【作者】 蔺静；

【导师】 杜建层；

【作者基本信息】 河北医科大学 ， 妇产科学， 2011， 硕士

【摘要】 宫颈癌是全球女性中仅次于乳腺癌的最常见的妇科恶性肿瘤,在一些发展中国家其发病率位居第一。近年来宫颈癌有年轻化的发展趋势,严重威胁女性的生育,甚至生命。研究宫颈癌发生的分子机制,为宫颈癌的早期诊断和靶向治疗提供理论依据是十分重要的。研究证实肿瘤的发生与细胞内信息的传递密切相关,丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号转导途径是细胞内重要的信号传导系统,是众多信号转导途径的汇聚点和共同通道。在哺乳动物细胞中已经发现3种不同的MAPK,即胞外信号调控激酶(extracellular signal- regulated kinase,ERK)、c-Jun N末端激酶(c-Jun N-terminal kinase, JNK)、P38丝裂原活化蛋白激酶(P38 mitogen-activated protein kinase,P38)。其中与ERK相关的细胞外信号转导通路是MAPK系统中主要和经典的通路,研究证实,ERK通路参与调节细胞增殖、分化、转移、血管发生、凋亡等过程。PERK即磷酸化的ERK,是ERK的活化形式,是ERK信号通路活化的标志。c-fos定位于人染色体14q21-31,是编码核蛋白的癌基因,属于立早基因的成员,编码的核蛋白c-fos的分子量为62kD,由380个氨基酸组成。单独的c-fos是没有功能的,必须和c-jun形成异源二聚体后具有转录激活活性,故又称转录因子AP-1。AP-1可以结合到多种与肿瘤发生、演进相关的基因启动子和增强子上,诱导靶基因的过表达而引起致癌作用。研究表明,AP-1与细胞增殖、分化相关,在肿瘤转化乃至逆转中均起关键作用。目的:近年来许多肿瘤中包括胃癌、喉癌、卵巢癌、皮肤鳞状细胞癌等均发现ERKl/2、PERK、c-fos蛋白的高表达及活性异常。但有关ERK信号通路在人宫颈癌中的作用机制报道较少,目前国内尚无宫颈癌组织中PERK蛋白表达的研究报道。本研究以PERK作为ERK信号通路活化的标志,应用免疫组化方法检测PERK与c-fos蛋白在人宫颈癌组织中的表达,探讨ERK信号通路、c-fos蛋白在宫颈癌中的活化状态及作用机制,为宫颈癌的生物靶向性治疗提供可靠的理论依据。方法:选取河北大学附属医院、保定市第一中心医院及河北医科大学第三医院2007～2010年间有完整临床资料并经病理确诊的石蜡包埋宫颈组织共100例,分为三组,A组为宫颈癌组40例,分为高分化20例(A1组)和低分化20例(A2组),有淋巴结转移18例(A3组)和无淋巴结转移22例(A4组),B组为宫颈上皮内瘤变(cervicalintraepithelia lneoplasia,CIN)组30例,C组为正常宫颈组30例。所有组织术前均未经化疗和放疗。应用免疫组化方法检测PERK与c-fos蛋白在人宫颈癌组织中的表达。应用SPSS 13.0统计软件进行数据处理。临床一般资料为计数资料,采用多个样本率比较的卡方检验及校正的卡方检验。三者之间比较,P<0.05有统计学意义;三者中两两比较,P<0.0125有统计学意义。相关性分析用等级资料的Spearman秩相关,以r=0作为判断相关性存在的标准。结果:1 PERK蛋白在宫颈组织中的定位及表达:PERK的表达位于细胞核及细胞浆,但主要位于细胞核中,呈棕黄色颗粒,弥漫性分布,有时呈团块状或条索状聚集。PERK在A组、B组、C组中的阳性表达率分别为70%、36.7%、3.3%,三组之间差异有统计学意义(P<0.05)。A组中PERK的表达率明显升高,与B组及C组相比,差异均有统计学意义(P<0.0125),且B组与C组相比,差异有统计学意义(P<0.0125)。A1组中PERK的阳性表达率85%高于A2组的55%,差异有统计学意义(P<0.05);A3组中PERK的阳性表达率为88.9%高于A4组的54.5%,差异有统计学意义(P<0.05)。2 c-fos在宫颈组织中的定位及表达:c-fos的表达位于细胞核,呈棕黄色颗粒,弥漫性分布,有时呈团块状聚集。c-fos在A组、B组、C组中阳性表达率分别为57.5%、23.3%、0%,三组之间差异有统计学意义(P<0.05)。A组中c-fos的表达率明显升高,与B组及C组相比,差异均有统计学意义(P<0.0125),但B组与C组相比,差异无统计学意义(P=0.016)。A1组中c-fos的阳性表达率95%高于A2组的45%,差异有统计学意义(P<0.05);A3组中c-fos的阳性表达率为77.8%高于A4组的40.9%,差异有统计学意义(P<0.05)。3宫颈癌组中PERK蛋白表达和c-fos蛋白表达的相关性:宫颈癌组中PERK蛋白表达与c-fos蛋白表达呈正相关(r_s=0.430 ,P<0.05)。结论:1宫颈癌组中PERK、c-fos蛋白的表达明显高于宫颈上皮内瘤变组和正常对照组,提示PERK、c-fos蛋白的高表达可能参与宫颈癌的发生,为宫颈癌的早期诊断提供理论依据。2 PERK、c-fos蛋白在宫颈癌高分化组的表达高于低分化组,有淋巴结转移组的表达高于无淋巴结转移组,提示PERK、c-fos蛋白的高表达在宫颈癌的分化、转移中可能起促进作用。3 PERK蛋白与c-fos蛋白在宫颈癌中的表达呈正相关,推测ERK信号转导通路可能通过调控c-fos的表达促进宫颈癌的发生,两者协同作用,可能是宫颈癌发生的分子机制之一。更多还原

【Abstract】 Cervical carcinoma is a very common gynecologic malignant tumor just inferior to breast carcinoma in women globally. Morbidity stands first on the list in some developing countries. In recent years, occurrence of cervical carcinoma shows younger trend. It threatens the fertility of women, even their lives. So it is very important to research the molecular mechanism which leads to the occurrence of cervical cancer to provide theoretical basis for the early diagnosis of cervical carcinoma and effective treatments.It is reported that the occurrence of tumor is closely correlated with information transfer in cells. MAPK(mitogen-activated protein kinase,MAPK) signal pathway is the important signal transmission system in cells, which is many signal transmission pathways’focal point and co-channel. There are three different types of MAPK:the extracellular-signalregulated kinases (ERK), c-Jun N-terminal kinase (JNK), P38 mitogen -activated protein kinase (P38 pathway). The ERK pathway is one of the most important and classical pathways of MAPK.Several lines of evidence indicate that overexpression and activation of ERK play an important role in growth, proliferation, differentiation, progression of cells. PERK is the phosphorylation of ERK, which is the activated form of ERK and the activation marker of the ERK pathway.c-fos’s fixed position is at the chromosome 14q21- 31, c-fos is a cancer gene which codes to check nuclear protein, belongs to the early gene.The protein outcome has 380 amino acids.c-fos protein has no function alone.c-fos protein must become a different source dimmer with c-jun protein,then it has transcribe activation, therefore is as well as called transcribe factor (AP-1).The AP-1 can integrate many genes’promoter and enhancer which are related with tumors’occurrence and development,it can case target genes’expression then induce the occurrence of tumor. It can promote cell growth and divide,even induce tumor conversion.Objective: recent studies showed that in the stomach cancer, laryngo carcinoma, ovarian cancer,skin squamous cell carcinoma,the expression and activity of PERK, ERK, c-fos were nomal. However, reports about ERK signal pathway in human cervical cancer cells are few. No report about the PERK expression in cervical carcinoma tissue was found in our nation so far. This research focuses on PERK as a marker of ERK pathway activation, immuno- histochemistry was adopted to detect the expression of PERK、c-fos protein in the human cervical carcinoma tissues ,to discuss the expression of c-fos protein in the human cervical carcinoma and its relationship with the ERK signal pathway, to further provide the theoretical basis for therapy of cervical carcinoma.Method:100 cases of paraffin-embeded cervical tissues were selected between 2007～2010 from the Hospital of HeBei University , the NO.1 Middle Hospital of BaoDing and the Third Hospital of HeBei Medical University , with a completed approvement of pathological diagnosis following the surgery. These include 40 cases of cervical carcinoma group (A group), 30 cases of CIN group (B group), 30 cases of normal cervix group (B group). The A group including 20 well differentiated cases(A1 group)and 20 poorly differentiated cases (A2 group).And there are 18 cases with lymphonodus metastasis (A3 group), 22 cases without lymphonodus metastasis (A4 group). All the patients did not receive chemotherapy before surgery. The expression of PERK and c-fos were detected by immunohistochemistry in all specimens.The experimental results were analyzed by comparing the rate of multiple samples to chi-square test and correction chi-square test with SPSS 13.0. It was statistically significant (P <0.05) between three groups; compare with each other, P<0.0125 statistically significant. Correlation analysis using Spearman’s rank correlation level of information , r = 0 for judging the relevance of existing standards. Results:1 The expression and localization of PERK protein in cervical tissues: PERK protein was located in the nucleus and cytoplasm, almost located in the nucleus .The expression rates of PERK in A group,B group and C group were 70%, 36.7%, 3.3% respectively. There were significant differences among the three groups (P<0.05). The expression rate of PERK in A group was significantly higher than those in B group and C group (P<0.0125), while the expression of PERK in B group was significantly higher than C group (P<0.0125). The expression rate of PERK in A1 group was 85%, which was significantly higher than those in A2 group’s 55% (P <0.05). The expression rate of PERK in A3 group was 88.9%, which was significantly higher than those in A4 group’s 54.5% (P <0.05).2 The expression and localization of c-fos protein in cervical tissues: c-fos protein was located in the nucleus .The expression rates of c-fos in A group,B group and C group were 57.5%, 23.3%, 0% respectively. There were significant differences among the three groups (P <0.05). The expression rate of c-fos in A group was significantly higher than those in B group and C group (P <0.0125), while there was no significant difference was found between the B group and C group (P=0.016). The expression rate of c-fos in A1 group was 95%, which was significantly higher than those in A2 group’s 45% (P <0.05). The expression rate of c-fos in A3 group’s was 77.8%, which was significantly higher than those in A4 group’s 40.9% (P <0.05).3 The correlation of the expression of PERK protein and c-fos protein in cervical carcinoma: the expression of PERK protein was positive correlated with c-fos protein in cervical carcinoma tissues (rs= 0.430 P<0.05).Conclusions:1 The expression rates of PERK and c-fos in cervical carcinoma tissues were significantly higher than those in CIN tissues and normal cervix tissues. It predieted that the over expression of PERK and c-fos protein might be related to the occurrence of invasive cervical carcinoma,which might provide the basis for early diagnosis of invasive cervical carcinoma.2 The expression rates of PERK and c-fos in well differentiated cervical carcinoma tissues were higher than those in poorly differentiated tissues, cervical carcinoma tissues with lymphonodus metastasis were higher than cervical carcinoma tissues without lymphonodus metastasis. The high expression of PERK and c-fos protein may be play a promoting role in cervical carcinoma differentiation and metastasis.3 The PERK protein was positive correlated with c-fos protein in cervical carcinoma tissues. ERK signal pathway might play an important role in the initiation and development of the cervical carcinoma through c-fos protein, their cooperation might be one of the molecular mechanisms of cervical carcinoma.更多还原