【作者】 陈文玲；

【导师】 朱严冰；

【作者基本信息】 河北医科大学 ， 肿瘤学， 2008， 硕士

【摘要】 目的:喉癌是危害人类健康和生命的恶性肿瘤,手术及放疗为其主要治疗手段,但手术后患者喉失去原有的发音功能,很大程度影响喉癌病人的生活质量及生存率。近年来随着化疗水平的提高,化疗在喉癌的综合治疗中越来越占有重要的地位,比如喉癌的新辅助化疗,其目的在于缩小肿瘤、使临床分期提前、与放疗或喉部分切除术结合尽可能保留喉的发音功能。喉癌因位置隐蔽,发现时往往已到中晚期,晚期喉癌或经手术后出现复发的患者再次手术较为困难,单纯放疗只针对局部病灶,难以收到理想的效果,临床上即给予化疗,以控制肿瘤的全身转移,缓解症状,延长生命。铂类是头颈部化疗的基础用药,以顺铂(cDDP)为基础的化疗在头颈部肿瘤治疗中研究较多,并取得了一定的疗效,由于顺铂严重的肾毒性和消化道副作用限制了它在临床的应用,因此开发高效低毒的铂类制剂一直是学者们研究的方向。洛铂是第三代铂类衍生物,洛铂以其较轻的毒副作用、与顺铂不完全交叉耐药以及较强的抗肿瘤活性,已初步显示了其在肿瘤治疗中的重要作用。本实验以喉癌细胞株Hep-2为模型,采用倒置显微镜、MTT比色法、流式细胞技术检测洛铂对喉癌细胞的增殖、周期阻滞、诱导细胞凋亡等作用,应用免疫细胞化学法检测凋亡相关蛋白bcl-2的表达,初步探讨洛铂对喉癌细胞的增殖抑制、细胞周期改变、诱导细胞凋亡及凋亡相关蛋白表达等方面的作用,为洛铂在头颈部肿瘤化疗中的应用提供实验依据。方法:1以对数生长期的喉癌细胞进行实验,将实验分组为:①空白对照组(培养液);②阴性对照组(细胞+培养液);③实验组(细胞+不同浓度洛铂+培养液);将各组分别培养24、48、72小时。2倒置显微镜下观察细胞生长情况及细胞形态学改变。3应用MTT比色法检测洛铂对喉癌细胞的增殖抑制情况。4应用流式细胞仪检测细胞周期和细胞凋亡率的改变。5应用免疫细胞化学法(S-P法)检测bcl-2蛋白表达的变化。6统计学处理:采用SPSS13.0统计软件包进行统计学处理。数据分析时先行正态性检验,符合正态分布计量资料用均数±标准差(x+s)表示。组间比较先进行方差齐性检验,符合方差齐性后采用单因素方差分析的方法进行,如差异有显著性,进一步LSD法进行两两比较。两样本均数的比较用t检验,显著性水平以p﹤0.05为有统计学意义。结果:1相差显微镜下每日观察对照组和各实验组细胞大小和形态的变化。阴性对照组Hep-2细胞为上皮样,贴壁生长于培养基质,轮廓清晰,细胞间结构紧密,生长旺盛。5μmol/L洛铂作用48h后可见少量细胞体积增大、变圆,仍贴壁生长。20μmol/L时贴壁生长细胞逐渐减少,变圆细胞逐渐增多,脱壁漂浮的小圆球细胞及细胞碎片逐渐增多,到80μmol/L高浓度药物作用后贴壁生长细胞极少,出现成堆漂浮的死亡细胞及细胞碎片2洛铂对细胞增殖的影响:不同浓度洛铂在同一时间A值与对照组比较, 5μmol/L洛铂作用24小时与对照组无统计学意义,其余各组与对照组比较差异均有统计学差异(p<0.05)。同一浓度洛铂作用24、48、72小时比较差异均有统计学意义(p<0.05)。洛铂能明显抑制喉癌细胞的增殖,抑制率随着药物浓度的增加而增加,有剂量依赖关系;抑制率并随着药物作用时间的延长而增加,有时间依赖关系。但是到达一定抑制率(80%左右)后,再增加洛铂药物浓度,抑制率增加并不明显,呈现平台效应。3洛铂对细胞周期的影响:同一浓度洛铂作用24、48、72小时S期及G0/G1期与对照组比较差异均有统计学意义(p<0.05)。5μmol/L洛铂作用24小时开始引起喉癌细胞S期比例增加,随着作用时间延长到72小时时S期细胞比例下降,G0/G1期细胞比例增加。20μmol/L洛铂作用24小时使细胞阻滞在S期,阻滞作用明显,随着作用时间延长,S期细胞比例下降,随后出现明显的G0/G1期阻滞,表明低浓度短时间内使喉癌细胞阻滞在S期,高浓度长时间可使喉癌细胞阻滞在G0/G1期。4洛铂对细胞凋亡率的影响:正常对照组细胞无凋亡峰出现,5μmol/L洛铂作用24小时后有小的凋亡峰出现,作用48、72小时后出现明显的凋亡峰,20μmol/L作用24、48小时均出现明显的凋亡峰。不同浓度洛铂作用于喉癌细胞24、48、72小时的凋亡率差异均有统计学意义(p<0.05),同一浓度洛铂对喉癌细胞作用24小时、48小时凋亡率差异有统计学意义(p<0.05)。结果表明洛铂可以诱导喉癌细胞凋亡,而且随着药物浓度和作用时间的增加,凋亡率逐渐增加,洛铂诱导喉癌细胞的凋亡呈时间和剂量依赖关系,低剂量洛铂(5μmol/L)24小时即可诱导喉癌细胞出现凋亡。5应用免疫细胞化学法(S-P法)检测各组细胞bcl-2蛋白表达的变化;20μmol/L洛铂作用48小时后,bcl-2表达减弱,经t检验p<0.05,差异有统计学意义。结论:1洛铂在体外具有抑制喉癌细胞增殖的作用,呈时间和剂量依赖性。2洛铂可以对喉癌细胞产生细胞周期阻滞,低浓度短时间阻滞在S期,高浓度长时间可产生G0/G1期阻滞。3洛铂能诱导喉癌细胞发生凋亡,呈时间和剂量依赖性, 5μmol/L浓度洛铂24小时即可诱导喉癌细胞凋亡。4洛铂可能是通过下调bcl-2的表达,而诱导喉癌细胞凋亡这可能是洛铂诱导凋亡的作用机制之一。更多还原

【Abstract】 Objective: The laryngeal carcinoma is the malignant tumor which endangers mankind’s health and life,Laryngeal carcinoma with surgical operation and put to treat for mainly cure means, But after operation sufferer’s larynx lose original pronunciation function, still very great patient’s existence quality and existence rate of the degree influence laryngeal carcinoma.In recent years along with the exaltation of chemotherapy level, the chemotherapy occupies an important position more and more in the comprehensive treatment of laryngeal carcinoma, for example laryngeal carcinoma of new assistance chemotherapy, its purpose lies in contract tumor and make clinical stage in advance, with radiotherapy or the partial laryngectomy combine to reserve a larynx voiced function. Laryngeal carcinoma because of position concealment, discover usually already arrive medium advanced stage, advanced stage laryngeal carcinoma or through radiotherapy or after partial laryngectomy again the surgical operation is more difficult, the simplicity radiotherapy to aim at local lesion, and hard to receive ideal result, clinically give chemotherapy, to control tumor of the whole body transfer, alleviating symptom, prolonging life.Laryngeal carcinoma With platinum is basic medication at head the neck cancer under chemotherapy treatment. With cisplatin (cDDP) for basic chemotherapy at head neck tumor the under medical treatment study more, and obtained certain curative effect, because of agreeable kidney toxicity and digest way side effect of cisplatin severity limited it in the clinical application,So develop efficiently low toxical platinum product has been the direction that the scholars study.Lobaplatin is the third generation platinum derivatives after cisplatin and carboplatin, the lobaplatin with its lighter side effect, with cross to drug fast incompletely, and stronger anti-tumor activity already initial show its important function in tumor under medical treatment.This experiment take the laryngeal carcinoma cell line Hep-2 as model, adopt MTT chromatometry and inverted -microscope and flow-cytometry to examine lobaplatinto to the Laryngeal carcinoma cell multiplication, cycle blockage,induce cell apopotosis etc.apply immunocy tochemical method to detect the expression of apoptosis related egg white bcl-2. Initially approach the effect lobaplatin to the Laryngeal carcino -ma cell multiplication, cycle blockage, induce cell apopotosis and the expression of apoptosis related egg white bcl-2. For lobaplatin can used for head and neck tumor ,s chemotherapy provide experiment basis.Methods:1 The laryngeal carcinoma cell line Hep-2 was was diviced into 3 groups:①blank contral(culture fluid);②negative control(cell+culture fluid);③experiment group (cell+ lobaplatin + culture fluid); each group cultivate 24, 48, 72 hours respectively2 To observe the cell growth and the change of cell morphology by inverted- microscope every day.3 Examine lobaplatin to repress the generation of the laryngeal carcinoma cell by MTT assay.4 The change of cell apoptosis and cell cycle was analyzed by the folw cytometry.5 Use the immunity cell chemistry method examine the express -ion of egg whites bcl-2.6 Statistic methods: The quantitative data were performed with one-way-ANOVA and the two sample data was performed with t-test. P<0.05 were considered statistically significant.Results:1 The observe results of cell morphology: negative control Hep-2 cell as endepidermis, adherence growth in nutritive medium, polygon,Fusiform shape ,edge sharpness,cell structue is closed, growth productive.after 5μmol/L effect 48h, a small quantity cell changes a circle, still adherence growth, along with giveing the aggrandizement o fdose of lobaplatin, become round cell amount to increase, arrive 80μmol/L, greatly parts of cells suspend in the nutritive medium. Becoming round cell to increase obviously is all suspended in nutritive medium, and appear a lot of fragments around the cell.2 The effect of lobaplatin to cell multiplication: Different dose lobaplatin at same time A value and matched control comparison, 5μmol/L function 24 hours and matched control comparison, the difference was not statistically significan (p>0.05),rest each group and matched control comparison, the difference all were statistically significan (p<0.05).Same dose lobaplatin function 24h,48h,72hcompare difference to all have statistically significan (p<0.05).The lobaplatin can repress the laryngeal carcinoma cell to propagate obviously, inhibition ratio rise along with the increment of dose of lobaplatin, having dose-dependent relation; inhibition ratio rise along with medicine function time of extension, having time-dependent relation. But arriving certain medicine dose and time ,increase again the lobaplatin dose, inhibition ratio increment was not obviously, presenting a platform effect.3 The influence of lobaplatin to cell cycle:Same dose of lobaplatin effect 24, 48, 72 hours S cycle and the G0/G1cycle all have statistical significance compare to control group (p<0.05). 5μmol/L lobaplatin start causing the laryngeal carcinoma cell’S cycle proportion increment for 24 hours, prolonging to 72 hours the S cycle proportion to descend, G0/G1 cycle cell proportion increment.20μmol/L lobaplatin make cell hold-up in S cycle for 24 hours,along with time extension, the S cycle proportion to descend, appearing obvious G0/G1 blockage later.Make while expressing low dose in a short time make cell hold-up in S cycle , high dose in a long time make cell hold-up in G0/G1 cycle.4 Lobaplatin influence to the cell apoptosis rate:The normal control cell has no apoptotic peak,5μmol/L lobaplatin appear small apoptotic peak after influence to the cell for 24 hours, appear obvious apoptotic peak after influence to the cell for 72 hours, 20μmol/L lobaplatin appear obvious apoptotic peak after influence to the cell for 24、48 hours.Different dose lobaplatin influence to laryngeal carcinoma cell 24,48,72 hours the difference all haave statistical significance (p<0.05), same dose lobaplatin influence to laryngeal carcinoma cell 24, 48,72 hours the difference all have statistical significance (p<0.05).The result expresses the lobaplatin can induce the laryngeal carcinoma cell apoptosis,and along with the increment of the medicine dose and time, the apoptosis rate increases gradually, the lobaplatin induce laryngeal carcinoma cell apoptosis of the time and dose-dependence relation,low dose lobaplatin (5μmol/L) can induce the laryngeal carcinoma cell apoptosis for 24 hours.5 Adopt immunocy tochemical method(S-P method) examines the expression of the egg whites bcl-2 in each group, the expressions of bcl-2 die down after 20μmol/L lobaplatin function 48hs,by Student’s t-test of significance,p<0.05,the difference has statistical significanceConclusion: 1 The lobaplatin can repress the laryngeal carcinoma cell to propagate obviously in vitro. having dose and time-dependent relation.2 Lobaplatin can make the laryngeal carcinoma cell cycle blockage, low dose in a short time make cell hold-up in S cycle,high dose in a long time make cell hold-up in G0/G1 cycle.3 The lobaplatin can induce laryngeal carcinoma cell apoptosis,it is of the time and dose-dependence relation,low dose lobaplatin (5μmol/L) can induce the laryngeal carcinoma cell apoptosis for 24 hours.4 Lobaplatin may be pass adjust the expression of bcl-2, induce laryngeal carcinoma cell apoptosis, this may be the function mechanism that lobaplatin induce cell apoptosis.更多还原