【作者】 汪雷；

【导师】 徐志云；

【作者基本信息】 第二军医大学 ， 外科学， 2011， 博士

【摘要】 早幼粒细胞白血病锌指蛋白(promyelocytic leukaemia zinc finger, PLZF),亦称为ZBTB16(zinc finger and BTB domain containing 16),作为一种转录抑制因子,具有参与调控细胞周期、增殖、分化和凋亡等多种功能。研究表明,PLZF表达下调所引起的细胞凋亡失控与粒系白血病以及前列腺癌、恶性黑色素瘤等实体瘤的发生和发展密切相关。但目前有关该基因在肺癌发生和发展中的作用尚未见文献报道。我们在前期通过基因芯片对5例非小细胞肺癌组织及其对应癌旁组织基因表达谱进行比较发现,肺癌组织中PLZF mRNA表达水平较癌旁组织明显下调。本研究扩大检测样本量,通过定量PCR (Quantitative PCR, Q-PCR)及免疫组化检测进一步发现,PLZF在非小细胞肺癌组织中的表达水平较对应癌旁组织显著下调,其表达水平变化与肿瘤病理类型、原发肿瘤大小及癌细胞凋亡率等存在明显的相关性。在此基础上,我们进一步通过细胞学和小鼠成瘤模型实验探讨了PLZF诱导肺鳞癌和腺癌细胞凋亡发生的机制及其对肺鳞癌和腺癌细胞生长的抑制作用。第一部分PLZF在非小细胞肺癌中表达与临床病理及凋亡的相关性研究目的：1、检测非小细胞肺癌及对应癌旁组织中PLZF表达。2、分析PLZF表达水平变化与患者年龄、性别、淋巴结转移、肿瘤大小、临床分期、病理类型及凋亡的关系。方法：1、抽提119对NSCLC样本(癌组织及其相应癌旁组织)总RNA,反转成cDNA后,采用Q-PCR方法对PLZF的mRNA水平表达进行检测。2、将119对NSCLC样本制成组织芯片,用免疫组化的方法(两步法)检测PLZF蛋白在NSCLC样本中的表达。结果的判定方法：阳性信号呈棕黄色或棕黑色颗粒状,主要位于细胞核内。根据肿瘤细胞染色程度分为4级：0(无着色),1(浅度染色),2(较深染色),3(深度染色)；再根据肿瘤细胞着色范围(阳性细胞占肿瘤细胞的百分比)分为4级：0(0～25%着色),1(26～50%着色),2(51～75%着色),3(76～100%着色)。两者乘积为最终结果,癌组织评分等于其对照组织为表达正常,低于其对照组织为表达下调。3、运用TUNEL法检测NSCLC样本的凋亡指数,凋亡指数定义为每张切片取5个高倍镜视野(×400),每个视野计数100个所需检测的细胞,计算凋亡细胞在500个细胞中比值。4、分析PLZF表达异常和临床病理及凋亡的相关性。结果：1、在所有119对NSCLC样本中,PLZF在mRNA水平及相应蛋白水平的表达变化趋势均一致,其中103例(86.6%)非小细胞肺癌组织中PLZF表达水平显著低于对应癌旁组织。2. PLZF的表达水平变化与患者年龄、性别、淋巴结侵袭、临床分期在统计学上无显著差异,但与原发肿瘤大小和病理类型显著相关。3、PLZF下调表达的肺鳞癌和腺癌组织凋亡指数较对应癌旁组织凋亡指数的下降程度显著高于PLZF表达正常的肺鳞癌和腺癌组织。结论：1、PLZF异常表达不仅参与肺癌发生,还参与肺癌发展。2、PLZF可能促进肺鳞癌和腺癌细胞凋亡。第二部分PLZF调控肺鳞癌及腺癌细胞凋亡的途径研究目的：1、以人肺鳞癌和腺癌细胞株为研究对象验证PLZF促体外细胞凋亡的功能。2、阐明PLZF调控肺鳞癌和腺癌细胞凋亡的机制。方法：1、PLZF表达质粒分别转染A549,Calu-3、H226和H520等肺癌细胞株,AnnexinV/PI双染色后通过流式细胞仪检测PLZF表达质粒转染组、空载体转染组和未转染组细胞凋亡情况,FITC-/PI-染色细胞为正常细胞,FITC+/PI-染色细胞为早期凋亡细胞,FITC+/PI+染色细胞为晚期凋亡细胞,FITC-/PI+染色为坏死细胞。2、分别通过Q-PCR及Western blot方法检测PLZF质粒转染组和其他两组细胞凋亡通路关键因子Fas、FasL、Bax、Bcl-2、caspase-3、8、9、XIAP及Survivin的mRNA和蛋白表达水平。3、挑选凋亡抑制基因作为假设靶基因,克隆其启动子连接DeRed红荧光蛋白开放阅读框构建重组报告质粒,与PLZF表达质粒共转染后观察PLZF对假设靶基因启动子的调控作用。结果：1、流式细胞仪检测发现转染PLZF表达质粒24小时后细胞凋亡明显多于空载体转染组和未转染组,随着时间增加,PLZF质粒转染组细胞凋亡率显著增加。2、Q-PCR及Western blot结果表明：PLZF表达在转染72小时后达到高峰,PLZF质粒转染组的Bax、Fas、caspase-3、8、9等凋亡相关基因表达显著高于空载体转染组和未转染组,Survivin、FasL和XIAP表达无显著差异,Bcl-2表达则显著低于其他两组。3、报告基因检测显示PLZF在转录水平上抑制Bcl-2的表达。结论：1、PLZF具有促进肺鳞癌和腺癌细胞凋亡的功能。2、PLZF可能主要通过抑制Bcl-2表达激活线粒体凋亡通路的方式调控肺鳞癌和腺癌细胞的凋亡。3、PLZF同时具有激活死亡受体Fas/FasL通路诱导肺鳞癌和腺癌细胞凋亡的功能,但具体机制仍有待进一步研究。第三部分PLZF调控肺鳞癌及腺癌细胞体内凋亡及增殖的实验研究目的：通过腺病毒介导的转基因方法研究PLZF高表达对肺鳞癌及腺癌细胞体内凋亡及增殖能力的影响,探讨其作为肺鳞癌及腺癌基因治疗靶点的可行性。方法：1、构建表达PLZF的重组腺病毒Ad.PLZF。2、以人肺鳞癌细胞株H226、H520及肺腺癌细胞株A549、Calu-3为研究对象,Ad.PLZF转染肺鳞癌和腺癌细胞后,通过测定细胞周期及细胞倍增时间明确PLZF高表达对体外细胞增殖活性的影响。3、制作小鼠移植瘤模型(皮下接种2×107个癌细胞),根据瘤体内注射物不同分为3组：Ad.PLZF组,Ad.GFP组和PBS组,通过计算相同时间各组瘤体体积、质量比较各组间肺鳞癌和腺癌细胞体内生长情况,并通过瘤体组织切片检测PCNA阳性细胞率和凋亡指数(TUNEL法)了解肿瘤细胞体内增殖及凋亡情况。结果：1、成功构建了重组腺病毒Ad.PLZF。2、Ad.PLZF转染组肺鳞癌及腺癌细胞的倍增时间显著延长,增殖指数显著降低。3、异种移植瘤体内注射重组腺病毒Ad.PLZF组的小鼠移植瘤体积及质量均显著低于对照组。4、瘤体组织切片检测结果表明Ad.PLZF组癌细胞的增殖能力显著下降,凋亡指数显著升高。结论：PLZF能抑制肺鳞癌和腺癌细胞的增殖并促进癌细胞凋亡,可能是肺鳞癌及腺癌基因治疗的潜在有效靶点。更多还原

【Abstract】 PLZF(promyelocytic leukaemia zinc finger), or ZBTB16 (zinc finger and BTB domain containing 16) is a transcription factor that plays a role in the signaling networks that regulate cell cycle, proliferation differentiation and apoptosis. Recent studies indicate that down regulation of PLZF gene expression not only contributes to acute promyelocytic leukemia but links to occurrence of malignant solid tumors such as prostatic csarcinoma and malignant melanoma. Evading apoptosis through down-regulated PLZF expression constitutes a mechanism of tumor growth. The function of PLZF in lung cancer is unknown. Microarray was used to examine the expression of PLZF mRNA in five non small cell lung carcinoma tissues and adjacent normal tissues. The microarray results showed that the level of PLZF mRNA expression in the lung cancer tissues was significantly lower than that in adjacent normal tissues. In this study,it was found PLZF was down-regulated in non small cell lung cancer tissue compared with that in adjacent normal tissue. In addition, there was a significant correlation between PLZF expression and apoptosis, tumor size and pathological types. The mechanism whereby PLZF induced apoptosis and growth inhibition in lung squamous cell carcinoma and adenocarcinoma was also investigated.Part 1 PLZF expression in non small cell lung cancer and its clinical significanceObjectives:To examine the expression level of PLZF in NSCLC tissuses, and determine associations between PLZF expression and age, gender, nodal involvement, clinical phasing, pathological types and apoptosis.Methods:1) Total RNA of 119 pairs of NSCLC samples (tumor tissues and their corresponding normal tissues) was extracted and reversely translated into cDNA. Using real-time quantitative polymerase chain reaction, the relative level of PLZF mRNA was detected.2) 119 pairs of NSCLC samples (tumor tissues and their corresponding normal lung tissues) were made into three tissue microarrays. PLZF protein expression was detected by immunohistochemistry assay using the monoclonal antibody against the extracellular domain of PLZF via the two-step immunohistochemical staining. All samples were examined by light microscopy and scored semi-quantitatively on the basis of the intensity of the staining reaction and the percentage of cells that displayed immunoreactivity. PLZF expression was predominantly in cell nuclei. The samples were divided into four grades according to the staining intensity:0 (negative staining),1 (weak staining),2 (medium staining), and 3 (strong staining). The percentage of positive staining cells was defined as 0 for 0-25%,1 for 26-50%,2 for 51-75%, and 3 for 76～100%. The final score was determined as the product of intensity score and the proportion. A cancer sample was defined as a reduced or a preserved type of PLZF expression if its final score was less than or the same as that of its corresponding normal tissue.3) TUNEL (TdT-mediated biotinylated-dUTP nick end labling method) was adopted to analyze apoptosis of lung squamous cell carcinoma and adenocarcinoma cells. In each case,100 cells which need to detected each in 5 different fields, totaling 500 cells, were evaluated at high magnification (×400), and the apoptotic index (AI) was determined as the number of apoptotic cells per 500 cells.4) The results were analyzed statistically.Results:1) 103 (86.6%) of these lung cancer tissues showed lower levels of PLZF mRNA compared with the corresponding normal tissues. Immunohistochemistry indicated that protein changes were coincident with the PLZF mRNA expression.2) There was no significant difference between PLZF expression and age, gender, nodal involvement and clinical phasing, but PLZF expression was significantly associated with advanced T stages and pathological types.3) AI of lung squamous cell carcinoma and adenocarcinoma tissues with downregulated PLZF expression was significantly lower than that of cancer tissues with normal PLZF expression.Conclusions:1) Abnormal expression of PLZF may participate in the carcinogenesis and progression of lung squamous cell carcinoma and adenocarcinoma.2) Down-regulation of PLZF gene expression may be the possible mechanism whereby cells evade apoptosis in lung squamous cell carcinoma and adenocarcinoma.Part 2 PLZF promotes the course of apoptosis in lung squamous cell carcinoma and adenocarcinoma cellsObjective:To verify the function and clarify the mechanism of PLZF in promoting the course of apoptosis in lung squamous cell carcinoma and adenocarcinoma cells in vitro.Methods:1) PLZF expression plasmid was transfected into lung cancer A549, Calu-3,H226 and H520 cells.2) Cells in untreated and treated groups were evaluated for apoptosis by flow cytometry (FCM). Annexin V binding was evaluated using bivariate FCM, and cell staining was determined by fluorescein isothiocyanate-labelled Annexin V, simultaneously with dye exclusion of propidium iodide (PI). The Annexin FITC-/PI- population was regarded as normal healthy cells, while Annexin FITC+/PI+were taken as late apoptosis, Annexin FITC+/PI-cells as early apoptosis, and Annexin FITC-/PI+ as necrosis.2) The level of the key genes mRNA and protein of apoptosis pathways (including Fas,FasL,Bax,Bcl-2, caspase-3,8,9, XIAP and Survivin) were detected by real-time quantitative polymerase chain reaction and Western blotting.3) Apoptosis inhibitor genes were selected as target genes. New recombinant plasmid was constructed by the Cloning of the target gene promoter, and the new recombinant plasmid and PLZF expression plasmids were co-transfected into lung squamous cell carcinoma and adenocarcinoma cells.Results:1) The highest rate of apoptosis in cells transfected by PLZF expression plasmid was observed by FCM. The apoptosis rate in PLZF gene transfected cells significantly increased compared with the void-vector transfected cells and untreated cells 24 h after transfection. With the transfection time prolonging, the apoptosis rate in PLZF gene transfected cells rose significantly.2) The expression level and activity of PLZF in transfected cells reached the peak 72 h after transfection. Fas, Bax, caspase-3,8,9mRNA expressions were significantly higher in PLZF gene transfected cells than those in void-vector transfected cells and untreated cells, and the expression of Bcl-2 mRNA was significantly lower in PLZF gene transfected cells than that in the other cells. There was no significant difference in FasL, Survivin and XIAP mRNA expression level between treated and untreated cells. The effects of protein expression of the above gene were similar to those of mRNA expression.3.Bcl-2 was a target gene of PLZF.Conclusions:PLZF could promote apoptosis of lung squamous cell carcinoma and adenocarcinoma cells in vitro, and could induce apoptosis of lung squamous cell carcinoma and adenocarcinoma cells through the mitochondrion pathway by down-regulating Bcl-2. In addition, PLZF could induce the death receptor Fas/FasL apoptotic pathway, but the specific mechanism is unknown.Part 3 Research of PLZF in regulating apoptosis and proliferation of lung squamous cell carcinoma and adenocarcinoma cells in vivoObjective:To investigate influences of PLZF on proliferation and apoptosis of lung cells via recombinant adeno-associated virus-mediated PLZF gene transfection and discuss the feasibility of lung cancer therapy with Ad.PLZF.Method:1) Recombinant adenovirus Ad.PLZF was first constructed, and then transfected into lung A549, Calu-3,H226 and H520 cells. Each lung cancer cell line was divided into 3 groups:Ad.PLZF group, Ad.GFP group, and untreated group.2) The effect of PLZF overexpression on the proliferation of lung cancer cells was observed by FCM.3) An animal model of lung adenocarcinoma and squamous cell carcinoma bearing nude mice was established by subcutaneous injection of 2×107 lung cancer cells. All mice were assigned to 3 groups:Ad.PLZF group, Ad.GFP group and PBS group. The length (A) and width (B) of tumor per 10 days were measured, and gross tumor volume was calculated based on the formula V=0.5xAxB2. After 30-day treatment, the mice were sacrificed and their tumors were excised for immunohistochemical staining of PCNA and TUNEL.Result:1) Recombinant adenovirus Ad.PLZF was constructed successfully.2) The reproductive activity of transfected pAd.PLZF group dropped significantly. The size and weight of the transplantable tumor in Ad.PLZF groups were significantly smaller and lower than those of the other groups.3) The proliferation index of the transplanted tumor in Ad.PLZF groups was significantly lower than that of Ad.GFP group and untreated group,but the apoptotic index of the transplanted tumor in Ad.PLZF groups was significantly higher than that of the other groups.Conclusion:PLZF could inhibit cell proliferation in lung squamous cell carcinoma and adenocarcinoma, and may prove to be a potential effective target for gene therapy.更多还原