發(fā)布時間：2018-02-06 03:38

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

【摘要】：背景與目的 宮頸癌（cervical carcinoma）是危害中國女性身體健康最常見的婦科惡性腫瘤，目前發(fā)病機制已明確，90%的宮頸癌患者都能檢測到人乳頭瘤病毒（Humanpapilloma virus，HPV）的表達，但是感染了HPV的患者僅有一小部分會發(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，真正促進其進一步惡化的機制尚待探討。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（細胞學、陰道鏡及病理組織學檢查）不能做到完全篩查高�；颊撸粚m頸癌的治療，采用手術治療適用范圍有限，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，一旦復發(fā)再次用藥效果較差。所以探討宮頸癌的發(fā)病機制，尋求一種靈敏性和特異性較高的早期輔助診斷的技術和找到一種更為有效的針對性治療方法依然是必要且迫切的。 最近國內(nèi)外一些研究發(fā)現(xiàn)，Wnt/β-catenin信號通路的改變可能是繼HPV感染之后促進宮頸癌的發(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)在細胞核的異常積累是該通路的核心環(huán)節(jié)。事實上，，近幾年有很多學者都開始著手研究Wnt/β-catenin信號通路和惡性腫瘤的相關性，但是多集中于乳腺癌、結腸癌、口腔鱗癌等，而對于宮頸癌的研究尚處于起步階段。目前許多學者已經(jīng)開始嘗試通過干預Wnt/β-catenin通路調(diào)整靶基因表達，以達到抑制宮頸癌發(fā)生發(fā)展的目的。 端粒酶在宮頸病變的早期都有表達，而且與宮頸病變的發(fā)生發(fā)展密切相關。國內(nèi)外很多學者通過端粒酶擴增法和熒光原位雜交法驗證了該結論，指出高達90%的宮頸癌組織中有端粒酶陽性表達，端粒酶的催化亞基對判斷宮頸病變的進展和轉歸有重大提示意義。目前多認為端粒酶不僅可作為宮頸癌的生物學標志，并能有效用于宮頸癌的早期篩查，輔助診斷以及判斷預后。 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，而且是介導端粒與端粒酶結合的關鍵端粒調(diào)控蛋白，能夠維持端粒平衡。2009年《Nature》雜志提出的一個化學小分子物質(zhì)XAV939，是利用化學遺傳學法專門制備的Tankyrase的特殊抑制劑，它可以通過穩(wěn)定軸蛋白Axin的表達特異性阻滯Wnt/β-catenin信號通路的活化從而限制腫瘤細胞增殖和生長。 本研究通過檢測端錨聚合酶抑制劑XAV939對宮頸癌Hela細胞增殖和凋亡的影響及對細胞核質(zhì)內(nèi)Tankyrase和β-catenin表達的調(diào)控情況，進一步探索宮頸癌相關發(fā)病機制和分析該藥物應用于臨床的可行性。 材料與方法 復蘇和培養(yǎng)宮頸癌Hela細胞至對數(shù)生長期，首先用MTT法分別檢測端錨聚合酶抑制劑XAV939、奈達鉑及兩者聯(lián)合作用下Hela細胞的抑制率；然后通過流式細胞儀測定不同濃度下XAV939對Hela細胞凋亡周期的影響；最后采用免疫印跡檢測Tankyrase和β-catenin在宮頸癌Hela細胞的表達情況，以及受到XAV939不同濃度作用48小時后的表達有無異常改變。 采用SPSS19.0統(tǒng)計分析相關實驗數(shù)據(jù)，以α=0.05作為檢驗水準。 結果 1、MTT法實驗結果顯示，隨著XAV939濃度的增加，藥物作用時間的延長，XAV939對Hela細胞生長抑制率不斷增加，呈現(xiàn)濃度和時間依賴性（P＜0.05），半數(shù)抑制率隨著時間的增加呈現(xiàn)下降趨勢；XAV939與奈達鉑的合用效應與藥物劑量和作用時間相關，短期小劑量時應用時表現(xiàn)為協(xié)同效應（CI＜1）,長期大劑量應用時表現(xiàn)為拮抗效應（CI＞1）。 2、流式細胞儀檢測結果顯示，隨著XAV939濃度的增加和作用時間的延長，Anterior G0/G1期的Hela細胞明顯增多（P＜0.05），S期的細胞百分比呈現(xiàn)下降趨勢（P＜0.05），而GO/G1期和G2/M期的百分比則無顯著性差異。 3、免疫印跡結果顯示，Tankyrase和β-catenin在宮頸癌Hela細胞中高表達；加入XAV939藥物作用后兩者表達均減少，且隨著藥物濃度的升高， Hela細胞中Tankyrase、β-catenin的表達逐漸減少。 結論 1、端錨聚合酶抑制劑XAV939對Hela細胞有較明顯的殺傷作用。XAV939短期可增加Hela細胞對奈達鉑敏感性，促使Hela細胞凋亡；隨著合用時間的延長、劑量的加大XAV939可能降低奈達鉑的藥效。 2、XAV939對Hela細胞的作用主要表現(xiàn)為阻止DNA合成和誘導細胞凋亡。 3、端錨聚合酶可作為治療宮頸癌潛在的分子靶點。
[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.

【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.33

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