發(fā)布時(shí)間：2018-06-19 19:32

  本文選題：PDCD5 + 表達(dá)��； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：實(shí)驗(yàn)?zāi)康?子宮內(nèi)膜癌被稱為是女性的生殖系統(tǒng)中最為常見(jiàn)的惡性腫瘤其中之一。流行病學(xué)研究表明,包括高齡、初潮提前、更年期推遲、未經(jīng)產(chǎn)、長(zhǎng)期使用雌激素、肥胖和糖尿病在內(nèi)的某些危險(xiǎn)因素與子宮內(nèi)膜癌的風(fēng)險(xiǎn)增加有關(guān)。絕大多數(shù)子宮內(nèi)膜癌是源于單層上皮細(xì)胞的癌癥。根據(jù)其臨床特征和發(fā)病機(jī)制,子宮內(nèi)膜癌大體可以分為兩種類型:Ⅰ型和Ⅱ型。Ⅰ型子宮內(nèi)膜癌最常發(fā)生于更年期之前和更年期左右,占子宮內(nèi)膜癌的75-90%。它們通常是子宮內(nèi)膜樣癌,依賴雌激素,藥物治療效果較好。Ⅱ型子宮內(nèi)膜癌通常發(fā)生于較年長(zhǎng)的絕經(jīng)后人群。它們通常是非子宮內(nèi)膜樣癌(例如漿液性癌、透明細(xì)胞癌和粘液癌等),這種類型不依賴雌激素并且預(yù)后較差。據(jù)報(bào)告,目前有很多腫瘤抑制基因和致癌基因(例如PTEN和K-ras)與子宮內(nèi)膜癌的發(fā)生有著一定的聯(lián)系。細(xì)胞程序性死亡5(PDCD5),一種新近發(fā)現(xiàn)的促凋亡基因,它是由北京大學(xué)的人類疾病基因研究中心從他們正常培養(yǎng)的白血病細(xì)胞株細(xì)胞中通過(guò)克隆而獲得到的,它也被稱為 TFAR19(TF-1 cell apoptosis related gene 19)。PDCD5 通過(guò)與Tip60(組蛋白乙酰轉(zhuǎn)移酶)的相互作用促進(jìn)DNA損傷誘發(fā)的細(xì)胞凋亡,由多功能激酶CK2誘導(dǎo)的PDCD5磷酸化是PDCD5發(fā)揮促凋亡作用的重要步驟。PDCD5的功能與抑癌基因P53有關(guān)。PDCD5與YY1相關(guān)因子2(YAF2)相互作用,促進(jìn)P53介導(dǎo)的基因毒性應(yīng)激反應(yīng)。依托泊苷治療時(shí)去泛素化酶OTU(OTUD5)與PDCD5結(jié)合,有效的介導(dǎo)了 PDCD5及P53的順序激活。PDCD5與P53結(jié)合后對(duì)P53途徑起正向調(diào)控作用。PDCD5選擇性介導(dǎo)組蛋白去乙�；�(HDAC3)與p53解離,誘導(dǎo)HDAC3的裂解及泛素依賴的蛋白酶體降解。PDCD5表達(dá)受DNAJB1負(fù)相調(diào)控,DNAJB1可以靶向PDCD5抑制P53依賴的癌細(xì)胞凋亡。此外,PDCD5可通過(guò)抑制人類骨肉瘤細(xì)胞系MG-63中的Ras/Raf/MEK/ERK信號(hào)通路來(lái)抑制腫瘤的發(fā)生,攜帶PDCD5基因的腺病毒可對(duì)人類白血病細(xì)胞系發(fā)揮有效的抗腫瘤作用。不僅如此,而且,目前已經(jīng)在多種癌癥中觀察到PDCD5的下調(diào),比如說(shuō)乳腺癌、卵巢癌、胃癌、肝細(xì)胞癌、急性和慢性粒細(xì)胞白血病、膠質(zhì)瘤和喉鱗狀細(xì)胞癌,PDCD5的下調(diào)與腫瘤的發(fā)展和預(yù)后密切相關(guān)。這些研究表明,PDCD5可能發(fā)揮了腫瘤抑制基因的作用,在癌癥的發(fā)病機(jī)制中扮演重要角色。然而,PDCD5在子宮內(nèi)膜癌的表達(dá)及其在臨床的應(yīng)用價(jià)值尚未得到充分闡述。本課題旨在利用qRT-PCR、western blot、免疫組化及免疫細(xì)胞化學(xué)檢測(cè)對(duì)照子宮內(nèi)膜組織、子宮內(nèi)膜樣子宮內(nèi)膜癌組織,以及對(duì)照子宮內(nèi)膜腺上皮細(xì)胞、子宮內(nèi)膜癌細(xì)胞系KLE中PDCD5的表達(dá)水平,分析其與子宮內(nèi)膜癌患者臨床與病理特征的相關(guān)性,初步探尋PDCD5在子宮內(nèi)膜癌發(fā)生、發(fā)展過(guò)程中所起到的作用。實(shí)驗(yàn)方法:一、采用qRT-PCR技術(shù)來(lái)檢測(cè)對(duì)照子宮內(nèi)膜組織與子宮內(nèi)膜樣子宮內(nèi)膜癌中的PDCD5mRNA表達(dá)的情況二、采用western blot技術(shù)來(lái)檢測(cè)對(duì)照子宮內(nèi)膜組織與子宮內(nèi)膜樣子宮內(nèi)膜癌中的PDCD5蛋白表達(dá)的情況三、采用免疫組織化學(xué)(IHC)技術(shù)來(lái)檢測(cè)子對(duì)照宮內(nèi)膜組織與子宮內(nèi)膜樣子宮內(nèi)膜癌中的PDCD5蛋白表達(dá)的情況四、采用免疫細(xì)胞化學(xué)(ICC)技術(shù)來(lái)檢測(cè)對(duì)照子宮內(nèi)膜腺上皮細(xì)胞與子宮內(nèi)膜癌細(xì)胞系KLE中的PDCD5表達(dá)的情況五、利用卡方檢驗(yàn)來(lái)分析PDCD5表達(dá)與子宮內(nèi)膜癌患者的臨床病理參數(shù)的相關(guān)性實(shí)驗(yàn)結(jié)果:一、qRT-PCR檢測(cè)對(duì)照子宮內(nèi)膜組織與子宮內(nèi)膜樣子宮內(nèi)膜癌中的PDCD5 mRNA表達(dá)水平17份對(duì)照子宮內(nèi)膜組織和16份新鮮冷凍的子宮內(nèi)膜樣子宮內(nèi)膜癌組織中的PDCD5 mRNA檢測(cè)顯示,正常對(duì)照子宮內(nèi)膜組織和子宮內(nèi)膜樣子宮內(nèi)膜癌組織之間的PDCD5 mRNA表達(dá)沒(méi)有顯著差異(P0.05)。二、Western blot檢測(cè)對(duì)照子宮內(nèi)膜組織與子宮內(nèi)膜樣子宮內(nèi)膜癌中的PDCD5蛋白表達(dá)水平17份對(duì)照子宮內(nèi)膜組織和16份新鮮冷凍的子宮內(nèi)膜樣子宮內(nèi)膜癌組織中的PDCD5蛋白檢測(cè)顯示,子宮內(nèi)膜樣子宮內(nèi)膜癌組織中的PDCD5蛋白的表達(dá)明顯低于對(duì)照子宮內(nèi)膜組織(P0.001),與qRT-PCR的結(jié)果不一致。三、IHC檢測(cè)正常對(duì)照子宮內(nèi)膜組織與子宮內(nèi)膜樣子宮內(nèi)膜癌中的PDCD5蛋白表達(dá)水平1、IHC結(jié)果顯示,PDCD5蛋白主要位于對(duì)照子宮內(nèi)膜腺細(xì)胞或子宮內(nèi)膜樣子宮內(nèi)膜癌細(xì)胞的細(xì)胞質(zhì)中,這些細(xì)胞的胞核中也有低水平的PDCD5表達(dá)。2、51份子宮內(nèi)膜樣子宮內(nèi)膜癌組織中的PDCD5蛋白IHC檢測(cè)結(jié)果明顯低于53份對(duì)照子宮內(nèi)膜組織中的水平(P0.01)。3、53份對(duì)照子宮內(nèi)膜組織的增殖期和分泌期之間的PDCD5表達(dá)水平?jīng)]有明顯差異(P0.05)。4、中、低分化的子宮內(nèi)膜樣子宮內(nèi)膜癌組織中的PDCD5蛋白表達(dá)明顯少于對(duì)照子宮內(nèi)膜組織(P0.001);高分化的子宮內(nèi)膜樣子宮內(nèi)膜癌組織和對(duì)照子宮內(nèi)膜組織之間沒(méi)有明顯差別。四、ICC技術(shù)檢測(cè)對(duì)照子宮內(nèi)膜腺上皮細(xì)胞與子宮內(nèi)膜癌細(xì)胞系KLE中的PDCD5表達(dá)水平1、子宮內(nèi)膜腺上皮細(xì)胞表達(dá)細(xì)胞角蛋白,子宮內(nèi)膜間質(zhì)細(xì)胞表達(dá)波形蛋白。2、PDCD5陽(yáng)性染色主要位于對(duì)照子宮內(nèi)膜腺上皮細(xì)胞和KLE細(xì)胞的細(xì)胞質(zhì)中,在上述細(xì)胞的胞核中也發(fā)現(xiàn)了 PDCD5的弱表達(dá)。KLE細(xì)胞中的PDCD5蛋白表達(dá)比對(duì)照子宮內(nèi)膜腺上皮細(xì)胞中的表達(dá)弱。五、子宮內(nèi)膜樣子宮內(nèi)膜癌組織中的PDCD5蛋白表達(dá)與患者臨床病理參數(shù)的相關(guān)性PDCD5蛋白水平和年齡、子宮肌層浸潤(rùn)、FIGO期、雌激素受體或孕激素受體之間沒(méi)有明顯的關(guān)聯(lián),但與腫瘤分化程度有顯著的相關(guān)性(P0.05)。與高分化子宮內(nèi)膜樣子宮內(nèi)膜癌樣本相比,中、低分化子宮內(nèi)膜樣子宮內(nèi)膜癌中的PDCD5的表達(dá)明顯降低(P0.05),這表明,PDCD5的表達(dá)可能與子宮內(nèi)膜癌的發(fā)生發(fā)展相關(guān)。實(shí)驗(yàn)結(jié)論:一、子宮內(nèi)膜樣子宮內(nèi)膜癌組織與對(duì)照子宮內(nèi)膜組織之間的PDCD5 mRNA表達(dá)沒(méi)有顯著差異,但子宮內(nèi)膜樣子宮內(nèi)膜癌組織中PDCD5蛋白比對(duì)照子宮內(nèi)膜組織中減少,提示子宮內(nèi)膜癌PDCD5蛋白表達(dá)可能存在轉(zhuǎn)錄后調(diào)控。二、子宮內(nèi)膜樣子宮內(nèi)膜癌組織中PDCD5表達(dá)在子宮內(nèi)膜的增生期和分泌期無(wú)明顯差別,提示PDCD5表達(dá)可能不受卵巢激素水平的調(diào)控。三、子宮內(nèi)膜樣子宮內(nèi)膜癌組織中PDCD5蛋白表達(dá)水平與腫瘤分化程度有關(guān),提示PDCD5表達(dá)可能在子宮內(nèi)膜樣子宮內(nèi)膜癌的發(fā)生發(fā)展過(guò)程中起了重要作用,并可能有助于改善預(yù)后。創(chuàng)新性及意義:一、本項(xiàng)研究首次發(fā)現(xiàn)PDCD5蛋白在子宮內(nèi)膜樣子宮內(nèi)膜癌組織中表達(dá)降低,并且表達(dá)水平與腫瘤分化程度有關(guān)。二、本項(xiàng)研究結(jié)果提示PDCD5可能在子宮內(nèi)膜癌的發(fā)生發(fā)展過(guò)程中發(fā)揮重要的作用,并有可能作為子宮內(nèi)膜癌診斷和治療的新靶點(diǎn)。本研究的局限性:本項(xiàng)研究所有結(jié)果均是臨床標(biāo)本及細(xì)胞的檢測(cè),缺乏體外實(shí)驗(yàn)和體內(nèi)實(shí)驗(yàn)的進(jìn)一步確證,有關(guān)PDCD5在子宮內(nèi)膜癌中的作用及其機(jī)制還有待于進(jìn)一步的探討。
[Abstract]:Objective: endometrial cancer is known as one of the most common malignant tumors in the female reproductive system. Epidemiological studies have shown that some dangerous risk factors, including estrogen, obesity, and diabetes, are associated with increased risk of endometrial cancer, including advanced age, early menarche, postponement of menopause, and non oestrus, and the risk factors for long-term use of estrogen, obesity and diabetes. Endometrial carcinoma is a cancer of single layer epithelial cells. According to its clinical characteristics and pathogenesis, endometrial carcinoma can be divided into two types: type I and type II. Type I endometrial carcinoma is most often occurring before and around menopause, and the 75-90%. of endometrium cancer is usually endometrioid carcinoma, depending on female excitation. Type II endometrial carcinoma usually occurs in older postmenopausal women. They are usually non endometrioid carcinomas (such as serous, transparent and mucous), which are not dependent on estrogen and have a poor prognosis. It is reported that there are many tumor suppressor genes and oncogenic genes (such as PTEN, for example. And K-ras) associated with the occurrence of endometrial cancer. Cell programmed death 5 (PDCD5), a newly discovered proapoptotic gene, was obtained by cloning of the human disease gene center of Peking University from their normal cultured leukemic cell lines, and also known as TFAR19 (TF-1 cell apoptosi). S related gene 19).PDCD5 can promote apoptosis induced by DNA damage through interaction with Tip60 (histone acetyltransferase). PDCD5 phosphorylation induced by multifunctional kinase CK2 is an important step for PDCD5 to play a role in promoting apoptosis; the function of.PDCD5 is associated with the interaction of.PDCD5 and associated factor 2, which is associated with the P53 of the tumor suppressor gene. Mediated gene toxicity stress response. Etoposide treated deubiquitinase OTU (OTUD5) combined with PDCD5, effectively mediated the sequence activation of PDCD5 and P53,.PDCD5 and P53 binding to P53 pathway..PDCD5 selective mediated histone deacetylase (HDAC3) and p53 dissociation, inducing HDAC3 fragmentation and ubiquitin dependence. The expression of proteasome degradation.PDCD5 is regulated by DNAJB1 negative phase, and DNAJB1 can target PDCD5 to inhibit the apoptosis of P53 dependent cancer cells. In addition, PDCD5 can inhibit the occurrence of tumor by inhibiting the Ras/Raf/MEK/ERK signaling pathway in human osteosarcoma cell line MG-63, and adenosoncosis with PDCD5 gene can play an effective role in human leukemia cell lines. Not only that, but also the downregulation of PDCD5, such as breast, ovarian, gastric, hepatocellular carcinoma, acute and chronic granulocytic leukemia, glioma and laryngeal squamous cell carcinoma, and the downregulation of PDCD5 are closely related to the development and prognosis of the tumor. These studies suggest that PDCD5 may play an important role. The role of tumor suppressor genes plays an important role in the pathogenesis of cancer. However, the expression of PDCD5 in endometrial cancer and its clinical application have not been fully explained. This topic aims to use qRT-PCR, Western blot, immunohistochemistry and immunocytochemical test to compare endometrium and endometrium in uterus. The expression level of PDCD5 in the endometrial adenocarcinoma cell line KLE and the endometrial carcinoma cell line, and the correlation between the clinical and pathological features of endometrial carcinoma, and the preliminary exploration of the use of PDCD5 in the development and development of endometrial carcinoma. The expression of PDCD5mRNA in endometrial tissue and endometrioid carcinoma two, Western blot technique was used to detect the expression of PDCD5 protein in endometrioid carcinoma and endometrioid endometrioid carcinoma. Immunohistochemistry (IHC) was used to detect endometrium and endometrium in the endometrium. The expression of PDCD5 protein in endometrial carcinoma (four), the expression of PDCD5 in endometrial adenocarcinoma and endometrial carcinoma cell line KLE was detected by immunocytochemistry (ICC). The correlation between PDCD5 expression and the clinicopathological parameters of endometrium cancer patients was analyzed by chi square test. The expression of PDCD5 mRNA in endometrial endometrial carcinoma and endometrioid endometrioid carcinoma by qRT-PCR and PDCD5 mRNA in 17 control endometrium tissues and 16 freshly frozen endometrioid endometrioid endometrioid carcinoma tissues; PDCD5 between normal endometrium and endometrioid endometrioid carcinoma tissue There was no significant difference in mRNA expression (P0.05). Two, Western blot detected the expression level of PDCD5 protein in endometrioid endometrioid carcinoma and endometrioid endometrioid carcinoma, PDCD5 protein detection in endometrioid endometrioid carcinoma tissues of 16 fresh frozen endometrioid endometrioid carcinoma tissues and endometrioid endometrial carcinoma tissues were detected. The expression of PDCD5 protein in the endometrium was significantly lower than that of the control endometrium (P0.001), which was not consistent with the results of qRT-PCR. Three, IHC was used to detect the expression of PDCD5 protein in endometrioid endometrioid carcinoma and endometrioid endometrioid carcinoma by IHC. The results of IHC showed that the PDCD5 protein was mainly in the endometrium or endometrioid uterus of the endometrium. In the cytoplasm of endometrial cancer cells, there are also low levels of PDCD5 expression in the nucleus of these cells, and the results of PDCD5 protein IHC in endometrioid endometrioid carcinoma tissues of.2,51 are significantly lower than that in 53 controls (P0.01).3,53 portion of PDCD5 expression water between the proliferative and secretory phases of the endometrial tissue. There was no significant difference (P0.05).4. The expression of PDCD5 protein in endometrioid endometrioid carcinoma tissues of low differentiated endometrioid endometrioid carcinoma was significantly less than that of the control endometrium (P0.001), and there was no significant difference between the highly differentiated endometrioid endometrioid carcinoma tissue and the control endometrium. Four, ICC technique was used to detect the endometrium epithelium. The expression level of PDCD5 in cell and endometrial carcinoma cell line KLE was 1, the epithelial cells of endometrium expressed cytokeratin, and the endometrial stromal cells expressed vimentin.2. The positive staining of PDCD5 was mainly in the cytoplasm of the endometrial gland epithelial cells and KLE cells of the control uterus. The weak table of PDCD5 was also found in the nuclei of the above cells. The expression of PDCD5 protein in the.KLE cells was weaker than that in the control of endometrial adenoepithelial cells. Five, the expression of PDCD5 protein in endometrioid endometrial carcinoma tissues was related to the level and age of the clinicopathological parameters of the patients, PDCD5 protein level and age, the uterine myometrium infiltration, the FIGO stage, the estrogen receptor or progesterone receptor. Correlation, but has a significant correlation with the degree of tumor differentiation (P0.05). Compared with highly differentiated endometrioid endometrioid carcinoma samples, the expression of PDCD5 in endometrioid endometrioid carcinoma in low differentiated endometrioid endometrioid carcinoma is significantly decreased (P0.05), which suggests that the expression of PDCD5 may be associated with the development of endometrial carcinoma. There was no significant difference in the expression of PDCD5 mRNA between endometrial carcinoma and control endometrium, but PDCD5 protein in endometrioid endometrioid carcinoma decreased in endometrium endometrium than in control endometrium, suggesting that the expression of PDCD5 protein in endometrial carcinoma may have post transcriptional regulation. Two, PDCD5 in endometrioid endometrium carcinoma tissue. There is no obvious difference in the expression of PDCD5 expression in endometrium, suggesting that the expression of PDCD5 may not be regulated by the level of ovarian hormone. Three, the expression of PDCD5 protein in endometrioid endometrium carcinoma is related to the degree of tumor differentiation, suggesting that the expression of PDCD5 may play a role in the development of endometrioid endometrial carcinoma. The important role, and may help to improve the prognosis, innovation and significance. First, this study first found that the expression of PDCD5 protein in endometrioid endometrial carcinoma is reduced, and the level of expression is related to the degree of tumor differentiation. Two, the results of this study suggest that PDCD5 may play an important role in the development of endometrial cancer. The role, and may be a new target for the diagnosis and treatment of endometrial cancer. The limitations of this study: all the results are clinical specimens and cells, the lack of further confirmation of in vitro and in vivo experiments. The role of PDCD5 in endometrial cancer and its mechanism remain to be further explored.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.33

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