本文選題：胃癌 + 細(xì)胞質(zhì)Rap1��； 參考：《鄭州大學(xué)》2016年碩士論文

【摘要】：目的胃癌是人類(lèi)消化系統(tǒng)中常見(jiàn)的惡性腫瘤,其發(fā)病率在我國(guó)惡性腫瘤中位居第二位,死亡率位居第三位,嚴(yán)重威脅人類(lèi)健康[1]。絕大部分患者在確診胃癌時(shí)已處于中晚期,胃癌的診斷治療及預(yù)后目前仍不樂(lè)觀,關(guān)于胃癌的基礎(chǔ)與臨床研究顯得非常重要。端粒是位于真核生物染色體末端的DNA-蛋白結(jié)構(gòu),由大量的端粒結(jié)合蛋白和許多TTAGGG重復(fù)序列組成。端粒結(jié)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)六個(gè)蛋白組成Shelterin復(fù)合體[2]。已經(jīng)證明端粒防止染色體末端發(fā)生融合、降解、丟失和重排等[3],在細(xì)胞發(fā)生分裂時(shí),端粒長(zhǎng)度會(huì)逐漸縮短,當(dāng)端粒長(zhǎng)度縮短到一定程度時(shí),染色體無(wú)法繼續(xù)進(jìn)行半保留復(fù)制,細(xì)胞開(kāi)始出現(xiàn)衰老死亡[4]。端粒結(jié)合蛋白參與維持端粒結(jié)構(gòu)和功能的穩(wěn)定,與細(xì)胞衰老和癌變密切相關(guān)[5]。最近研究發(fā)現(xiàn)端粒結(jié)合蛋白除了在端粒發(fā)揮作用外,也具有端粒外功能[6-11]。Rap1與TRF2在端粒組成復(fù)合體,敲除TRF2會(huì)導(dǎo)致細(xì)胞核Rap1明顯減少,人類(lèi)細(xì)胞的細(xì)胞質(zhì)中也存在一定數(shù)量Rap1,這部分Rap1不與TRF2結(jié)合且不受TRF2表達(dá)水平的影響[12]。細(xì)胞質(zhì)Rap1通過(guò)結(jié)合端粒外TTAGGG位點(diǎn)調(diào)控基因的轉(zhuǎn)錄[6]。另外Teo等發(fā)現(xiàn)細(xì)胞質(zhì)Rap1蛋白與IκB激酶(IκB kinases,IKKs)相互作用,磷酸化NF-κB的p65亞基,激活NF-κB信號(hào)通路。在乳腺癌組織中細(xì)胞質(zhì)Rap1表達(dá)增加,抑制Rap1會(huì)促進(jìn)乳腺癌細(xì)胞凋亡[8],但細(xì)胞質(zhì)Rap1在胃癌發(fā)生發(fā)展中的作用仍不清楚。本研究采用免疫組化技術(shù)檢測(cè)細(xì)胞質(zhì)Rap1、細(xì)胞核NF-κB p65在胃癌組織、癌旁組織以及正常胃粘膜組織中的表達(dá)及相關(guān)性,探討其在胃癌的發(fā)生發(fā)展中可能的作用及臨床意義。材料與方法選取2013年4月至2015年7月在鄭州大學(xué)第一附屬醫(yī)院經(jīng)手術(shù)切除胃癌、癌旁(距離腫瘤組織邊緣小于3 cm以?xún)?nèi))及正常胃粘膜組織(距離腫瘤組織邊緣大于3cm)各90例,且術(shù)前均未接受放、化療及免疫治療。其中男47例,女43例;年齡32~80歲,50歲的48例,≤50歲的42例,中位年齡50.3歲。TNM分期:TNMⅠ~Ⅱ期35例,Ⅲ~Ⅳ期55例;病理分型均為腺癌:高分化腺癌30例,中分化腺癌28例,低分化腺癌32例;淋巴結(jié)轉(zhuǎn)移:不伴淋巴結(jié)轉(zhuǎn)移者38例,伴淋巴結(jié)轉(zhuǎn)移者52例。浸潤(rùn)深度:未超過(guò)漿膜層者28例,超過(guò)漿膜層者62例。全部患者均知情同意且所有標(biāo)本均通過(guò)了鄭州大學(xué)第一附屬醫(yī)院倫理委員會(huì)的批準(zhǔn)。使用40 g/L甲醛固定所需標(biāo)本,將固定好的標(biāo)本包埋在石蠟中,然后經(jīng)過(guò)切片機(jī)切成厚約4μm切片,脫蠟水化,最后高溫高壓修復(fù),采用免疫組織化學(xué)染色法SP,嚴(yán)格按照試劑盒說(shuō)明書(shū)的具體步驟進(jìn)行操作。以已知的陽(yáng)性胃癌組織標(biāo)本切片作為陽(yáng)性對(duì)照,以PBS緩沖液代替一抗作為陰性對(duì)照。采用SPSS17.0統(tǒng)計(jì)軟件包進(jìn)行統(tǒng)計(jì)學(xué)分析,采用χ2檢驗(yàn)進(jìn)行組間差異比較,采用Spearman檢驗(yàn)進(jìn)行相關(guān)分析,P0.05認(rèn)為差異具有統(tǒng)計(jì)學(xué)意義。結(jié)果(1)Rap1蛋白為細(xì)胞質(zhì)與細(xì)胞核著色,以細(xì)胞質(zhì)著色判斷為陽(yáng)性,陽(yáng)性染色呈深棕色。Rap1蛋白在正常胃粘膜組織主要為細(xì)胞核染色,細(xì)胞質(zhì)染色較弱,細(xì)胞質(zhì)Rap1在胃癌組織中表達(dá)增加。細(xì)胞質(zhì)Rap1蛋白在正常胃組織、癌旁組織及胃腺癌組織中的陽(yáng)性表達(dá)率逐漸升高,分別是22.2%、37.8%、75.6%,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。(2)NF-κBp65蛋白為細(xì)胞質(zhì)與細(xì)胞核著色,以細(xì)胞核著色判斷為陽(yáng)性,陽(yáng)性染色呈深棕色。細(xì)胞核NF-κBp65蛋白在正常胃粘膜組織、癌旁組織及胃腺癌組織中的陽(yáng)性表達(dá)率也是逐漸升高,分別是24.5%、46.7%、70.0%,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。(3)Rap1蛋白在胃腺癌組織中的表達(dá)與癌組織的分化程度、淋巴結(jié)轉(zhuǎn)移、浸潤(rùn)深度及TNM分期相關(guān)(P0.05),與患者性別、年齡無(wú)關(guān)(P0.05)。NF-κBp65蛋白在胃腺癌組織中的表達(dá)與癌組織的分化程度、淋巴結(jié)轉(zhuǎn)移、浸潤(rùn)深度及TNM分期相關(guān)(P0.05),與患者性別、年齡無(wú)關(guān)(P0.05)。(4)細(xì)胞質(zhì)Rap1蛋白與細(xì)胞核NF-κBp65蛋白在胃腺癌中表達(dá)呈正相關(guān)(r=0.241,P0.05)。結(jié)論(1)細(xì)胞質(zhì)Rap1蛋白在胃癌組織中的表達(dá)高于其在癌旁組織、正常胃粘膜組織中的表達(dá)。Rap1蛋白在胃腺癌組織中的表達(dá)與癌組織的分化程度、淋巴結(jié)轉(zhuǎn)移、浸潤(rùn)深度及TNM分期相關(guān)。提示細(xì)胞質(zhì)Rap1蛋白可能參與胃癌的發(fā)生發(fā)展。(2)細(xì)胞核NF-κBp65蛋白在胃癌組織中的表達(dá)高于其在癌旁組織、正常胃粘膜組織中的表達(dá)。NF-κBp65蛋白在胃腺癌組織中的表達(dá)與癌組織的分化程度、淋巴結(jié)轉(zhuǎn)移、浸潤(rùn)深度及TNM分期相關(guān)。提示NF-κBp65蛋白可能參與胃癌的發(fā)生發(fā)展。(3)細(xì)胞質(zhì)Rap1與細(xì)胞核NF-κB p65在胃癌中的表達(dá)呈明顯正相關(guān),提示Rap1細(xì)胞質(zhì)表達(dá)可能與NF-κB所介導(dǎo)的信號(hào)轉(zhuǎn)導(dǎo)通路相關(guān),共同參與胃癌的發(fā)生發(fā)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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R735.2

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