细胞质Rap1与细胞核NF-κB p65在胃癌组织中的表达及相关性分析

发布时间：2018-05-22 09:19

本文选题：胃癌 + 细胞质Rap1　；参考：《郑州大学》2016年硕士论文

【摘要】：目的胃癌是人类消化系统中常见的恶性肿瘤,其发病率在我国恶性肿瘤中位居第二位,死亡率位居第三位,严重威胁人类健康[1]。绝大部分患者在确诊胃癌时已处于中晚期,胃癌的诊断治疗及预后目前仍不乐观,关于胃癌的基础与临床研究显得非常重要。端粒是位于真核生物染色体末端的DNA-蛋白结构,由大量的端粒结合蛋白和许多TTAGGG重复序列组成。端粒结合蛋白TRF1(telomeric-repeat binding factor-1)、TRF2(telomeric-repeat binding factor-2)、POT1(protection of telomeres 1)、TPP1(adrenocortical dysplasia protein homolog isoform1)、TIN2(TRF1-interacting nuclear protein 2)和Rap1(repressor activator protein 1)六个蛋白组成Shelterin复合体[2]。已经证明端粒防止染色体末端发生融合、降解、丢失和重排等[3],在细胞发生分裂时,端粒长度会逐渐缩短,当端粒长度缩短到一定程度时,染色体无法继续进行半保留复制,细胞开始出现衰老死亡[4]。端粒结合蛋白参与维持端粒结构和功能的稳定,与细胞衰老和癌变密切相关[5]。最近研究发现端粒结合蛋白除了在端粒发挥作用外,也具有端粒外功能[6-11]。Rap1与TRF2在端粒组成复合体,敲除TRF2会导致细胞核Rap1明显减少,人类细胞的细胞质中也存在一定数量Rap1,这部分Rap1不与TRF2结合且不受TRF2表达水平的影响[12]。细胞质Rap1通过结合端粒外TTAGGG位点调控基因的转录[6]。另外Teo等发现细胞质Rap1蛋白与IκB激酶(IκB kinases,IKKs)相互作用,磷酸化NF-κB的p65亚基,激活NF-κB信号通路。在乳腺癌组织中细胞质Rap1表达增加,抑制Rap1会促进乳腺癌细胞凋亡[8],但细胞质Rap1在胃癌发生发展中的作用仍不清楚。本研究采用免疫组化技术检测细胞质Rap1、细胞核NF-κB p65在胃癌组织、癌旁组织以及正常胃粘膜组织中的表达及相关性,探讨其在胃癌的发生发展中可能的作用及临床意义。材料与方法选取2013年4月至2015年7月在郑州大学第一附属医院经手术切除胃癌、癌旁(距离肿瘤组织边缘小于3 cm以内)及正常胃粘膜组织(距离肿瘤组织边缘大于3cm)各90例,且术前均未接受放、化疗及免疫治疗。其中男47例,女43例;年龄32~80岁,50岁的48例,≤50岁的42例,中位年龄50.3岁。TNM分期:TNMⅠ~Ⅱ期35例,Ⅲ~Ⅳ期55例;病理分型均为腺癌:高分化腺癌30例,中分化腺癌28例,低分化腺癌32例;淋巴结转移:不伴淋巴结转移者38例,伴淋巴结转移者52例。浸润深度:未超过浆膜层者28例,超过浆膜层者62例。全部患者均知情同意且所有标本均通过了郑州大学第一附属医院伦理委员会的批准。使用40 g/L甲醛固定所需标本,将固定好的标本包埋在石蜡中,然后经过切片机切成厚约4μm切片,脱蜡水化,最后高温高压修复,采用免疫组织化学染色法SP,严格按照试剂盒说明书的具体步骤进行操作。以已知的阳性胃癌组织标本切片作为阳性对照,以PBS缓冲液代替一抗作为阴性对照。采用SPSS17.0统计软件包进行统计学分析,采用χ2检验进行组间差异比较,采用Spearman检验进行相关分析,P0.05认为差异具有统计学意义。结果(1)Rap1蛋白为细胞质与细胞核着色,以细胞质着色判断为阳性,阳性染色呈深棕色。Rap1蛋白在正常胃粘膜组织主要为细胞核染色,细胞质染色较弱,细胞质Rap1在胃癌组织中表达增加。细胞质Rap1蛋白在正常胃组织、癌旁组织及胃腺癌组织中的阳性表达率逐渐升高,分别是22.2%、37.8%、75.6%,差异具有统计学意义(P0.05)。(2)NF-κBp65蛋白为细胞质与细胞核着色,以细胞核着色判断为阳性,阳性染色呈深棕色。细胞核NF-κBp65蛋白在正常胃粘膜组织、癌旁组织及胃腺癌组织中的阳性表达率也是逐渐升高,分别是24.5%、46.7%、70.0%,差异具有统计学意义(P0.05)。(3)Rap1蛋白在胃腺癌组织中的表达与癌组织的分化程度、淋巴结转移、浸润深度及TNM分期相关(P0.05),与患者性别、年龄无关(P0.05)。NF-κBp65蛋白在胃腺癌组织中的表达与癌组织的分化程度、淋巴结转移、浸润深度及TNM分期相关(P0.05),与患者性别、年龄无关(P0.05)。(4)细胞质Rap1蛋白与细胞核NF-κBp65蛋白在胃腺癌中表达呈正相关(r=0.241,P0.05)。结论(1)细胞质Rap1蛋白在胃癌组织中的表达高于其在癌旁组织、正常胃粘膜组织中的表达。Rap1蛋白在胃腺癌组织中的表达与癌组织的分化程度、淋巴结转移、浸润深度及TNM分期相关。提示细胞质Rap1蛋白可能参与胃癌的发生发展。(2)细胞核NF-κBp65蛋白在胃癌组织中的表达高于其在癌旁组织、正常胃粘膜组织中的表达。NF-κBp65蛋白在胃腺癌组织中的表达与癌组织的分化程度、淋巴结转移、浸润深度及TNM分期相关。提示NF-κBp65蛋白可能参与胃癌的发生发展。(3)细胞质Rap1与细胞核NF-κB p65在胃癌中的表达呈明显正相关,提示Rap1细胞质表达可能与NF-κB所介导的信号转导通路相关,共同参与胃癌的发生发展。
[Abstract]:Objective gastric cancer is a common malignant tumor in the human digestive system. Its incidence is the second place in the malignant tumor of our country. The mortality rate is the third place. Most of the patients who have serious threat to human health [1]. have been in the middle and late stage of diagnosis of gastric cancer. The diagnosis and treatment of gastric cancer are still not optimistic. The research is very important. Telomere is the structure of DNA- protein at the end of eukaryotic chromosomes, consisting of a large number of telomere binding proteins and many TTAGGG repeats. The telomere binding protein TRF1 (telomeric-repeat binding factor-1), TRF2 (telomeric-repeat binding factor-2), POT1 (protection) L dysplasia protein homolog isoform1), TIN2 (TRF1-interacting nuclear protein 2) and Rap1 (repressor activator) six proteins have proved that telomere prevents chromosomal end fusion, degradation, loss and rearrangement, and the telomere length will gradually shorten when cell division occurs, when telomere is telomere. When the length is shortened to a certain extent, the chromosome can not continue to be semi preserved, and the cells begin to appear senescence and death [4]. telomere binding protein to maintain the stability of telomere structure and function, which is closely related to cell senescence and carcinogenesis. [5]. recently found that telomere binding protein also has telomere function except for telomere. [6-11].Rap1 and TRF2 in the telomere composition complex, knockout TRF2 can lead to a significant decrease in the nucleus Rap1, and a certain number of Rap1 in the cytoplasm of human cells. This part of Rap1 does not bind to TRF2 and is not affected by the expression level of TRF2, and [12]. cytoplasm Rap1 is transcriptional [6]. in addition to the transcriptional [6]. by binding to the TTAGGG loci of the telomere. The cytoplasmic Rap1 protein is interacting with the I kappa B kinases (IKKs) kinase (I kappa B kinases, IKKs), and the p65 subunit of NF- kappa B phosphorylation activates the NF- B signal pathway. The expression of cytoplasm is increased in breast cancer tissue, and the inhibitory effect on the apoptosis of breast cancer cells is promoted, but the role of cytoplasm in the development of gastric cancer is still unclear. The expression and correlation of cytoplasmic Rap1 and nuclear NF- kappa B p65 in gastric cancer tissue, para cancer tissue and normal gastric mucosa were examined by chemical technique. The possible role and clinical significance in the development of gastric cancer were discussed. Materials and methods were selected from April 2013 to July 2015 at the First Affiliated Hospital of Zhengzhou University, and the cancer was excised by surgery and cancer. 90 cases (less than 3 cm within the margin of tumor tissue) and normal gastric mucosa (distance to the edge of tumor tissue were greater than 3cm) were not accepted, chemotherapy and immunotherapy before operation, including 47 men and 43 women; 48 cases of age 32~80, 48 cases of 50 years old, 42 cases younger than 50 years, 35 cases of TNM I ~ II, 55 cases in stage III ~ IV; All types were adenocarcinoma: 30 cases of highly differentiated adenocarcinoma, 28 cases of middle differentiated adenocarcinoma, 32 cases of low differentiated adenocarcinoma, 38 cases of lymph node metastasis, 38 cases without lymph node metastasis, 52 cases with lymph node metastasis, 28 cases without serous layer, 62 cases exceeding serous layer. All patients informed consent and all specimens passed the first attached Zhengzhou University It was approved by the ethics committee of the hospital. The specimens were fixed by 40 g/L formaldehyde, and the fixed specimens were embedded in the paraffin. Then the slice machine was cut into thick 4 Mu slices, dewaxing and hydrating, and finally repaired at high temperature and high pressure. The immunohistochemical staining method, SP, was used strictly in accordance with the specific steps of the kit instructions. The positive gastric cancer tissue specimens were taken as positive control and PBS buffer solution was replaced by PBS as negative control. Statistical analysis was carried out by SPSS17.0 statistical package. The difference was compared between groups by x 2 test and correlation analysis was carried out by Spearman test. P0.05 thought the difference was statistically significant. The results (1) Rap1 protein was cytoplasm and The positive staining of cytoplasmic staining was positive. The positive staining was dark brown.Rap1 protein in normal gastric mucosa, and the cytoplasmic staining was weak. Cytoplasmic Rap1 was expressed in gastric cancer tissue. The positive expression rate of cytoplasmic Rap1 protein in normal gastric tissue, para cancer tissues and gastric adenocarcinoma tissues gradually increased. The difference was 22.2%, 37.8% and 75.6% respectively. The difference was statistically significant (P0.05). (2) NF- kappa Bp65 protein was cytoplasm and nucleus coloring, and the positive staining was dark brown. The positive expression rate of NF- kappa Bp65 protein in normal gastric mucosa, and in para cancer tissue and gastric adenocarcinoma tissue was gradually increased, respectively. It was 24.5%, 46.7%, 70%, and the difference was statistically significant (P0.05). (3) the expression of Rap1 protein in gastric adenocarcinoma tissue was associated with the degree of differentiation, lymph node metastasis, infiltration depth and TNM staging (P0.05), and the expression of.NF- kappa Bp65 protein in gastric adenocarcinoma tissue and the degree of differentiation of cancer tissue, lymph node, and lymph node. Metastasis, depth of infiltration and TNM staging (P0.05) were not related to sex and age of the patients (P0.05). (4) the expression of cytoplasmic Rap1 protein and nuclear NF- kappa Bp65 protein in gastric adenocarcinoma was positively correlated (r=0.241, P0.05). Conclusion (1) the expression of cytoplasmic Rap1 protein in gastric cancer tissues is higher than that in the para cancer tissues and the expression of.Rap1 in normal gastric mucosa. The expression of protein in gastric adenocarcinoma tissue is related to the degree of differentiation, lymph node metastasis, depth of infiltration and TNM staging. It is suggested that cytoplasmic Rap1 protein may be involved in the development of gastric cancer. (2) the expression of nuclear NF- kappa Bp65 protein in gastric cancer tissues is higher than that in the paracancerous tissue, and the expression of.NF- kappa Bp65 protein in normal gastric mucosa tissue The expression in gastric adenocarcinoma tissue is related to the degree of differentiation, lymph node metastasis, depth of infiltration and TNM staging. It is suggested that NF- kappa Bp65 protein may be involved in the development of gastric cancer. (3) the expression of cytoplasmic Rap1 and nuclear NF- kappa B p65 in gastric cancer is positively correlated, suggesting that the cytoplasm expression of Rap1 may be related to the signal transduction mediated by NF- kappa B. The guide pathway is associated with the development of gastric cancer.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.2

【相似文献】