化疗药物的使用而得到显著提高,而且有些化疗药物严重的毒副作用限制了它们在临床上的应用。探索低剂量多药联合应用,是肿瘤药物研究的一个重要方向。全反式维甲酸(all-trans retinoic acid, ATRA)是维生素A的主要代谢产物,是维持生长发育不可缺少的物质,是一种公认的诱导分化剂。奥沙利铂(Oxaliplatin, L-OHP)是第三代铂类抗癌化合物,主要用于晚期大肠癌的治疗。由于其与第一代顺铂和第二代卡铂均无交叉耐药性,并且耐受性好、毒副作用小,因而受到广泛重视。ATRA与L-OHP联合作用于胃癌是否能够提高疗效,目前未见报道。本实验从体外细胞学角度观察全反式维甲酸(ATRA)与奥沙利铂(L-OHP)对人胃癌BGC-823细胞凋亡的诱导作用,从而为进一步研究提供理论基础。1.用ATRA和L-OHP单独及联合作用于BGC-823细胞,倒置显微镜下观察细胞形态变化；2.采用MTT法检测ATRA及L-OHP分别作用24h、48h、72h后BGC-823细胞的增殖抑制率；3.采用流式细胞仪检测ATRA及L-OHP作用48h后BGC-823细胞的凋亡率及周期变化；4.采用免疫细胞化学技术检测ATRA及L-OHP作用48h后BGC-823细胞中Bcl-2和Survivin蛋白的表达情况。1.药物作用24h后,细胞间隙增宽,胞内颗粒增多,出现空泡；作用48h后,多数细胞漂浮于培养液中,少量细胞仍贴壁生长,并可见部分细胞的细胞核固缩、断裂成碎片、核膜消失,细胞膜完整且有突起,呈凋亡晚期改变；作用72h后,凋亡细胞数量逐渐增多。随着作用时间的延长,与对照组相比,实验组细胞碎片数量明显增多,贴壁细胞数量显著减少。2. ATRA对BGC-823细胞的抑制作用呈现时间依赖性,24h后对细胞的抑制作用不显著,48h和72h后抑制作用相对明显,与对照组相比差异有统计学意义(P<0.05)。联合用药后抑制作用更加显著,与ATRA组及L-OHP组相比差异有统计学意义(P<0.05),最佳作用时间为加药后48h。3. ATRA作用细胞后,G0/G1期和G2/M期的细胞比率增加,S期的细胞比率下降；L-OHP作用细胞后,G0/G1期的细胞比率下降,S期和G2/M期的细胞比率增加；联合用药后G0/G1期和G2/M期的细胞比率增加,S期的细胞比率下降；联合用药组与ATRA组、L-OHP组相比,凋亡率差异均有统计学意义(P<0.05)。4.对照组中可以见到细胞质中较多的Survivin蛋白和Bcl-2蛋白表达,呈浅黄色到棕黄色颗粒。ATRA组和L-OHP组阳性表达细胞数减少,而联合用药组的细胞表达数更少。联合用药组与ATRA组、L-OHP组相比,蛋白阳性率差异均有统计学意义(P<0.05)。1. ATRA对人胃癌BGC-823细胞存在增殖抑制作用,且具有时间依赖性,最佳作用时间为48h,与L-OHP联用后对BGC-823细胞的增殖抑制作用更显著。2. ATRA可诱导人胃癌BGC-823细胞分化,形成G0/G1期阻滞；L-OHP可促进细胞凋亡,形成G2/M期和S期阻滞。两者联用可发挥协同作用,加强对BGC-823细胞凋亡的促进作用。3. ATRA与L-OHP可诱导人胃癌BGC-823细胞凋亡,机制可能与下调Bcl-2蛋白和Survivin蛋白的表达有关。更多还原

【Abstract】 Gastric cancer is a common malignancy worldwide, accounts for the forth most common malignant tumors and the first two of the total cancer death rate. In our country,the incidence and mortality of gastric cancer account for the first 1 and 3 respectly. Medicines for the treatment of gastric cancer emerging in the short term showed good clinical efficacy. However, large-scale follow-up show that long-term survival of cancer patients has not increased significantly as the use of chemotherapy and serious side effects of some chemotherapy drugs limit their clinical application. Low-dose and multi-drug combinatioand is an important cancer drug research direction.All-trans retinoic acid (all-trans retinoic acid, ATRA) is the major metabolite of vitamin A,and is essential to maintain growth and development.It is a recognized differentiation inducer. Oxaliplatin (Oxaliplatin, L-OHP) is the third generation platinum anti-cancer compounds, mainly used for advanced colorectal cancer. It has no cross-resistance with the first generation cisplatin and the second generation carboplatin and its good tolerance, low toxicity, therefore it is recognized.Whether ATRA combined with L-OHP used in gastric cancer can increase efficacy has currently not reported. In this study, we observe the induction of apoptosis of retinoic acid (ATRA) comibined with oxaliplatin (L-OHP) in human gastric cancer BGC-823 cells in vitro observation of cellular, thus provide a theoretical basis for further study.1. Treat BGC-823 cells with ATRA、L-OHP respectively and then combined, observe the morphologieal changes of BGC-823 cells by inverted microscope.2. Assesse the proliferative inhibition rate of BGC-823 cells after treated with ATRA and L-OHP respectively for 24h,48h,72h by MTT assay.3. Analyze the changes of apoptosis rate and cell cycle of BGC-823 cells after treated with ATRA and L-OHP for 48h by flow cytometry(FCM).4. Detect the expressions of Bcl-2 and Survivin protein of BGC-823 cells after treated with ATRA and L-OHP for 48h by immunohistochemistry method.1.24h after treated with drugs, cell gaps widened, intracellular granules increased and became vacuoles.48h later,most of cells floated in the culture medium and few cells still shew adherent growth. The nucleus in some cells were pyknosis and broken.The nuclear membrane disappeard but the cell membrane kept integrity and grew apophysises.All of the changes shew the advanced stage of apoptotisis.72h later, the number of apoptotic cells increased.Compared with the control group,more cells became debris and the number of adherent cells reduced significantly in experimental groups.2. The proliferative inhibition effect of the cells treated with ATRA shew time dependence and the effect was not obvious at 24h.48h and 72h later,the effect was relatively obvious.Compared with the control group,the difference was statistically significant(P<0.05).The inhibition effect was more significant in the combined group. The best time of action was 48h.Compared with the ATRA group and L-OHP group,the difference was statistically significant(P<0.05). 3.48h after treated with ATRA, the rate of G0/G1 and G2/M phase cellls increased and the rate of S phase decreased. After treated with L-OHP, the rate of G0/G1 phase declined, while phase S and G2/M increased.In the combined group, the rate of G0/G1 phase and G2/M phase cells increased and S phase decreased. The differences of the apoptosis rate among the three groups was statistically significant(P<0.05).4. Among the four groups,the expression of Survivin protein and Bcl-2 protein in cytoplasm were the most in the control group and the least in the combined group.The differences of the positive rate among the four groups was statistically significant (P<0.05).1. ATRA could inhibit the proliferation of human gastric cancer BGC-823 cells and the effect shew time dependence.The best results appeared at 48h. Associated with L-OHP, the proliferative inhibition effect of the cells reinforced.2. ATRA could induce the differentiation of human gastric cancer BGC-823 cells and arouse G0/G1 phase arrest.L-OHP could promote cell apoptosis and arouse G2/M phase and S phase arrest.The effect of inducing apoptosis were enhanced when ATRA and L-OHP were combined.3. ATRA and L-OHP could induce the apoptosis of human gastric cancer BGC-823 cells and the probable mechanism might be related to the down regulation of the expression of Bcl-2 and Survivin protein.更多还原