【作者】 杨成梁；

【导师】 樊青霞； 张明智；

【作者基本信息】 郑州大学 ， 肿瘤学， 2007， 硕士

【摘要】 背景和目的：食管癌是常见的消化道恶性肿瘤之一，由于早期诊断率低，既使手术切除，预后仍较差，全世界每年约有30万人死于此疾病。寻找有效的化疗药物成为食管癌治疗领域研究的热点。维甲酸是一大类化合物，包括维生素A及其代谢产物，和具有类似结构或生物活性的天然及人工合成的衍生物。维甲酸可在人体的正常生理活动中发挥重要的功能，如生长、胚胎形成、视觉、免疫等。大量研究证实，维甲酸对多种恶性肿瘤有诱导分化、抑制增殖、诱导凋亡等作用，在皮肤鳞癌、颈癌、乳腺癌等多种恶性肿瘤中已进人临床研究阶段，促进了抗肿瘤治疗的研究。维甲酸类药物调节细胞增殖、分化及凋亡从而达到抑制肿瘤细胞生长的分子机制仍未完全阐明。ATRA是否可能成为治疗食管癌的有效药物?为了回答这一问题，本实验在体外实验中观察ATRA对食管癌细胞系EC109的作用，研究ATRA是否对食管癌细胞系EC109具有增殖抑制作用，并进一步探讨ATRA在体外诱导食管癌细胞凋亡的作用及其作用过程中相关基因表达的变化，为其进一步研究提供实验依据。材料与方法：（1）MTT法测定ATRA对EC109细胞增殖的影响常规培养人食管癌细胞系EC109细胞，将对数生长期的EC109细胞调整细胞浓度为1×10⁴／mL，待培养24h细胞贴壁生长后分为对照组和实验组，对照组加入RPMI-1640培养液，实验组加ATRA至终浓度分别为10μmol／L、5μmol／L、1μmol／L、0.5μmol／L、0.1μmol／L，每一浓度设6个复孔。分别于加药后培养24h、48h、72h、96h检测各组细胞增殖抑制率的差异。（2）流式细胞仪检测细胞凋亡及周期分布常规培养人食管癌细胞系EC109细胞，对照组加入RPMI-1640培养液，实验组加入ATRA使其终浓度为1μmol／L。分别于更换新培养液后24h、48h、72h、96h收集各对照组和实验组的3瓶EC109细胞，用流式细胞仪检测细胞凋亡及周期分布情况。（3）RT-PCR法检测凋亡相关基因mRNA水平常规培养人食管癌细胞系EC109细胞，对照组加入RPMI-1640培养液，实验组加入ATRA使其终浓度为1μmol／L。分别于更换新培养液后24h、48h、72h、96h收集各对照组和实验组的3瓶EC109细胞，采用RT-PCR技术检测凋亡相关基因bcl-2和bax mRNA表达情况。（4）实验数据经SPSS11.0统计软件处理，采用单因素方差分析，以α=0.05为差异显著性标准。结果：（1）ATRA对EC109细胞的抑制作用呈现明显的剂量依赖效应关系和时间依赖效应关系，实验组与对照组之间及相邻实验组之间的A值和抑制率相比差异有统计学意义（P＜0.05）。（2）EC109细胞经过1.0μmol／L的ATRA作用后，与对照组相比细胞出现凋亡峰，细胞周期发生明显变化。随药物作用时间的增加凋亡率增高，G₀／G₁期细胞比例增高，S+G₂／M期细胞比例下降，实验组各时间点与对照组的比较和相邻实验组间的比较，差异均有统计学意义（P＜0.05）。（3）EC109细胞经过1.0μmol／L的ATRA作用后，随着药物作用时间的不断增加，bcl-2基因的mRNA水平表现出明显的逐渐减弱趋势，而bax基因的mRNA水平则随着药物作用时间的不断增加表现出明显的逐渐增强趋势，实验组与对照组及相邻实验组间半定量结果差异有统计学意义（P＜0.05）。结论：1．全反式维甲酸对人食管癌细胞株EC109具有明显的增殖抑制作用。2．全反式维甲酸可以诱导人食管癌细胞株EC109发生凋亡。3．全反式维甲酸诱导人食管癌细胞株EC109凋亡的分子机制可能与下调bcl-2基因转录和上调bax基因转录有关。更多还原

【Abstract】 Background and Objective:Esophageal cancer is a common gastrointestinal cancer. Owing to the low rate of early diagnosis, the prognosis remains poor even if surgery. About 0.3 million people died from the disease annually throughout the world. To find effective chemotherapy treatment of esophageal cancer drugs have become a hot research field. Retinoic acid is a major compounds, including vitamin A and its metabolites, with a similar structure and biological activity of natural and synthetic derivatives. Retinoic acid plays an important role in the normal physiology of human activities, such as the growth of embryo formation, visual and immunization. Numerous studies have shown that retinoic acid affects a variety of malignant tumors including induction of differentiation, inhibition of proliferation and induction of apoptosis, and so on. Squamous cell carcinoma of the skin, cervical cancer, breast cancer and other malignant tumors had entered clinical study phase, which promoted anti-tumor therapy. Retinoic acid drugs regulate cell proliferation, differentiation and apoptosis thereby inhibit tumor cell growth ,the mechanisms of which are still not fully explained. Whether ATRA may be an effective drug on the treatment of esophageal cancer or not? To answer this question, the experiments observing ATRA on esophageal cancer cell line EC109 in vitro study whether ATRA inhibit the esophageal cancer cell line EC109 and induce apoptosis or not, in the process of which the gene expression changes are detected and provide experimental evidence for further study.Materials and Methods: (1) The cell proliferation activity was detected by MTT assay after treated with ATRA The human esophageal cancer cell line EC 109 was cultured conventionally to log period and adjusted to concentration of 1×10~4/mL. EC 109 cell in adherent growth was divided into control and experimental groups 24 hours later. The control group was cultured with RPMI-1640 medium and the experimental groups was cultured with ATRA concentration of 10μmol/L、5μmoI/L、1μmol/L、0.5μmol/L and 0.1μmol/L etc, each of which had six repeated holes. The differences of cell proliferation inhibition rate between different groups was detected after cultured with ATRA 24h、48h、72h and 96h etc.(2) The cell apoptosis and cell cycle distribution was detected by flow cytometry machine The human esophageal cancer cell line EC 109 was cultured conventionally. The control group was cultured with RPMI-1640 medium and the experimental groups was cultured with 1μmol/L ATRA. Three bottles of EC109 cells was collected from the control and experimental groups respectively after replacing the new medium 24h、48h、72h and 96h. The cell apoptosis and cell cycle distribution was detected by flow cytometry machine.(3) The apoptosis-related gene mRNA levels were detected by RT-PCR The human esophageal cancer cell line EC109 was cultured conventionally. The control group was cultured with RPMI-1640 medium and the experimental groups was cultured with 1μmol/L ATRA. Three bottles of EC109 cells was collected from the control and experimental groups respectively after replacing the new medium 24h、48h、72h and 96h. The mRNA levels of apoptosis-related gene bcl-2 and bax were detected by RT-PCR technology.(4) Experimental data are disposed by SPSS 11.0 statistics soft ware, using single-factor analysis of variance , with significance of difference standard :α=0.05.RESULTS:(1) The inhibition effects of ATRA on EC109 cells were significantly dose-dependent and time-dependent. The difference of value A and inhibition rate between experimental groups and the control group and the adjacent experimental groups was statistically significant (P< 0.05) .(2) Compared with the cell of control group, there were apoptosis peak and significant cell cycle change in experimental groups after disposal by ATRA of 1.0μmol/L. With the disposal time increasing , the apoptosis rate increased ,G0/G1 cells ratio increased and S+G2/M phase cell ratio decreased. The difference of between experimental groups and the control group and the adjacent experimental groups was statistically significant (P<0.05) .(3) With the disposal time increasing ,the mRNA level of bcl-2 gene showed a significant gradual weakening trend but the mRNA level of bax gene showed a significant gradual increasing trend after EC109 cell disposal by ATRA of 1.0μmol/L. The difference of semi-quantitative results between and the control group and adjacent experimental groups were statistically significant (P<0.05) .CONCLUSION:1. ATRA inhibits the proliferation of human esophageal cancer cell line EC109 significantly.2. The apoptosis in EC109 cells could be induced by ATRA.3. The molecular mechanism of inducing apoptosis of EC109 may be related to down-regulation of bcl-2 transcription and up-regulation of bax transcription.更多还原