【作者】 葛晓晴；

【导师】 任景丽； 杨鲲鹏；

【作者基本信息】 郑州大学 ， 外科学， 2012， 硕士

【摘要】 1研究背景肺癌是当今常见的恶性肿瘤,在所有癌症致死的疾病中占居前位。肺癌的发病率和病死率在工业发达国家明显高于工业不发达国家。目前中国癌症发病类型中,肺癌在男性占居第一位,在女性位居于乳腺癌之后。肺癌严重危害着人类的健康和生命,经过专家学者的多年努力,在肺癌的预防和治疗方取得了较大成就,患者的生存率和生活质量有了明显的提高,据估计目前肺癌患者的五年生存率已提高了15%左右。目前肺癌的主要治疗手段仍以外科手术治疗为主,化疗、放疗、靶向治疗及其他生物治疗等综合治疗为辅,但治疗效果仍不满意,总体五年生存率仍较低。影响治疗效果的因素很多,影响肺癌临床治疗效果、预后的最主要因素之一是其恶性生物学行为。目前我国的肺癌患者就诊时大多处于中晚期,已有局部侵犯或远处淋巴及脏器转移,部分肺癌患者还合并有严重的合并症,早期诊断和治疗可能有助于肺癌的病死率下降。如何早期诊断肺癌是目前许多专家和学者所追录和摸索的方向。现在的研究中仅见报道有个别基因,如EGFR基因突变的肯定研究报道,是否存在其它基因共同作用于肺癌,不可得知。在王立东等的研究中,发现PLCE1与食管癌的高风险密切相关,并随病变程度加重,PLCE1表达呈显著升高趋势。食管鳞状细胞癌是最常见的组织分型(占95%以上),近年来的研究提示食管鳞状细胞癌与肺鳞状细胞癌有许多相似的分子机制。磷脂酶CE1(phospholipase C epsilon-1, PLCE1)基因是最近这几年所发现的磷脂酶C(PLC)家族的新成员,它也是一种新的磷脂酶C同功酶,它的分子结构比较复杂,作为转导通路中的关键酶,调节磷酯酰肌醇信号,在细胞膜到细胞核的转导过程中起着较为重要作用。它不仅参与细胞的信号传导,还参与基因调控的表达,参与细胞凋亡和血管新生以及调节细胞的生长、分化等过程。本研究采用免疫组织化学方法及逆转录-聚合酶链反应(RT-PCR)方法检测并探讨PLCE1在肺鳞状细胞癌中的表达及其与临床病理特征的关系,分析比较PLCE1与肺癌生物学行为之间的关系,探讨PLCE1在肺鳞状细胞癌发生发展中的作用和意义。2材料及方法2.1免疫组织化学法检测PLCE1在肺鳞癌中的表达及意义2.1.1研究对象收集手术切除并经病理证实的肺鳞状上皮细胞癌标本48例,其中男性44例,女性4例,年龄31-77岁,平均年龄59.34岁。按肺癌的国际TNM分期标准(第七版),Ⅰ+Ⅱ期28例,Ⅲ期20例,所有病例术前未经过任何相关治疗(放疗、化疗及生物学治疗等),选取48例癌组织旁正常肺组织作为对照组。一抗为Sigma公司所生产的兔抗人磷脂酶CE1多克隆抗体,EnVision试剂盒购自DAKO公司,每批实验都设阳性和阴性对照。2.1.2统计学方法实验结果采用SPSS17.0统计软件对数据进行处理,采用χ2检验,α=0.05。2.2RT-PCR检测PLCE1在肺鳞癌中的表达及意义2.2.1研究对象收集手术切除并经病理证实的肺鳞状上皮细胞癌组织50例及相应癌旁正常组织50例。其中男性39例,女性11例；年龄33-74岁,平均58.62岁。TNM分期：Ⅰ期13例,Ⅱ期19例,Ⅲ期18例,所有病例术前未经过任何相关治疗(放疗、化疗及生物学治疗等)。2.2.2研究方法采用RT-PCR的方法检测50例肺鳞状上皮细胞癌组织以及50例相应癌旁正常肺组织中PLCE1基因的表达,并结合患者的临床病理特征表现,分析50例肺鳞状上皮细胞癌组织中PLCE1基因的表达与临床病理特征间的关系。2.2.3统计学方法数据采用SPSS17.0统计软件进行处理,两组定量资料的比较采用t检验,不满足t检验条件的t’检验。多组定量资料之间的比较采用单因素方差分析,组间的比较则采用LSD-t检验,α=0.05。3结果3.1免疫组织化学法检测结果3.1.1肺鳞状细胞癌组织中PLCE1的表达率(39/48,81.3%),高于正常肺组织12.5%(6/48)(P<0.01)。3.2.1PLCE1的表达与肿瘤淋巴结转移和肿瘤分期相关,差异有统计学意义(P<0.01),与年龄、性别和肿瘤分化程度差异无统计学意义(P>0.05)。3.2RT-PCR检测!PLCE1在肺鳞癌中表达的结果3.2.1肺鳞癌组织中PLCE1mRNA表达量较癌旁正常肺组织高(P<0.01)。3.2.2PLCE1mRNA的表达强度主要与肺鳞癌的组织学分级、淋巴结转移、TNM分期有关,分期越晚、淋巴结出现转移,则表达量越高(P<0.05)。PLCE1mRNA的表达与年龄、性别无关(P>0.05)。4结论4.1PLCE1免疫组化结果提示,PLCE1在肺鳞状细胞癌癌变发展过程中可能起着重要作用。4.2PLCE1的检测可能对肺鳞状细胞癌的预后判断有一定帮助。更多还原

【Abstract】 1BackgroundLung cancer is common malignancy, the death of lung cancer patients occupy a primary position in all cancer. The incidence and mortality of lung cancer in industrialized countries is significantly higher than the industrial developing countries. The incidence of cnacer type of China, lung cancer in male occupies the first and the female ranks after breast cancer. Lung cancer have seriously affect human health and lift, with experts and scholars’great efforts, the achievements have been made in lung cancer curation and prevention, the survival rate and quality of life has been significantly improved. It is estimated that the five-year survival rate of patients with lung cancer has increased15%. Currently, the main treatment of lung cancer is resection, chemotherapy, radiotherapy and targeted therapy, but the result of treatment is not satisfied, the five-year survival rate is still in low level. Many factors affect the treatment, the malignant biological behavior is the main factor affect the clinical treatment and prognostic of lung cancer. When most patients visit the patients, they already in the later period of the lung cancer, there maybe local invasion or distant lymph node and visceral metastasis, some patients with lung cancer is also associated with serious complications, early diagnosis and treatment may contribute to decrease the mortality rate of lung cancer. Many experts and scholars are pursuing the early diagnosis of lung cnacer. Currently, experts and scholars have found EGFR gene affect the lung cancer,but still not found the key gene to determine the therapy and prognosis of lung cancer.Wang LD et al. found PLCE1gene is closely related to the high risk of esophageal cancer, the expression of PLCE1was increased with the disease become much more serious. Esophageal squamous cell carcinoma is the most common histological type (above95%), recent studies suggest that esophageal squamous cell carcinoma and lung squamous cell carcinoma have similar molecular mechanisms. Phospholipase C epsilon-1(PLCE1) gene is discovered in recent years, it is a new member of phospholipase C (PLC) family, also found it was a new PLC isozyme. The molecular structure is complicated, it is a key enzyme in phosphatidylinositol signal transduction pathway, its plays an important role in the cell membrane transduce to the nucleus, it is involved in cell signaling, regulation of gene expression and regulate the cell growth, differentiation, participate in the process of apoptosis and angiogenesis.In this study, immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) method were use to detect the expression of PLCE1in lung squamous cell carcinoma and its relationship with clinicopathological features, analysis the biology of lung cancer, explore the function and significance of the PLCE1gene in lung squamous cell carcinoma.2Materials and Methods2.1Immunohistochemical analysis2.1.1PatientsThis study enrolled96surgically resected lung tissue samples, including48cases lung squamous cell carcinoma,48cases adjacent normal tissues. The male have44cases and4cases female, the mean age is59.34. All cases have not preoperative radiotherapy, chemotherapy and other treatment.The antibody for PLCE1was buy form American Sigma company, it is polyclonal rabbit anti-human. EnVision kits were bought from DAKO Company. Every batch had control and negative groups.2.1.2Statistical MethodsThe data were analyzed by SPSS17.0. The x2test was applied for different groups comparison (a=0.05).2.2RT-PCR analysis2.2.1PatientsOne-hundred surgically resected tissue samples were enrolled in this study, including50cases lung squamous cell carcinoma,50cases normal controls. All cases have not preoperative radiotherapy, chemotherapy and other treatment.2.2.2MethodsDetecting the expression of PLCE1gene in50cases lung squamous cell carcinoma and50cases adjacent normal lung tissue with RT-PCR, and analysis the gene expression combine with the clinical pathological characteristics and features.2.2.3Statistical MethodsThe data were analyzed by SPSS17.0software. The t test, t’ test, one-way ANOVA and LSD-t test were applied for different groups comparison (a=0.05).3Results3.1Expression of PLCE1in lung squamous cell carcinomaThe immunohistochemistry positive rates of PLCE1expression in lung squamous cell carcinoma were83%(39/48), higher than the normal controls (6/48,12.5%)(P<0.01). The expression of PLCE1has relationship with lymph node metastasis and tumor staging (P＜0.01), non-relationship with age, sex and tumor differentiation (P>0.05).3.2RT-PCR in detecting the expression of PLCE1The expression of PLCE1mRNA in cancer tissues was higher than adjacent normal lung tissues (P<0.01). TNM staging of lung squamous cell carcinoma was related with the tumor histological classification, lymph node metastasis and tumor staging (P<0.05), non-relationship with sex, age (P>0.05).4Conclusion4.1Immunohistochemistry results suggest:PLCE1may play an important role in the development of lung squamous cell carcinoma.4.2The detection of PLCE1may contribute to prognosis lung squamous cell carcinoma.更多还原