端锚聚合酶抑制剂XAV939对宫颈癌Hela细胞增殖和凋亡的影响

发布时间：2018-02-06 03:38

本文关键词： 端锚聚合酶宫颈癌 XAV939 beta-链蛋白　出处：《郑州大学》2014年硕士论文　论文类型：学位论文

【摘要】：背景与目的宫颈癌（cervical carcinoma）是危害中国女性身体健康最常见的妇科恶性肿瘤，目前发病机制已明确，90%的宫颈癌患者都能检测到人乳头瘤病毒（Humanpapilloma virus，HPV）的表达，但是感染了HPV的患者仅有一小部分会发展成宫颈癌，持续性高危型HPV感染只是宫颈癌发生发展的第一步，真正促进其进一步恶化的机制尚待探讨。此外，宫颈腺癌和一小部分鳞癌患者早期不易发现,仅依靠宫颈病变三阶梯诊断法（细胞学、阴道镜及病理组织学检查）不能做到完全筛查高危患者；对宫颈癌的治疗，采用手术治疗适用范围有限，而且有可能损伤年轻患者的生育功能，放化疗副作用大且容易耐受，一旦复发再次用药效果较差。所以探讨宫颈癌的发病机制，寻求一种灵敏性和特异性较高的早期辅助诊断的技术和找到一种更为有效的针对性治疗方法依然是必要且迫切的。最近国内外一些研究发现，Wnt/β-catenin信号通路的改变可能是继HPV感染之后促进宫颈癌的发生发展的“第二次重大事件”，其中β-catenin (又称β-链蛋白，β-连接蛋白)在细胞核的异常积累是该通路的核心环节。事实上，，近几年有很多学者都开始着手研究Wnt/β-catenin信号通路和恶性肿瘤的相关性，但是多集中于乳腺癌、结肠癌、口腔鳞癌等，而对于宫颈癌的研究尚处于起步阶段。目前许多学者已经开始尝试通过干预Wnt/β-catenin通路调整靶基因表达，以达到抑制宫颈癌发生发展的目的。端粒酶在宫颈病变的早期都有表达，而且与宫颈病变的发生发展密切相关。国内外很多学者通过端粒酶扩增法和荧光原位杂交法验证了该结论，指出高达90%的宫颈癌组织中有端粒酶阳性表达，端粒酶的催化亚基对判断宫颈病变的进展和转归有重大提示意义。目前多认为端粒酶不仅可作为宫颈癌的生物学标志，并能有效用于宫颈癌的早期筛查，辅助诊断以及判断预后。其中端锚聚合酶（TRF1-interacting ankyrin-related ADP-ribose polymerase，Tankyrase）不仅是Wnt经典通路的正向调控因子，而且是介导端粒与端粒酶结合的关键端粒调控蛋白，能够维持端粒平衡。2009年《Nature》杂志提出的一个化学小分子物质XAV939，是利用化学遗传学法专门制备的Tankyrase的特殊抑制剂，它可以通过稳定轴蛋白Axin的表达特异性阻滞Wnt/β-catenin信号通路的活化从而限制肿瘤细胞增殖和生长。本研究通过检测端锚聚合酶抑制剂XAV939对宫颈癌Hela细胞增殖和凋亡的影响及对细胞核质内Tankyrase和β-catenin表达的调控情况，进一步探索宫颈癌相关发病机制和分析该药物应用于临床的可行性。材料与方法复苏和培养宫颈癌Hela细胞至对数生长期，首先用MTT法分别检测端锚聚合酶抑制剂XAV939、奈达铂及两者联合作用下Hela细胞的抑制率；然后通过流式细胞仪测定不同浓度下XAV939对Hela细胞凋亡周期的影响；最后采用免疫印迹检测Tankyrase和β-catenin在宫颈癌Hela细胞的表达情况，以及受到XAV939不同浓度作用48小时后的表达有无异常改变。采用SPSS19.0统计分析相关实验数据，以α=0.05作为检验水准。结果 1、MTT法实验结果显示，随着XAV939浓度的增加，药物作用时间的延长，XAV939对Hela细胞生长抑制率不断增加，呈现浓度和时间依赖性（P＜0.05），半数抑制率随着时间的增加呈现下降趋势；XAV939与奈达铂的合用效应与药物剂量和作用时间相关，短期小剂量时应用时表现为协同效应（CI＜1）,长期大剂量应用时表现为拮抗效应（CI＞1）。 2、流式细胞仪检测结果显示，随着XAV939浓度的增加和作用时间的延长，Anterior G0/G1期的Hela细胞明显增多（P＜0.05），S期的细胞百分比呈现下降趋势（P＜0.05），而GO/G1期和G2/M期的百分比则无显著性差异。 3、免疫印迹结果显示，Tankyrase和β-catenin在宫颈癌Hela细胞中高表达；加入XAV939药物作用后两者表达均减少，且随着药物浓度的升高， Hela细胞中Tankyrase、β-catenin的表达逐渐减少。结论 1、端锚聚合酶抑制剂XAV939对Hela细胞有较明显的杀伤作用。XAV939短期可增加Hela细胞对奈达铂敏感性，促使Hela细胞凋亡；随着合用时间的延长、剂量的加大XAV939可能降低奈达铂的药效。 2、XAV939对Hela细胞的作用主要表现为阻止DNA合成和诱导细胞凋亡。 3、端锚聚合酶可作为治疗宫颈癌潜在的分子靶点。
[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.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

【参考文献】