【作者】 王玉明；

【导师】 金克炜；

【作者基本信息】 昆明医学院 ， 外科学， 2009， 博士

【摘要】 目的探讨puma基因突变、mRNA及其功能蛋白表达与肺癌的相关性;PUMA与p53的依赖性关系。方法通过对实验条件的探讨,建立了扩增高GC碱基的PCR方法,使GC含量高达82%的模板成功扩增;建立了实时荧光定量RT-PCR检测puma mRNA的方法。应用所建立的方法和免疫印迹法（Western Blot,WB）检测40例肺癌患者的癌组织、癌旁组织和远癌组织及10例肺良性病变组织中DNA和BH3结构域的突变:puma mRNA及其编码蛋白表达的情况;其次,TUNEL检测其中20例肺癌患者的癌组织、癌旁组织和远癌组织的细胞凋亡状态。结果1.通过对实验条件的探讨,确立了puma基因三个外显子的最终扩增条件,分别如下:（1）外显子2（Exon2）的反应体系为dNTP 200μmol/L,MgSO4 3.0mmol/L,引物500nmol/L,TaqDNA聚合酶0.1U/μL,模板量不低于4 000ng,10×缓冲液5μL,增强液10μL,总体积50μL。反应条件为95℃预变性5min,95℃45Sec、64℃45sec、72℃45sec,循环33次,最后72℃延伸5min。（2）外显子3（Exon3）的反应体系MgSO4 2.0mmol/L,外显子4（Exon4）的反应体系MgSO4 1.0mmol/L,其余组分和扩增程序与外显子2相同。2.应用优化后的扩增体系和条件对40例肺癌患者的癌组织,癌旁组织和远癌组织以及10例肺良性病变患者的肺组织的puma基因2、3和4共3个外显子进行了PCR扩增,扩增产物经纯化后测序检测突变及小的删除片段,经Blast 2与GeneBank序列比对,未发现一例外显子突变及小的删除片段,HB3结构域也没有变异。3.通过对实验条件的探讨,检测puma mRNA实时荧光定量RT-PCR反应体系为cDNA 1.0μl,dNTP 800μmol/L,Taq DNA聚合酶5.0 u,Mg²⁺1.5mmol/L,1×Enhancersolution 5.0μl,10×buffer 5.0μl,引物各500nmol/L,0.5×SYB Green I 1.0μl,总体积50μl。内参基因GAPDH体系中不加1×Enhancer solution,其余成分同上。扩增条件:94℃预变性5min,94℃变性45see,56℃退火45sec,72℃延伸45sec,共40个循环,终末72℃延伸5 min。4.应用所建立的实时荧光定量RT-PCR检测puma mRNA表达的相对量,pumamRNA在癌组织、癌旁组、远癌组及肺良性病变对照组中均有表达,癌组织1.17±0.08、癌旁组织1.16±0.07、远癌组织1.18±0.07、肺良性病变对照组织1.17±0.11。经方差分析,puma mRNA的表达在同一病例的癌组织、癌旁组织和远癌组织之间差异无统计学意义（F=0.416,P＞0.05）,puma mRNA在肺癌组织和肺良性病变的表达量差别无统计学意义（F=0.264,P＞0.05）。而且鳞癌、腺癌及腺鳞癌的癌组织、癌旁组织和远癌组织中puma mRNA水平差异无统计学意义（F=0.311,P＞0.05;F=0.338,P＞0.05:F=0.329,P＞0.05）。5.PUMA蛋白检测显示:癌组织、癌旁组织及远癌组织中的表达量分别为1.30（0.98～1.74）、1.11（0.72～1.69）和1.01（0.71～1.49）;10份肺良性组织标本的均值为1.17±0.11。经秩和检验分析,在同一病例的癌组织、癌旁组织和远癌组织的PUMA蛋白表达差异无统计学意义（x²（H）=4.918,P＞0.05）;PUMA蛋白在肺癌组织和肺良性病变的表达量差别无统计学意义（x²（H）=5.092,P＞0.05）。WB结果还显示鳞癌、腺癌及腺鳞癌的癌组织、癌旁组织和远癌组织中PUMA表达水平差异无统计学意义（F=0.101,P＞0.05;F=0.599,P＞0.05;F=1.054,P＞0.05）,表明PUMA表达水平与肺癌的病理分型无关。6.P53蛋白检测显示:癌组织P53蛋白的表达量与癌旁组织、远癌组织和肺良性病变组织之间,蛋白表达差异有统计学意义,癌旁组织和远癌组织之间差异无统计学意义。7.TUNEL结果显示:癌组织、癌旁组织和远癌组织AI分别是:4.96%±1.63%,1.45%±0.46%和1.26%±0.42%。癌组织的AI高于癌旁组织和远癌组织,AI差异有统计学意义,Z=-0.-3.920,P=0.000;同时癌旁组织与远癌组织之间AI的差异也有统计学意义,Z=-0.-3.469,P=0.001。8.P53与PUMA关系分析:PUMA蛋白表达与P53蛋白表达之间没有直接关系,其可能的原因是由于肺癌患者P53的突变很高,突变的P53失去来对PUMA的调控作用。结论puma在肺癌发生发展过程中基因的结构很稳定,没有发生有意义的突变和BH3结构域的变异;puma mRNA、蛋白表达和细胞凋亡等研究表明,PUMA表达可能不是伴随肺癌发生和发展过程的重要因素;mRNA和蛋白表达水平与肺癌的组织类型无关。P53没有介导PUMA的表达,同时肺癌发生发展的各种因素也没有诱导PUMA的过表达,因此,在肺癌发生发展过程中,细胞凋亡的异常,引起细胞的过度增殖和对放化疗的敏感性减低,导致肺癌一直是全球发病率和死亡率都很高的恶性肿瘤。本研究结果与体外细胞株的研究结果不一致,而与其他肿瘤组织的研究结果一致,说明PUMA在肿瘤的发生发展中,不同的组织有不同的表现,其在肺癌发生发展中的作用还有待进一步的研究,是否能作为肺癌诊断和治疗的新靶点还有待进一步的研究。更多还原

【Abstract】 Objective to explore the correlation of the mutation and expression of puma gene to tumorigenesis and development of lung cancer and the relation between PUMA and p53.Methods Explored the condition of PCR and optimized amplification system and reaction conditions of three high GC content Exon of puma,it make the exon 2 that GC content 82%was amplificationed succeefully.Explored the reaction conditions of fluorescence quantitative reverse transcript-polymerase chain reaction,the FQ- RT-PCR assay was established to examine the puma mRNA.The quantitative fluorogenic real-time PCR and Western Blot Analysis were used to detect the mutation of puma,the expression puma mRNA and its protein in all 40 cases of primary lung cancer tissues,para-cancer lung tissues and far-cancer lung tissues,10 control cases of non-cancer lung tissues were also detected. Apoptosis of 20 NSCLC samples were detected by TUNEL technique.Results1.By explored the experiment conditions of PCR,the ultimate amplification system and react contions of three exon of puma were eatablished.（1） The optimized reaction system of exon2 were:dNTP 200μmol/L,enhancer system MgSO4 3.0mmol/L,each primers 500nmol/L,Taq DNA polymerase 0.1U/μl,template more than 4000ng,10×buffer 5μl,ehancer solution 10μl,the total volume was 50μl.The amplificat condition was:After 5 min at 95℃,reactions were cycled through 45s denaturation at 95℃,45s annealing at 64℃,and 45s extension at 72℃for 33 cycles;followed for 5 min.（2） Exon3 and exon 4 conditions were:MgSO4 2.0mmol/L and 1.0mmol/L,other components were same as exon2.2.By using the optimized amplification systems and reaction conditions,the exon 2, exon 3 and exon 4 of puma gene in all 40 cases of lung cancer tissues,paracancer lung tissues and farcancer lung tissues,10 cases of lung benign disease were also amplificationed.The amplification productions were depurated and sequenced both mutation and small deleted fragments.The sequencing results compare to GeneBank sequence by Blast 2,non of mutation and small fragments were found,and BH3 domain is normaol.3.By exploring the conditions of fluorescence quantitative RT-PCR,we showed that the optimized fluorescence quantitative RT-PCR reaction mixtures contained the forward and reverse primers at 500nmol/L each;dNTP 800μmol/L;1.5mmol/L MgCl₂;1×Enhancer solution 5.0μl;10×PCR buffer 5.0μl;0.5×SYB GREEN I 1.0μl;Taq DNA polymerase 0.1U/μl; template 1.0μl;the total reaction volume was 50μl.The RT-PCR was performed under the following conditions:94℃for 5 minutes;followed by 40 cycles of 94℃for 45 seconds,56℃for 45 seconds and 72℃for 45 seconds;and 72℃for 5 minutes.4.The means of the relative rate of puma mRNA and GAPDH mRNA in lung cancer tissue,paracancer lung tissue,farcancer lung tissue and lung benign disease tissues were 1.17±0.08,1.16±0.07,1.18±0.07 and 1.17±0.11 which detected by fluorescence quantitative RT-PCR.The data were evaluated by analysis of variance.There was no any significant difference among lung cancer tissue,paracancer lung tissue and farcancer lung tissue （F=0.416,P＞0.05）.Also,there was no significant difference among squamous cell carcinoma, adenocarcinoma and adenosquamous carcinoma in the group of lung cancer tissue,paracancer lung tissue and farcancer lung tissue（F=0.311,P＞0.05;F=0.338,P＞0.05;F=0.329,P＞0.05）.5.In the 40 cases of primary lung cancer tissues,the medians of PUMA protein in lung cancer tissue,paracancer lung tissue and farcancer lung tissue were 1.30（0.98～1.74）, 1.11（0.72～1.69） and 1.01（0.71～1.49） which detected by Western Blot Analysis.The mean of PUMA protein in benigh disease lung tissues was 11.17±0.11 The data were evaluated by analysis of Krusdal-Wallis H test.There were no any significant difference among lung cancer tissue,paracancer lung tissue and farcancer lung tissue（x²（H） =4.918,P＞0.05）.Also,there was no significant difference among squamous cell carcinoma,adenocarcinoma and adenosquamous carcinoma in the group of lung cancer tissue,paracancer lung tissue and farcancer lung tissue（F=0.101,P＞0.05;F=0.599,P＞0.05;F=1.054,P＞0.05）.6.Western Blot Analyzed p53 protein:the p53 protein in cancer tissue is significant difference in paracancer lung tissue,farcancer lung tissue and lung benign disease tissue; there was no any significant difference among paracancer lung tissue and farcancer lung tissue.7.The results of TUNEL were:The apoptosic index（AI） in lung cancer tissue,paracancer lung tissue and farcancer lung tissue were 4.96%±0.1.63%,1.45%±0.46%and 1.26%±0.42%. AI in lung cancer was significant difference between lung cancer tissue with paracancer lung tissue and farcancer lung tissue（Z=-3.920,P=0.000）,AI of paracancer lung tissue was higher than farcancer lung tissue,and has significant difference of them（Z=-3.469,P=0.001）.8 The relationship of PUMA and p53:PUMA protein expression was not correlate with p53 protein expression.The cause of it may be that the p53 has high mutation in lung cancer and the mutated p53 can not imediate expression of PUMA.Conclusions Mutational analysis revealed that there was no PUMA BH3 domain and exon mutation in the 40 NSCLC,suggesting that PUMA BH3 domain mutation and exon mutation are not a direct target of inactivation in NSCLC development.PUMA expression may be not play a role in NSCLC initiation and progression of cancer.There was not relationship between puma mRNA and protein with different pathological types of NSCLC. PUMA expression was not mediates by p53,and also induced from various apoptotic stimuli. In the course of the occurrence and development of lung cancer,abnormal apoptosis,caused by excessive cell proliferation and to reduce the sensitivity of radiotherapy and chemotherapy, leading to lung cancer has been the global morbidity and mortality are highly malignant.This findings are inconsistent with in vitro cell line findings,but is consistent with other tumor organization findings,explained that PUMA in the tumor occurrence development,different organizations have different performance,and its occurrence in lung cancer development remains to be further research,whether as a lung cancer diagnosis and treatment have yet to be a new target for further study.更多还原