發(fā)布時(shí)間：2018-02-06 03:38

  本文關(guān)鍵詞： 端錨聚合酶 宮頸癌 XAV939 beta-鏈蛋白　出處：《鄭州大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：背景與目的 宮頸癌（cervical carcinoma）是危害中國(guó)女性身體健康最常見的婦科惡性腫瘤，目前發(fā)病機(jī)制已明確，90%的宮頸癌患者都能檢測(cè)到人乳頭瘤病毒（Humanpapilloma virus，HPV）的表達(dá)，但是感染了HPV的患者僅有一小部分會(huì)發(fā)展成宮頸癌，持續(xù)性高危型HPV感染只是宮頸癌發(fā)生發(fā)展的第一步，真正促進(jìn)其進(jìn)一步惡化的機(jī)制尚待探討。此外，宮頸腺癌和一小部分鱗癌患者早期不易發(fā)現(xiàn),僅依靠宮頸病變?nèi)A梯診斷法（細(xì)胞學(xué)、陰道鏡及病理組織學(xué)檢查）不能做到完全篩查高危患者；對(duì)宮頸癌的治療，采用手術(shù)治療適用范圍有限，而且有可能損傷年輕患者的生育功能，放化療副作用大且容易耐受，一旦復(fù)發(fā)再次用藥效果較差。所以探討宮頸癌的發(fā)病機(jī)制，尋求一種靈敏性和特異性較高的早期輔助診斷的技術(shù)和找到一種更為有效的針對(duì)性治療方法依然是必要且迫切的。 最近國(guó)內(nèi)外一些研究發(fā)現(xiàn)，Wnt/β-catenin信號(hào)通路的改變可能是繼HPV感染之后促進(jìn)宮頸癌的發(fā)生發(fā)展的“第二次重大事件”，其中β-catenin (又稱β-鏈蛋白，β-連接蛋白)在細(xì)胞核的異常積累是該通路的核心環(huán)節(jié)。事實(shí)上，，近幾年有很多學(xué)者都開始著手研究Wnt/β-catenin信號(hào)通路和惡性腫瘤的相關(guān)性，但是多集中于乳腺癌、結(jié)腸癌、口腔鱗癌等，而對(duì)于宮頸癌的研究尚處于起步階段。目前許多學(xué)者已經(jīng)開始嘗試通過(guò)干預(yù)Wnt/β-catenin通路調(diào)整靶基因表達(dá)，以達(dá)到抑制宮頸癌發(fā)生發(fā)展的目的。 端粒酶在宮頸病變的早期都有表達(dá)，而且與宮頸病變的發(fā)生發(fā)展密切相關(guān)。國(guó)內(nèi)外很多學(xué)者通過(guò)端粒酶擴(kuò)增法和熒光原位雜交法驗(yàn)證了該結(jié)論，指出高達(dá)90%的宮頸癌組織中有端粒酶陽(yáng)性表達(dá)，端粒酶的催化亞基對(duì)判斷宮頸病變的進(jìn)展和轉(zhuǎn)歸有重大提示意義。目前多認(rèn)為端粒酶不僅可作為宮頸癌的生物學(xué)標(biāo)志，并能有效用于宮頸癌的早期篩查，輔助診斷以及判斷預(yù)后。 其中端錨聚合酶（TRF1-interacting ankyrin-related ADP-ribose polymerase，Tankyrase）不僅是Wnt經(jīng)典通路的正向調(diào)控因子，而且是介導(dǎo)端粒與端粒酶結(jié)合的關(guān)鍵端粒調(diào)控蛋白，能夠維持端粒平衡。2009年《Nature》雜志提出的一個(gè)化學(xué)小分子物質(zhì)XAV939，是利用化學(xué)遺傳學(xué)法專門制備的Tankyrase的特殊抑制劑，它可以通過(guò)穩(wěn)定軸蛋白Axin的表達(dá)特異性阻滯Wnt/β-catenin信號(hào)通路的活化從而限制腫瘤細(xì)胞增殖和生長(zhǎng)。 本研究通過(guò)檢測(cè)端錨聚合酶抑制劑XAV939對(duì)宮頸癌Hela細(xì)胞增殖和凋亡的影響及對(duì)細(xì)胞核質(zhì)內(nèi)Tankyrase和β-catenin表達(dá)的調(diào)控情況，進(jìn)一步探索宮頸癌相關(guān)發(fā)病機(jī)制和分析該藥物應(yīng)用于臨床的可行性。 材料與方法 復(fù)蘇和培養(yǎng)宮頸癌Hela細(xì)胞至對(duì)數(shù)生長(zhǎng)期，首先用MTT法分別檢測(cè)端錨聚合酶抑制劑XAV939、奈達(dá)鉑及兩者聯(lián)合作用下Hela細(xì)胞的抑制率；然后通過(guò)流式細(xì)胞儀測(cè)定不同濃度下XAV939對(duì)Hela細(xì)胞凋亡周期的影響；最后采用免疫印跡檢測(cè)Tankyrase和β-catenin在宮頸癌Hela細(xì)胞的表達(dá)情況，以及受到XAV939不同濃度作用48小時(shí)后的表達(dá)有無(wú)異常改變。 采用SPSS19.0統(tǒng)計(jì)分析相關(guān)實(shí)驗(yàn)數(shù)據(jù)，以α=0.05作為檢驗(yàn)水準(zhǔn)。 結(jié)果 1、MTT法實(shí)驗(yàn)結(jié)果顯示，隨著XAV939濃度的增加，藥物作用時(shí)間的延長(zhǎng)，XAV939對(duì)Hela細(xì)胞生長(zhǎng)抑制率不斷增加，呈現(xiàn)濃度和時(shí)間依賴性（P＜0.05），半數(shù)抑制率隨著時(shí)間的增加呈現(xiàn)下降趨勢(shì)；XAV939與奈達(dá)鉑的合用效應(yīng)與藥物劑量和作用時(shí)間相關(guān)，短期小劑量時(shí)應(yīng)用時(shí)表現(xiàn)為協(xié)同效應(yīng)（CI＜1）,長(zhǎng)期大劑量應(yīng)用時(shí)表現(xiàn)為拮抗效應(yīng)（CI＞1）。 2、流式細(xì)胞儀檢測(cè)結(jié)果顯示，隨著XAV939濃度的增加和作用時(shí)間的延長(zhǎng)，Anterior G0/G1期的Hela細(xì)胞明顯增多（P＜0.05），S期的細(xì)胞百分比呈現(xiàn)下降趨勢(shì)（P＜0.05），而GO/G1期和G2/M期的百分比則無(wú)顯著性差異。 3、免疫印跡結(jié)果顯示，Tankyrase和β-catenin在宮頸癌Hela細(xì)胞中高表達(dá)；加入XAV939藥物作用后兩者表達(dá)均減少，且隨著藥物濃度的升高， Hela細(xì)胞中Tankyrase、β-catenin的表達(dá)逐漸減少。 結(jié)論 1、端錨聚合酶抑制劑XAV939對(duì)Hela細(xì)胞有較明顯的殺傷作用。XAV939短期可增加Hela細(xì)胞對(duì)奈達(dá)鉑敏感性，促使Hela細(xì)胞凋亡；隨著合用時(shí)間的延長(zhǎng)、劑量的加大XAV939可能降低奈達(dá)鉑的藥效。 2、XAV939對(duì)Hela細(xì)胞的作用主要表現(xiàn)為阻止DNA合成和誘導(dǎo)細(xì)胞凋亡。 3、端錨聚合酶可作為治療宮頸癌潛在的分子靶點(diǎn)。
[Abstract]:Background and purpose
Cervical cancer (cervical carcinoma) China is harmful to the health of women the most common gynecologic malignant tumor, the pathogenesis is clear, 90% of the patients with cervical cancer can be detected by the human papilloma virus (Humanpapilloma, virus, HPV) expression of HPV infected patients, but only a small part will develop into cervical cancer, continued of high-risk HPV infection is only the first step in the development of cervical cancer, and promote the further deterioration of the mechanism is still to be explored. In addition, a small part of cervical adenocarcinoma and squamous cell carcinoma patients is not easy to find, only rely on the three step method for the diagnosis of cervical lesions (cytology, colposcopy and pathological examination) can not be fully screening high-risk patients; the treatment of cervical cancer, the surgical treatment of limited scope, and may damage the reproductive function for young patients, chemotherapy side effects and easy to tolerate, once again relapse Therefore, it is still necessary and urgent to explore the pathogenesis of cervical cancer, find a sensitive and specific early diagnosis technology and find a more effective targeted therapy.
Some study found recently, Wnt/ beta -catenin signaling pathway may be changed after HPV infection after promoting the occurrence and development of cervical cancer "second major events, including beta -catenin (also known as beta chain protein, beta catenin) is the core part of this pathway in the nucleus. In fact the abnormal accumulation there is correlation, many scholars have begun to study Wnt/ beta -catenin signaling pathway and malignant tumor in recent years, but more focused on breast cancer, colon cancer, oral squamous cell carcinoma, and for cervical cancer research is still in its infancy. At present, many scholars have begun to try through the intervention of Wnt/ beta -catenin pathway regulating target gene expression and to inhibit the occurrence and development of cervical cancer.
Telomerase is expressed in the early stage of cervical lesions, and closely related with the occurrence and development of cervical lesions. Many scholars at home and abroad by telomerase amplification and fluorescence in situ hybridization method to verify the conclusion, pointed out that the telomerase expression is as high as 90% of cervical cancer tissues, the catalytic subunit of telomerase in judging the progression of cervical lesions the outcome and major implications. It is considered that telomerase not only can be used as a biological marker of cervical cancer, and can be used for early screening of cervical cancer, diagnosis and prognosis.
Which tankyrase (TRF1-interacting ankyrin-related ADP-ribose polymerase, Tankyrase) is not only a positive regulatory factor Wnt classic pathway, and is mediated by telomere and telomerase with critical telomere regulatory protein, able to maintain a small chemical molecules XAV939.2009 Journal of telomere balance is proposed, a special inhibitor specifically prepared by Tankyrase chemical genetics method, it can activate the expression of Axin protein stability axis specific blocking Wnt/ beta -catenin signaling pathway to limit the proliferation and growth of tumor cells.
Regulation of expression of the study on the proliferation and apoptosis of cervical cancer Hela cells by detecting tankyrase inhibitors XAV939 and Tankyrase on the cell nucleus and beta -catenin, to further explore the mechanism of cervical cancer pathogenesis and drug analysis of the feasibility of clinical application.
Materials and methods
Recovery and cultivation of cervical cancer Hela cells in logarithmic growth phase, firstly were detected by MTT assay of tankyrase inhibitors XAV939, nedaplatin and their combination inhibition rate of Hela cells; and then through the determination of effects of different concentrations of XAV939 on apoptosis of Hela cell cycle by flow cytometry; finally, by Western blot detection of Tankyrase and beta expression of -catenin in cervical cancer Hela cells, and the expression of XAV939 by different concentrations for 48 hours after there is no abnormal change.
The relevant experimental data were statistically analyzed by SPSS19.0, and alpha =0.05 was used as the test level.
Result
1, MTT method, experimental results show that with the increase of XAV939 concentration, prolong the time of drug action, XAV939 growth inhibition rate of Hela cells increased, showing a time and concentration dependent (P < 0.05), half inhibition rate decreased with time increasing; XAV939 and Nedaplatin Combined with drug dose and effect the application of short action time, low dose showed a synergistic effect (CI < 1), long-term high-dose application showed antagonistic effects (CI > 1).
2, flow cytometry results showed that, with the increase of XAV939 concentration and the prolongation of action time, the Anterior G0/G1 phase Hela cells increased significantly (P < 0.05), the percentage of S phase cells showed a decreasing trend (P < 0.05), but the percentage of GO/G1 phase and G2/M phase had no significant difference.
3, Western blotting showed that Tankyrase and beta -catenin were highly expressed in cervical cancer Hela cells. The expression of both XAV939 and XAV939 decreased after drug treatment, and the expression of Tankyrase and beta -catenin gradually decreased with the increase of drug concentration.
conclusion
1, the terminal anchor polymerase inhibitor XAV939 has a significant killing effect on Hela cells..XAV939 can increase the sensitivity of Hela cells to nedaplatin in a short time, and promote the apoptosis of Hela cells. With the prolongation of the time of combination, the dose of XAV939 may decrease the efficacy of nedaplatin.
2, the effect of XAV939 on Hela cells is mainly to prevent DNA synthesis and induce cell apoptosis.
3, the end anchor polymerase can be used as a potential molecular target for the treatment of cervical 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】：2014
【分類號(hào)】：R737.33

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