【作者】 刘永安；

【导师】 李兵； 修清玉； 石昭泉； 陈吉泉； 方正；

【作者基本信息】 第二军医大学 ， 内科学， 2008， 硕士

【摘要】 【研究背景及目的】据世界卫生组织（WHO）预测:至2025年,我国患肺癌人数将居世界之首,每年新增的肺癌病死例数将超过100万。目前治疗肺癌的主要方法是多学科综合治疗,主要包括手术、化疗、放疗及生物靶向治疗等;这些治疗均是通过不同途径杀伤肿瘤细胞达到治疗目的,但效果不尽如人意,在恶性肿瘤中的肺癌病死率是最高的,5年生存率在我国仅为10%左右。寻找有效的治疗药物和治疗手段是摆在人们面前的一项迫不及待的任务。三氧化二砷是中药砒霜的有效成份,过去中药利用其“以毒攻毒”的原则来治疗牙髓病以及银屑病、梅毒、风湿等疾病,因为有剧毒,临床应用范围十分狭窄;然而自从20世纪七十年代三氧化二砷被用来治疗急性早幼粒细胞性白血病并且获得了较高的缓解率以来;三氧化二砷的抗肿瘤作用同益引起国人的重视。三氧化二砷不仅对急性早幼粒细胞性白血病有效果,对其它类型的白血病以及其他许多恶性肿瘤,如胃癌、前列腺癌、乳腺癌等都有一定的治疗效果。肿瘤的产生与细胞分化异常有关;三氧化二砷可以诱导急性早幼粒细胞分化成为成熟粒细胞,通过阻滞细胞周期影响细胞生长与分化,达到抑制肿瘤的作用;诱导分化治疗副作用小,疗效不比常规化疗差,因此诱导肿瘤分化正成为治疗肿瘤的一种新的热点。如果三氧化二砷对肺癌细胞也有一定的诱导分化作用,则有可能转变肺癌的治疗模式。目前国内外有相关研究证实三氧化二砷对肺癌细胞有一定的细胞周期阻滞和诱导凋亡作用,但研究结果不尽相同,有报道三氧化二砷可以使肺癌细胞周期阻滞于G₂/M期也有报道三氧化二砷可以使肺癌细胞周期阻滞于G₁期;细胞周期中G₁期称为细胞分化期,G1期停滞可作为细胞分化的指标,若能使细胞周期阻滞于G₁期,则有可能诱导肺癌细胞分化。与其它细胞毒性药物不同的是,三氧化二砷在部分低氧的实体肿瘤组织中作用仍较明显;而且低氧可以加强三氧化二砷对白血病的诱导分化作用。低氧及厌氧环境对三氧化二砷肺癌细胞周期阻滞作用将产生怎样的影响呢?是否更有利于肺癌细胞分化呢?依托泊苷（VP-16）是白血病和肺癌治疗中常用的药物,可以使肿瘤细胞周期阻滞于G2/M期,低浓度的VP—16能通过丝裂原活化蛋白激酶（MAPK）途径诱导慢性粒急性变的K562细胞分化。本实验首先在常氧环境下利用三氧化二砷对肺癌A549细胞体外诱导培养,与细胞周期特异性药物依托泊苷（VP-16）作对照,研究三氧化二砷在常氧环境下对肺癌A549细胞周期的作用特点;然后在微缺氧（5%）、厌氧环境观察缺氧对其细胞周期阻滞作用的影响;从G1期阻滞角度来探讨三氧化二砷对肺癌A549细胞可能存在的诱导分化作用,为临床上三氧化二砷治疗肺癌提供理论依据。【实验方法】一、实验分组:根据处理药物不同,将消化后的细胞随机分成1μmol/L三氧化二砷与1.0μmol/L VP-16及空白对照组3个组,接种于24孔板;每组设6个复孔,每孔细胞接种密度约为3*10⁵个/孔。二、细胞培养:分别在常氧环境下体外诱导培养12小时、24小时、48小时;微缺氧、厌氧环境下体外诱导培养24小时,采用厌氧袋发生法制造微缺氧及厌氧环境。三、细胞收取:在各种氧环境下诱导培养至相应时段以后,用含0.25%胰蛋白酶消化液消化各组细胞,收集、离心、弃上清后PBS洗涤、离心、弃上清两次,加入70%冰乙醇固定,4℃过夜。四、流式细胞仪（FCM）检测细胞DNA含量:固定过夜后的细胞,加入PBS振荡洗涤后离心弃上清,加入碘化丙啶（PI）染色,室温避光孵育后进行流式细胞仪检测;每个流式细胞管检测细胞数不低于8000个,低于G₁期DNA含量主峰（亚G₁期峰）的细胞为凋亡细胞。五、细胞周期分析:采用CellQuest软件进行细胞周期分析,结果以直方图进行表示;并计算出各期细胞比例。六、统计学分析:细胞周期分析结果为百分比形式,以均数±标准差表示,采用SPSS13.0统计软件进行分析,具体分析方法详见各部分。【结果】（一）、常氧环境As₂O₃对肺癌A549细胞周期分布及细胞凋亡的作用1、随作用时间的延长,1μmol/L As₂O₃组G1期细胞比例增加,S期细胞比例减少,作用48小时,G₁期细胞比例为47.04%,S期细胞比例为50.54%;与空白对照组相比较,G₁期细胞比例明显增加（P＜0.05）,S期细胞比例明显减少（P＜0.05）;与VP-16组比较:G₁期细胞比例无明显差异,S期细胞比例较高（P＜0.05）,G2/M期细胞比例明显减少（P＜0.05）。2、1μmol/L As₂O₃组各时段细胞凋亡率与空白对照组相应时段均无明显差异,但各时段均较同等浓度VP-16组明显减少（P＜0.05）。（二）、不同氧环境As₂O₃对肺癌A549细胞周期分布及细胞凋亡的作用1、随缺氧加重,1μmol/LAs₂O₃组G₁期细胞比例增加,S期细胞比例减少,与常氧、微缺氧环境相比,厌氧环境G₁期、G₂/M期细胞比例显著增加（P＜0.05）S期细胞比例显著降低（P＜0.05）;与空白对照组比较,微缺氧环境G₁期、S期细胞比例无明显差异,厌氧环境G₁期细胞比例明显增加（P＜0.05）、S期细胞比例明显减少（P＜0.05）;与VP-16组相比较:三种氧环境下各期细胞比例均有明显差异,G₁期、G2/M期细胞比例减少（P＜0.05）,S期细胞比例增加（P＜0.05）。2、与1μmol/LVP-16组相比较,As₂O₃组各种氧环境下细胞凋亡率均明显减少（P＜0.05）;与空白对照组相比较,常氧及微缺氧环境无明显差异,厌氧环境反而降低（P＜0.05）。【结论】一、低浓度三氧化二砷对肺癌A549细胞G₁期的阻滞作用具有一定的时-效效应;随缺氧加重,G₁期阻滞作用增强;低氧环境下延长作用时间有可能诱导A549细胞分化。二、低浓度三氧化二砷对肺癌A549细胞周期的特异性阻滞作用不及同一浓度VP-16强,对A549细胞无明显诱导凋亡作用。更多还原

【Abstract】 【Backgrounds and Objective】The case fatality rate of lung cancer is highest among malignant tumor,five-year survial of which is only between 10%and 13%.According to the World Health Organnization prediction:to 2025,the number of lung cancer patients in our country will exceed one millon.The present tragedy of lung cancer is interdiscipline combined therapy, these approaches will attain the goal of therapy through different ways to kill tumor cells, but the consequent is not satisfying.A new direction for tumor treatment is differentiation therapy.The tumor is associated with cell abnormal differentiation,to induce tumor cell differentiation is a new focus on the tumor therapy.Arsenic trioxides（As₂O₃） can induce acute promyelocytic leukemia（APL） into mature granulocytes.The further study has find that it has effect not only on the APL but also on other types of leukemia,even on the solid tumors such as gastric cancer、lung cancer、prostatic carcinoma etal.It inhibits the tumor through influencing the growth and differentiation of tumor cells with different mechanism.Different with other cytotoxic drugs,it can even have apparent inhibitory effect in mionectic tumor tissues.But there has few study about As₂O₃ ’s effect on lung cancer cell line in mionectic surroundings.Our objective is to study the features of Arsenic trioxides ’ effect on the cell cycle of A549 cell lines in normoxia,explore the influence of hypoxia on the Arsenic trioxides’ effect on the cell cycle of A549 cell line.【Materials and Methods】1.Groups of the experiment:there are three cell groups in this study,which comprises 1μmol/L Arsenic trioxides and 1.0μmol/LVP- 16 and the blanket control.2.Cells cultivating:In normoxia microenviroment.theA549 cell lines in are cultivating for 12、24,48hours respectively.In hypoxia and anerobic microenviroment the cell lines are cultivating for 24 hours.3.Collect cells of different groups,and wash them twice with PBS,centrifuge with 1500RPM,fix them with 70%cold alcohol,put them in the refrigerator overnight.4.Dye the cells of different groups with PI solely,then put them in the normal environment for about thirty minutes away from light. 5.Investigate cells DNA contents of different groups respectively through flow cytometry;then analyze the cell cycle and apoptosis results with CellQuest software.6.Analyze the results with SPSS11.0.【Results】1.In normoxia microenvironment,Arsenic Trioxide can prolong A549 G₁ phase rate in cell cycle and decrease A549 S phase rate with the cultivating time increasing,after exploration for about 48 hours,Arsenic groups can make almost half of the cells arresting in the G1 phase,compared with the blanket control groups,the difference is significant （P＜0.05）;the S phase cell lines rate is less（P＜0.05）.Compared with the VP-16 groups, Arsenic trioxide’s effect onf G₁ phase S phase and G₂/M phase is less（P＜0.05）,and the apoptosis rate of Arsenic trioxide group is not apparent which is much lower than VP-16 （P＜0.05）.2.In hypoxia and anerobic Microenviroment,exploring for 24 hours,the G₁ phase rate is increasing with the severity of oxygen scarcity increasing in three groups;compared with the blanket control groups,the arrest effect on G₁ phase of Arsenic trioxide group is more apparent in anerobic enviroment（P＜0.05）,there is no apparent difference between them in nonnoxiaand hypoxia environment.Compared with the VP-16 groups,there are apparent difference between them in three oxygen microenvironment respectively,the G₁ phase and G₂/M phase rate decrease（P＜0.05）,and the S phase increase accordingly（P＜0.05）.【Conclusion】1.In normoxia microenviroment Arsenic trioxide has a time-dependent effect on the G1 phase arrest to some extent,In hypoxia and anerobic microenviroment,the G1 phase arrest effect of arsenic trioxide.becomes more apparent with the more scarcity of oxygen.but the effect of apoptosis is becoming weaker.2.Low concentration of arsenic trioxide has less apparent effect on A549 cell line cell cycle arrest and apoptosis than VP-16.更多还原