【作者】 李莹；

【导师】 吴翠环；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2009， 硕士

【摘要】 【目的】1、探讨HPV感染在NSCLC发生中的病因学意义并分析HPV感染与临床病理特征的关系;2、研究NSCLC中HPV感染与Rb、CDK-2蛋白表达的关系,并初步探讨HPV导致NSCLC发生的分子机制;3、分析Rb、CDK-2蛋白表达与NSCLC发生及临床病理特征的关系。【方法】60例标本均取自华中科技大学同济医学院附属同济医院手术切除标本,其中NSCLC组织47例,肺良性病变组织13例(肺结核4例、炎性假瘤5例、肺大疱4例),所有患者术前未经放疗和化疗。47例NSCLC中鳞癌29例,腺癌18例;高、中、低分化肺癌分别为12、18、17例;淋巴结转移28例;吸烟者33例。肺良性病变13例作为对照组。所有标本选定后均收集并记录其年龄、性别、吸烟史、淋巴结转移、组织学类型等资料。1、采用PCR方法选用HPV16型E6、HPV18型E7特异性引物分别检测47例NSCLC及13例肺良性病变组织HPV感染情况。2、免疫组化SP方法检测Rb蛋白与CDK-2蛋白在47例NSCLC及13例肺良性病变组织中的表达情况。IHC染色结果均采用图像处理系统进行图像采集,所有数据由SPSS for windows 13.0统计分析软件进行统计学处理。【结果】1、47例NSCLC和13例肺良性病变组织中HPV感染和Rb蛋白、CDK2蛋白表达结果采用PCR方法对HPV16/18亚型进行检测,肺癌组织中HPV DNA检出率为42.55%(20/47),肺良性病变组中,HPV DNA检出率为7.69%(1/13),二者差异有统计学意义(P<0.05)。采用免疫组化SP法检测Rb蛋白和CDK2蛋白表达情况,肺癌组织中Rb蛋白阳性表达率为68.09%(32/47),而13例肺良性病变组织中则全部表达Rb蛋白,差异有统计学意义(P<0.05),提示Rb蛋白失活可能是肺癌发生过程中的因素之一;肺癌组织中CDK2蛋白阳性表达率为82.98%(39/47),而CDK2蛋白在肺良性病变组织中则不表达,两组比较差异有统计学意义。2、47例NSCLC中HPV阳性组和HPV阴性组的Rb蛋白和CDK2蛋白表达情况HPV阳性组中Rb蛋白阳性表达率为45.00%(9/20),而HPV阴性组中Rb蛋白阳性表达率为85.19%(23/27),两组比较差异有统计学意义(P<0.05),此结果提示HPV感染可能直接或间接的影响Rb蛋白的表达及其活性; HPV阳性组中全部表达CDK2蛋白,HPV阴性组中CDK2阳性表达率为70.37%(19/27),差异有统计学意义(P<0.05)。3、47例NSCLC中HPV感染、Rb蛋白及CDK2蛋白表达与临床病理参数的关系NSCLC中HPV感染与患者年龄、性别、淋巴结转移均无明显相关性,但与NSCLC组织学类型、分化程度及吸烟有关(P<0.05)。HPV感染率在肺鳞癌中为55.17%(16/29)显著高于腺癌中的感染率22.22%(4/18);HPV感染在NSCLC高、中、低分化组中感染率分别为16.67%(2/12)、66.67%(12/18)、35.29%(6/17),三组差异有统计学意义(P<0.05),而且中、低分化组感染率较高;吸烟患者HPV感染率为54.54%(18/33)显著高于不吸烟患者14.29%(2/14)。利用IHC方法分别检测47例NSCLC组织中Rb蛋白和CDK2蛋白表达情况,并分析其与临床病理参数(年龄、性别、吸烟史、组织学类型、分化程度、淋巴结转移)的相关性。NSCLC组中Rb蛋白阳性表达率与上述临床病理参数无关;CDK-2蛋白在高、中、低分化程度肺癌组中阳性表达率分别为58.33%(7/12)、83.33%(15/18)、100%(17/17),差异有统计学意义(P<0.05),提示CDK2蛋白阳性表达与肺癌分化程度相关,而经分析CDK2蛋白表达与其他临床病理参数无相关性。【结论】1、NSCLC组织中高危型HPV16/18型感染率显著高于良性对照组,且与NSCLC组织类型、分化程度及吸烟史有关,提示高危型HPV感染可能在NSCLC发生发展中发挥一定的作用;2、NSCLC组织中Rb蛋白表达程度与良性病变组比较有显著差异,且在肺癌组中Rb蛋白异常表达与HPV感染相关,其机制可能为HPV通过E7蛋白与PRb形成E7-PRb复合体而引起PRb的降解失活,从而参与NSCLC的发生发展过程;3、NSCLC组织中CDK2蛋白表达程度与良性病变组比较有显著差异,且在肺癌组CDK-2蛋白高表达与HPV感染有关,提示HPV可能通过作用于细胞周期调节因子,从而使细胞周期调节失控,诱发肿瘤细胞的增殖和发展;4、Rb、CDK-2蛋白表达水平均与HPV阳性的NSCLC密切相关,它们可能与HPV在NSCLC发生发展中起到协同作用。更多还原

【Abstract】 【Objection】1、To investigate the etiological role of HPV on the carcinogenesis of NSCLC and to study the relationship between HPV infection and clinical pathological characteristics.2、To study the relationship between HPV infection and the expression of Rb、CDK-2 protein, and to elucidate the possible molecular mechanisms that HPV infection induced carcinogenesis of NSCLC.3、To study the relationship between the expression of Rb、CDK-2 protein and carcinogenesis of NSCLC and clinical pathological characteristics.【Methods】60 cases of specimens were taken from Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. All patients didn’t receive preoperative radiotherapy and chemotherapy. There were 29 cases of squamous cell carcinoma, 18 cases of adenocacinoma in 47 cases of NSCLC and high, medium and poorly differentiated lung cancer cases were 12, 18, 17; 28 cases of lymph node metastasis; 33 cases of smokers. There were 13 cases of benign pulmonary lesions (4 cases of pulmonary tuberculosis, 5 cases of inflammatory pseudotumor, 4 cases of bullous lung) as the control group. All specimens were selected to collect and record their age, sex, smoking history, lymph node metastasis and histological type and so on.1、The PCR methods were used to detect the HPV positive rate and the gene type by HPV16、18 type-specific primers in 47 cases of NSCLC and 13 cases of lung benign diseases.2、The immunohistochemical SP method was used to measure the expression of protein Rb and CDK-2 The protein expression levels of Rb、CDK-2 in 47 cases of NSCLC and 13 cases of lung benign diseases. Pictures for the result of IHC staining were gathered by system for study pictures. All data was dealt with SPSS for Windows 13.0 statistical software.【Results】1、The detection of HPV infection and protein Rb in 47 cases of lung cancer and 13 cases of benign pathological change lung tissues The PCR methods were used to detect the HPV positive rate and the gene type16/18. Detection rate of HPV DNA in lung cancer group was 42.55 %( 20/47) and that in benign disease group was 7.69 %( 1/13). There was a significant difference between the two groups (P<0.05).The immunohistochemical SP method were used to detect the expression of Rb protein and CDK2 protein。Negative expression rate of Rb protein was 68.09% (32/47) in lung cancer group, and there was significantly different with that in benign diseases 100% (13/13)(P<0.05), which suggested that the decrease of Rb protein might be one of the reasons for lung cancer. The expression of CDK-2 protein in lung cancer group was 82.98% ( 39/47), but there was no expression in the lung benign pathological change group. There was statistical difference between the two groups (P<0.05).2、The expression of protein Rb and CDK2 between HPV positive group and HPV negative group in 47 cases of lung cancerThe positive expression level of protein Rb in HPV positive lung cancers group was 45%(9/20). However, the positive expression rate of protein Rb in HPV negative of lung cancers was 85.19% (23/27). There was statistical difference between the two groups (P <0.05), from which we could imagine that HPV infection might directly or indirectly influence the expression and the effect of protein Rb.The expression level of protein CDK2 in HPV positive lung cancers group was 100%, and the expression rate of protein CDK2 in HPV negative of lung cancers was 70.37% (19/27). There was statistical difference between the two groups (P <0.05).3、The relationship among the infection of HPV and the expression of protein Rb and CDK2 with their clinic pathologic characteristics in 47 cases of lung cancer No correlation was found between HPV infection rate and age、sex、lymph node metastasis of patient. But there was a significant correlation among HPV infection rate with histological type、degree of differentiation、history of smoking(P<0.05). The HPV infection rate in squamous cell carcinoma 55.17% (16/29) was significantly higher than that in adenocarcinoma 22.22% (4/18). HPV infection rate in high、moderately and poorly differentiated NSCLC groups was 16.67%(2/12)、66.67% (12/18) and 35.29%( 6/17). There was statistical difference among the three groups (P <0.05) and it was higher in moderately and poorly differentiated NSCLC groups. HPV infection rate in smoking lung cancer patients’was 54.54%( 18/33) which was significantly higher than that in non-smokers 14.29% (2/14).IHC methods were used to detect the expression of Rb protein and CDK2 protein in 47cases of NSCLC tissues and analysis of their relationship with clinical pathological parameters (age, sex, smoking history, histological type, differentiation degree, lymph node metastasis). There was no relationship between Rb negative expression and the clinical pathological characteristics of NSCLC. The relationship was found between the expression of CDK-2 protein and degree of carcinoma differentiation. Expression rate of CDK-2 protein in high differentiation lung cancer was 58.33% (7/12) and 83.33% (15/18) in middle differentiation lung cancer, which was 100% (17/17) in low differentiation lung cancer. It prompted that there was a relationship between the expression of protein CDK2 and the differentiation of the lung cancer (p<0.05). But there was no relationship between the expression of CDK-2 protein and the other clinical pathological characteristics.【Conclusion】1. HPV16/18 high-risk type infection of NSCLC tissue was significantly higher than benign pathological change lung tissues, and HPV infection was related to histological type、degree of carcinoma differentiation and history of smoking. It suggested that HPV infection was one of important etiological factors in carcinogenesis of NSCLC, especially squamous cell carcinoma. 2. The expression of Rb protein was significantly different between NSCLC tissues and benign pathological change lung tissues, and in the lung cancer group the abnormal expression of Rb protein associated with HPV infection, there was a relationship between aberrant expression of Rb in lung cancer group and HPV infection. HPV might form a complex with Rb protein namely E7-PRb compound, and then made PRb degradation and inactivation, consequently HPV participate in progression of NSCLC.3. The expression of CDK2 protein was significantly different between NSCLC tissues and benign pathological change lung tissues, and there was a relationship between high expression of CDK-2 protein in lung cancer group and HPV infection. This suggested that HPV might act on cell cycle regulators and induce carcinogenesis of NSCLC by breaking the balance the cell cycle.4. There was relationship between expression of Rb、CDK-2 protein and NSCLC with HPV positive. They might have a cooperation role in carcinogenesis of NSCLC.更多还原