【作者】 赵雅娟；

【导师】 殷飞；

【作者基本信息】 河北医科大学 ， 内科学， 2008， 硕士

【摘要】 目的:Raf/MEK/ERK通路在周期调控、细胞增殖、细胞凋亡、细胞的生长抑制及分化中起着重要的调控作用,是一种重要的肿瘤生物学进程。Cyclin D1是一种作用于G1期的细胞周期相关蛋白,与多种蛋白或癌基因协同作用而发生过表达。Raf/MEK/ERK通路激活后,促进Cyclin D1与CDK4/6结合形成复合体而磷酸化并激活CDK,加速细胞G1/S期的转化而促进细胞增殖,进而促进肿瘤发生。有研究提示Raf/MEK/ERK通路的激活及Cyclin D1的过表达与肝细胞肝癌的发生密切相关。本实验用免疫组织化学技术比较Raf-1、MEK-1、ERK-1及Cyclin D1在肝细胞癌、癌旁硬化肝组织及正常肝组织中的表达情况,探讨各蛋白间相关性、临床意义及对预后的影响。方法:1研究对象:50例肝细胞癌癌组织、17例癌旁硬化肝组织及14例肝血管瘤周边正常肝组织(作为对照)均来自河北医科大学第四医院肝胆外科2001年11月~2006年5月手术切除标本。所有组织类型均经河北医科大学第四医院病理科确诊。所有患者术前均未进行任何形式的治疗。2研究方法:组织切除后经10%中性甲醛固定后石蜡包埋备用。应用免疫组织化学法检测Raf-1、MEK-1、ERK-1及Cyclin D1在不同肝组织中的表达情况。对肝癌患者的临床资料进行汇总,探讨各蛋白间的相互关系及其与肝细胞癌患者临床表现间的关系。随访患者生存情况,比较各蛋白表达对不同临床分期肝癌患者预后的影响。结果:1临床病理资料对肝癌患者术后生存的影响:年龄、癌栓、病灶大小、HBsAg、HBeAb、癌灶数目及包膜完整性等对肝细胞癌患者的预后的影响无显著性差异,而临床分期(χ~2=7.636,P=0.006)、病理分级(χ~2=6.296,P=0.043)及AFP(χ~2=4.07,P=0.044)对肝细胞癌患者的预后的影响有显著性差异。临床分期较早、病理分化较好、AFP较低者,生存率较高,平均生存时间较长,预后较好。2 Raf-1在各组肝组织均有表达,表现为细胞浆及细胞膜黄染。Raf-1在癌组织的表达(弱17例、中20例、强9例/50例)显著高于癌旁硬化肝组织(弱5例/17例)和正常肝组织(弱2例/14例)(Z=-5.079和Z=-5.082,P=0.000),癌旁硬化肝组织表达和正常组织表达统计学差异无显著性。Raf-1在肝癌组织的表达与性别、癌灶数目、AFP、HBeAb阴阳性、乙肝有无及包膜是否完整均无关;在肝癌临床分期较早的癌组织Raf-1的表达强于分期较晚者(χ~2=13.496,P=0.009;r=-0.452,P=0.001);在肝癌病理分级高分化的癌组织Raf-1的表达强于低分化者(χ~2=14.904,P=0.001;r=-0.547,P=0.000);在年龄大于或等于50岁者癌组织Raf-1的表达强于小于50岁者(Z=-2.109,P=0.035;r=0.301,P=0.034);在肿瘤小于5cm者癌组织Raf-1的表达强于大于或等于5cm者(Z=-2.502,P=0.012;r=-0.357,P=0.011);在不伴癌栓者癌组织Raf-1的表达强于伴癌栓者(Z=-3.246,P=0.001;r=-0.464,P=0.001)。Raf-1表达(-~+)及(++~+++)者累积生存率分别为14.3%和54.3%,平均生存时间分别为12.93月和28.23月。Raf-1表达越强,生存率越高,平均生存时间越长,预后越好(χ~2=6.793,P=0.009)。3 MEK-1在各组肝组织均有表达,表现为细胞浆及细胞膜黄染。MEK-1在肝癌组织的表达(弱14例、中24例、强7例/50例)显著高于癌旁硬化肝组织(弱6例/17例)和正常肝组织(弱3例/14例)(Z=-4.909和Z=-4.873,P=0.000),癌旁硬化肝组织表达和正常组织表达统计学差异无显著性。MEK-1在肝癌组织的表达与性别、癌灶数目、AFP、HBeAb阴阳性、乙肝有无及包膜是否完整均无关;在肝癌临床分期较早的癌组织MEK-1的表达强于分期较晚者(χ~2=9.869,P=0.043;r=-0.433,P=0.002);在肝癌病理分级高分化的癌组织MEK-1的表达强于低分化者(χ~2=17.569,P=0.000;r=-0.599,P=0.000);在年龄大于或等于50岁者癌组织MEK-1的表达强于小于50岁者(Z=-2.375,P=0.018;r=0.339,P=0.016);在肿瘤小于5cm者癌组织MEK-1的表达强于大于或等于5cm者(Z=-2.635,P=0.008;r=-0.376,P=0.007);在不伴癌栓者癌组织MEK-1的表达强于伴癌栓者(Z=-2.891,P=0.004;r=-0.413,P=0.003)。MEK-1表达(-~+)及(++~+++)者累积生存率分别为15.4%和51.5%,平均生存时间分别为13.77月和27.64月。MEK-1表达越强,生存率越高,平均生存时间越长,预后越好(χ~2=6.581,P=0.01)。4 ERK-1在各组肝组织均有表达,表现为细胞浆和(或)细胞核黄染,以细胞浆为主。ERK-1在肝癌组织的表达(弱13例、中22例、强7例/50例)显著高于癌旁硬化肝组织(弱7例/17例)和正常肝组织(弱2例/14例)(Z=-4.264和Z=-4.635,P=0.000),癌旁硬化肝组织表达和正常组织表达统计学差异无显著性。ERK-1在肝癌组织的表达与性别、癌灶数目、AFP、HBeAb阴阳性、乙肝有无及包膜是否完整均无关;在肝癌临床分期较早的癌组织ERK-1的表达强于分期较晚者(χ~2=10.227,P=0.037;r=-0.422,P=0.002);在肝癌病理分级高分化的癌组织ERK-1的表达强于低分化者(χ~2=18.946,P=0.000;r=-0.615,P=0.000);在年龄大于或等于50岁者癌组织ERK-1的表达强于小于50岁者(Z=-2.266,P=0.023;r=0.324,P=0.022);在肿瘤小于5cm者癌组织ERK-1的表达强于大于或等于5cm者(Z=-2.769,P=0.006;r=-0.396,P=0.004);在不伴癌栓者癌组织ERK-1的表达强于伴癌栓者(Z=-2.953,P=0.003;r=-0.422,P=0.002)。ERK-1表达(-~+)及(++~+++)者累积生存率分别为9.5%和55%,平均生存时间分别为11.71月和28.92月。ERK-1表达越强,生存率越高,平均生存时间越长,预后越好(χ~2=9.869,P=0.002)。5 Cyclin D1在各组肝组织均有表达,表现为细胞核和(或)细胞浆黄染,以细胞核为主。Cyclin D1在肝癌组织的表达(弱14例、中17例、强8例/50例)显著高于癌旁硬化肝组织(弱6例/17例)和正常肝组织(弱3例/14例)(Z=-3.853和Z=-3.987,P=0.000),癌旁硬化肝组织表达和正常组织表达统计学差异无显著性。Cyclin D1在肝癌组织的表达与临床分期、性别、年龄、癌灶数目、肿瘤大小、AFP、HBeAb阴阳性、乙肝有无、有无癌栓及包膜是否完整均无关;在肝癌病理分级高分化的癌组织Cyclin D1的表达强于低分化者(χ~2=6.227,P=0.044;r=-0.352,P=0.012)。Cyclin D1表达(-~+)及(++~+++)者累积生存率分别为14.3%和55.6%,平均生存时间分别为15.55月和27.26月。Cyclin D1表达越强,生存率越高,平均生存时间越长,预后越好(χ~2=4.219,P=0.04)。6肝癌组织Raf-1、MEK-1、ERK-1与Cyclin D1表达情况的相关性:肝癌组织中Raf-1与MEK-1的表达呈正相关(r=0.747,P=0.000),与ERK-1的表达呈正相关(r=0.691,P=0.000),与Cyclin D1的表达呈正相关(r=0.341,P=0.015);MEK-1与ERK-1的表达呈正相关(r=0.858,P=0.000),与Cyclin D1的表达呈正相关(r=0.429,P=0.002);ERK-1与Cyclin D1的表达呈正相关(r=0.378,P=0.007)。结论:1临床分期、病理分级及AFP对肝细胞癌患者的预后的影响有显著性差异,临床分期较早、病理分化较好、AFP较低者,生存率较高,生存期较长,预后较好。2 Raf-1、MEK-1、ERK-1在肝癌组织中的表达显著升高;青壮年患者、伴有癌栓、分化较差、肿瘤较大、属临床分期较晚期者,Raf-1、MEK-1、ERK-1阳性表达较弱,生存率较低,生存期较短,预后较差。3 Cyclin D1在肝癌组织中的表达显著升高;在肝癌病理分级为高分化的癌组织中Cyclin D1的表达较强,生存率较高,生存期较长,预后较好。4肝癌组织中Raf-1、MEK-1、ERK-1、Cyclin D1的表达两两之间均呈正相关。更多还原

【Abstract】 Objective: Raf/MEK/ERK pathway played an important regulatory role in cell cycle regulation, cell proliferation, cell apoptosis, cellular growth inhibiting and differentiation. This cascade was an essential oncogenic progression. Cyclin D1 was a cell cycle associated protein that affected on the G1 phase of the cell cycle. It could be overexpressed when it played coorperation with lots of proteins or oncogenes. After the Raf/MEK/ERK pathway was actived, Cyclin D1 was promoted to form an active enzyme complex with cyclin dependent kinase 4/6 (CDK4/6) that phosphorylated and activated CDK and leaded to G1/S transition. Thus, it enhanced cell proliferation and promoted tumorigenesis. There were studies about the activation of Raf/MEK/ERK pathway and the Cyclin D1 overexpression in human hepatocellular carcinoma. To identify whether different Raf-1, MEK-1, ERK-1 and Cyclin D1 expressional levels predicts different clinical outcome of hepatocellular carcinoma (HCC), we perform immunostaining of these oncogenes in cancerous, non-cancerous and normal hepatic tissues. Our research indicates new clinical target for tumor theraphy. Methods: 1 Samples collection and patients follow up: 50 specimens of HCC, 17 specimens of adjacent non-cancerous cirrhosis hepatic tissue and 14 specimens of normal hepatic tissue obtained from the fourth hospital of Hebei Medical University without clinical treatment before operation. All patients were followed up for 2 years.2 Imunostaining: The samples were fixed by 10% formaldehyde, embeded in paraffin after excision. Raf-1, MEK-1, ERK-1 and Cyclin D1 were stained, and two pathologist without backgroud on these samples graded these oncogenes expression and proliferation on each tissue.Resluts: 1 Survival analysis of the clinical and pathological data in postoperative HCC patients: Age, the portal vein tumor thrombosis, the size of tumor, HBsAg, HBeAb, the number of tumor foci and the integrity of the tumor’s envelope were no statistical difference in postoperative HCC patients. The clinical stage (χ~2=7.636, P=0.006), the pathological classification (χ~2=6.296, P=0.043) and AFP (χ~2=4.07, P=0.044) were the significant prognostic factors in postoperative HCC patients.2 Raf-1 mostly expressed in cytoplasm and cellular membrane. The Raf-1 expressed not differently refered to sex, the number of tumor foci, AFP, HBeAb, HBsAg and the integrity of the tumor’s envelope based on our statistical analysis. In the 50 HCC samples, Raf-1 weakly expressed in 17 samples, moderately expressed in 20 samples and strongly expressed 9 samples, repectively. Whereas it weakly expressed in 5/17 of adjacent non-cancerous cirrhosis hepatic tissues and 2/14 of normal hepatic tissues. Raf-1 expression frequency in HCC was significantly higher than that in adjacent non-cancerous hepatic tissue and in normal hepatic tissue (Z=-5.079 and Z=-5.082, P=0.000), and no statistical difference existed bwteen in adjacent non-cancerous hepatic tissue and in normal hepatic tissue. Our data demonstrated that Raf-1 expression was positively correlated with hepatic carcinogenesis. Raf-1 expression in early clinical stage in HCC was stronger than that in late clinical stage (χ~2=13.496, P=0.009. r=-0.452, P=0.001). Raf-1 expression in the well-differentiated HCC about the pathological classification was stronger than that in poorly differentiated tissue (χ~2=14.904, P=0.001. r=-0.547, P=0.000). Raf-1 expression in older than or equal to 50 years old of HCC patients was stronger than that in younger than 50 years old (Z=-2.109, P=0.035. r=0.301, P=0.034). Raf-1 expression in less than 5cm about the size of tumor was stronger than that in larger than or equal to 5cm (Z=-2.502, P=0.012. r=-0.357, P=0.011). Raf-1 expression in the tissue without the portal vein tumor thrombosis was stronger than that with the portal vein tumor thrombosis (Z=-3.246, P=0.001. r=-0.464, P=0.001). The patients in whom Raf-1 negatively and weakly expressed had 12.93 months’mean survival time, and the cumulative survival rate was 14.3%. The patients in whom Raf-1 moderately and strongly expressed had 28.23 months’ mean survival time, and the cumulative survival rate was 54.3%. Raf-1 expression was stronger, the survival rate was higher, and the prognosis was better (By single variable Log rank test,χ~2=6.793, P=0.009).3 MEK-1 mostly expressed in cytoplasm and cellular membrane. The MEK-1 expressed not differently refered to sex, the number of tumor foci, AFP, HBeAb, HBsAg and the integrity of the tumor’s envelope based on our statistical analysis. In the 50 HCC samples, MEK-1 weakly expressed in 14 samples, moderately expressed in 24 samples and strongly expressed 7 samples, repectively. Whereas it weakly expressed in 6/17 of adjacent non-cancerous cirrhosis hepatic tissues and 3/14 normal hepatic tissues. MEK-1 expression frequency in HCC was significantly higher than that in adjacent non-cancerous hepatic tissue and in normal hepatic tissue (Z=-4.909 and Z=-4.873, P=0.000), and no statistical difference existed bwteen in adjacent non-cancerous hepatic tissue and in normal hepatic tissue. Our data demonstrated that MEK-1 expression was positively correlated with hepatic carcinogenesis. MEK-1 expression in early clinical stage in HCC was stronger than that in late clinical stage (χ~2=9.869, P=0.043. r=-0.433, P=0.002). MEK-1 expression in the well-differentiated HCC about the pathological classification was stronger than that in poorly differentiated tissue (χ~2=17.569, P=0.000. r=-0.599, P=0.000). MEK-1 expression in older than or equal to 50 years old of HCC patients was stronger than that in younger than 50 years old (Z=-2.375, P=0.018. r=0.339, P=0.016). MEK-1 expression in less than 5cm about the size of tumor was stronger than that in larger than or equal to 5cm (Z=-2.635, P=0.008. r=-0.376, P=0.007). MEK-1 expression in the tissue without the portal vein tumor thrombosis was stronger than that with the portal vein tumor thrombosis (Z=-2.891, P=0.004. r=-0.413, P=0.003). The patients in whom MEK-1 negatively and weakly expressed had 13.77 months’mean survival time, and the cumulative survival rate was 15.4%. The patients in whom MEK-1 moderately and strongly expressed had 27.64 months’mean survival time, and the cumulative survival rate was 51.5%. MEK-1 expression was stronger, the survival rate was higher, and the prognosis was better (By single variable Log rank test,χ~2=6.581, P=0.01).4 ERK-1 mostly expressed in cytoplasm and cellular nucleus. The ERK-1 expressed not differently refered to sex, the number of tumor foci, AFP, HBeAb, HBsAg and the integrity of the tumor’s envelope based on our statistical analysis. In the 50 HCC samples, ERK-1 weakly expressed in 13 samples, moderately expressed in 22 samples and strongly expressed 7 samples, repectively. Whereas it weakly expressed in 7/17 of adjacent non-cancerous cirrhosis hepatic tissues and 2/14 normal hepatic tissues. ERK-1 expression frequency in HCC was significantly higher than that in adjacent non-cancerous hepatic tissue and in normal hepatic tissue (Z=-4.264 and Z=-4.635, P=0.000), and no statistical difference existed bwteen in adjacent non-cancerous hepatic tissue and in normal hepatic tissue. Our data demonstrated that ERK-1 expression was positively correlated with hepatic carcinogenesis. ERK-1 expression in early clinical stage in HCC was stronger than that in late clinical stage (χ~2=10.227, P=0.03. r=-0.422, P=0.002). ERK-1 expression in the well-differentiated HCC about the pathological classification was stronger than that in poorly differentiated tissue (χ~2=18.946, P=0.000. r=-0.615, P=0.000). ERK-1 expression in older than or equal to 50 years old of HCC patients was stronger than that in younger than 50 years old (Z=-2.266, P=0.023. r=0.324, P=0.022). ERK-1 expression in less than 5cm about the size of tumor was stronger than that in larger than or equal to 5cm (Z=-2.769, P=0.006. r=-0.396, P=0.004). MEK-1 expression in the tissue without the portal vein tumor thrombosis was stronger than that with the portal vein tumor thrombosis (Z=-2.953, P=0.003. r=-0.422, P=0.002). The patients in whom ERK-1 negatively and weakly expressed had 11.71 months’mean survival time, and the cumulative survival rate was 9.5%. The patients in whom ERK-1 moderately and strongly expressed had 28.92 months’mean survival time, and the cumulative survival rate was 55%. ERK-1 expression was stronger, the survival rate was higher, and the prognosis was better (By single variable Log rank test,χ~2=9.869, P=0.002).5 Cyclin D1 mostly expressed in cellular nucleus and cytoplasm. The Cyclin D1 expressed not differently refered to clinical stage, sex, age, the size of tumor, the number of tumor foci, AFP, HBeAb, HbsAg, the portal vein tumor thrombosis and the integrity of the tumor’s envelope based on our statistical analysis. In the 50 HCC samples, Cyclin D1 weakly expressed in 14 samples, moderately expressed in 17 samples and strongly expressed 8 samples, repectively. Whereas it weakly expressed in 6/17 of adjacent non-cancerous cirrhosis hepatic tissues and 3/14 normal hepatic tissues. Cyclin D1 expression frequency in HCC was significantly higher than that in adjacent non-cancerous hepatic tissue and in normal hepatic tissue (Z=-3.853 and Z=-3.987, P=0.000), and no statistical difference existed bwteen in adjacent non-cancerous hepatic tissue and in normal hepatic tissue. Our data demonstrated that Cyclin D1 expression was positively correlated with hepatic carcinogenesis. Cyclin D1 expression in the well-differentiated HCC about the pathological classification was stronger than that in poorly differentiated tissue (χ~2=6.227, P=0.044. r=-0.352, P=0.012). The patients in whom Cyclin D1 negatively and weakly expressed had 15.55 months’mean survival time, and the cumulative survival rate was 14.3%. The patients in whom Cyclin D1 moderately and strongly expressed had 27.26 months’mean survival time, and the cumulative survival rate was 55.6%. Cyclin D1 expression was stronger, the survival rate was higher, and the prognosis was better (By single variable Log rank test,χ~2=4.219, P=0.04).6 The correlation among Raf-1, MEK-1, ERK-1 and Cyclin D1 expression in HCC: Raf-1 expressional level was positively correlated to MEK-1 expressional level in HCC (r=0.747, P=0.000), and was positively correlated ERK-1 expressional level in HCC (r=0.691, P=0.000), and was positively correlated to Cyclin D1 expressional level in HCC (r=0.341, P=0.015). MEK-1 expressional level was positively correlated to ERK-1 expressional level in HCC (r=0.858, P=0.000), and was positively correlated to Cyclin D1 expressional level in HCC (r=0.429, P=0.002). ERK-1 expressional level was positively correlated to Cyclin D1 expressional level in HCC (r=0.378, P=0.007).Conclusions: 1 The clinical stage, the pathological classification and AFP were the significant prognostic factors in postoperative HCC patients. In the patients in early clinical stage with low level of AFP whose hepatic tissue was well-differentiated, the prognosis was better.2 Raf-1, MEK-1, ERK-1 expression was positively correlated with hepatic carcinogenesis. Raf-1, MEK-1, ERK-1 weakly expressed in the younger patients, late clinical stage, poorly differentiated tissue, the big size of tumor and with the portal vein tumor thrombosis. While the survival rate was shorter, the prognosis was poorer.3 Cyclin D1 expression was positively correlated with hepatic carcinogenesis. Cyclin D1 strongly expressed in well-differentiated tissue. While the survival rate was higher, the prognosis was better. 4 There were positively correlations among Raf-1, MEK-1, ERK-1 and Cyclin D1 expression.更多还原