Sp1对宫颈癌放疗敏感性的影响及其机制研究

发布时间：2018-03-12 23:12

  本文选题：宫颈癌　切入点：放疗敏感性　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景和目的宫颈癌是女性生殖系统中最常见的恶性肿瘤。而放疗是宫颈癌的治疗手段之一,80%患者需接受放疗,而30-50%的晚期患者出现了放疗抵抗。因此,提高放疗敏感性对晚期宫颈癌患者而言十分重要。如今对宫颈癌的研究发现一些癌基因、转录因子等与宫颈癌的放疗敏感性密切相关,但仍缺乏公认的分子标志物。因此,寻找相关分子并研究其调控机制,对改善宫颈癌放疗敏感具有重要的意义。转录调控因子Sp1是一个具有序列特异性的DNA结合蛋白,参与调控了一些肿瘤的放疗敏感性,且其与宫颈癌的进展密切相关。因此,Sp1可能是影响宫颈癌放疗敏感的一个重要因子。本研究发现放疗可上调宫颈癌细胞中Sp1的表达,通过沉默/过表达SiHa、HeLa中Sp1的表达,揭示了 Sp1通过上调CDK1的表达减少放疗相关的G2/M期阻滞从而下调宫颈癌的放疗敏感性。本研究将分四部分进行。第一章Sp1与宫颈癌放疗的相关性研究目的研究Sp1与宫颈癌放疗的相关性方法利用western blot和RT-PCR检测放疗后宫颈癌细胞中Sp1的变化。结果宫颈癌细胞中Sp1的表达水平随照射剂量的增加而增加。结论Sp1与宫颈癌放疗密切相关。第二章Sp1与宫颈癌细胞放疗敏感性的关系目的探讨Sp1对宫颈癌放疗敏感性的影响方法构建Sp1沉默/过表达的宫颈癌细胞株,利用克隆形成及CCK8实验检测Sp1对宫颈癌放疗敏感性的影响。结果沉默Sp1可显著上调宫颈癌的放疗敏感性,过表达Sp1则相反。结论Sp1可下调宫颈癌的放疗敏感性。第三章Sp1通过调控放疗相关的细胞周期阻滞影响宫颈癌细胞放疗敏感性目的研究Sp1对宫颈癌细胞周期的影响方法利用流式细胞术检测Sp1对宫颈癌放疗相关细胞周期阻滞的影响结果抑制Sp1的表达可显著上调宫颈癌的G2/M期阻滞(p0.05),而过表达Sp1则显著减少宫颈癌的G2/M期阻滞(p0.05)结论Sp1可通过下调放疗相关的G2/M期阻滞从而下调宫颈癌的放疗敏感性。第四章Sp1通过调控CDK1影响宫颈癌细胞的放疗敏感性目的研究Sp1影响宫颈癌放疗敏感性的分子机制。方法1.利用实时荧光定量PCR及蛋白免疫印迹检测沉默/过表达Sp1后G2/M周期相关的基因变化2.利用恢复试验验证Sp1通过调控CDK1影响宫颈癌的放疗敏感性3.利用双荧光素酶实验验证Sp1是否作用于CDK1的启动子结果1.Sp1可上调CDK1的表达(P0.05)2.下调CDK1的表达后,Sp1对宫颈癌放疗敏感性的下调作用减小(P0.05)3.Sp1显著上调CDK1启动子的荧光活性(P0.01)结论Sp1可通过上调CDK1的表达下调宫颈癌的放疗敏感性。全文总结1.Sp1与宫颈癌放疗敏感性密切相关,沉默/过表达Sp1可显著上调/下调宫颈癌的放疗敏感性2.Sp1通过减少辐射相关的G2/M期阻滞下调宫颈癌的放疗敏感性3.Sp1通过上调CDK1的表达下调宫颈癌的放疗敏感性本项目的创新之处1.验证了 Sp1对宫颈癌放疗敏感性的作用;2.揭示Sp1通过调控CDK1下调宫颈癌放疗敏感性的分子机制。
[Abstract]:Background and objective Cervical cancer is the most common malignant tumor in the female reproductive system. Radiotherapy is one of the treatments for cervical cancer. 80% of the patients need radiotherapy, while 30-50% of the advanced patients have radiation resistance. Increasing radiosensitivity is important for patients with advanced cervical cancer. Research on cervical cancer has found that some oncogenes, transcription factors, and so on are closely related to the radiosensitivity of cervical cancer, but still lack of recognized molecular markers. It is important to search for the related molecules and study its regulatory mechanism to improve the radiosensitivity of cervical cancer. The transcription regulator Sp1 is a sequence-specific DNA binding protein involved in regulating the radiosensitivity of some tumors. Sp1 may be an important factor affecting the sensitivity of cervical cancer to radiotherapy. In this study, we found that radiotherapy can up-regulate the expression of Sp1 in cervical cancer cells, and by silencing / overexpressing the expression of Sp1 in SiHa-HeLa, it may be an important factor affecting the radiosensitivity of cervical cancer. It is revealed that Sp1 down-regulates the radiosensitivity of cervical cancer by up-regulating the expression of CDK1 and reducing the radiotherapy-associated G _ 2 / M block. This study will be conducted in four parts. Chapter 1: study on the correlation between Sp1 and radiotherapy for cervical cancer objective to study Sp1. Methods western blot and RT-PCR were used to detect the changes of Sp1 in cervical cancer cells after radiotherapy. Results the expression of Sp1 in cervical cancer cells increased with the increase of irradiation dose. Conclusion Sp1 is closely associated with radiotherapy for cervical cancer. Chapter 2 relationship between Sp1 and radiosensitivity of cervical cancer cells objective to investigate the effects of Sp1 on the radiosensitivity of cervical cancer cells. The effects of Sp1 on the radiosensitivity of cervical cancer were detected by clone formation and CCK8 assay. Results silencing Sp1 could significantly up-regulate the radiosensitivity of cervical cancer. Conclusion Sp1 can down-regulate the radiosensitivity of cervical cancer cells by overexpression of Sp1. Chapter 3: to study the effect of Sp1 on the radiosensitivity of cervical cancer cells by regulating the cell cycle block associated with radiotherapy objective to study the effect of Sp1 on the radiosensitivity of cervical cancer cells. Methods flow cytometry was used to detect the effect of Sp1 on cell cycle arrest associated with radiotherapy for cervical cancer. Inhibiting the expression of Sp1 could significantly up-regulate the G _ 2 / M phase block of cervical cancer, while overexpression of Sp1 significantly decreased the G _ 2 / M phase block of cervical cancer. Conclusion Sp1 can down-regulate the radiosensitivity of cervical cancer by down-regulating the G _ 2 / M phase block associated with radiotherapy. Chapter 4th Sp1 studies the effects of Sp1 on the radiosensitivity of cervical cancer by regulating the radiosensitivity of cervical cancer cells by regulating CDK1. Methods 1. Real-time fluorescent quantitative PCR and Western blotting were used to detect the G 2 / M cycle related gene changes after silencing / overexpression of Sp1 2.Using recovery test to verify the effect of Sp1 on the radiosensitivity of cervical cancer by regulating CDK1. 2. 3. Double luciferase assay was used to verify whether Sp1 acted on the promoter of CDK1. 1. Sp1 could up-regulate the expression of CDK1 and P0.05 ~ 2. After down-regulating the expression of CDK1, the down-regulation of pSp1 on radiosensitivity of cervical cancer decreased P0.05 ~ (3) Sp1 significantly up-regulated the fluorescence of CDK1 promoter. Conclusion Sp1 can down-regulate the radiosensitivity of cervical cancer by up-regulating the expression of CDK1. 1. Sp1 is closely related to the radiosensitivity of cervical cancer. Silencing / overexpression of Sp1 could significantly up-regulate / down-regulate the radiosensitivity of cervical cancer 2.Sp1 down-regulates the radiosensitivity of cervical cancer by reducing radiation-related G _ 2 / M block 3.Sp1 down-regulates the radiosensitivity of cervical cancer by up-regulating the expression of CDK1. The role of Sp1 in the radiosensitivity of cervical cancer 2.The molecular mechanism of Sp1 down-regulating the radiosensitivity of cervical cancer by regulating CDK1. 2.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

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