兰州大学机构库 >学院待认领
细胞内自噬与前列腺癌细胞PC3放射敏感性关系的实验研究
Alternative TitleChanges in Radiosensitivity of PC3 Prostate Cancer Cells Induced by Alteration of the Autophagy Process
贺振华
Thesis Advisor陈一戎
2011-12-07
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name博士
Keyword自噬 放射敏感性 前列腺癌 PC3 细胞 Gamma射线
Abstract目的 前列腺癌是男性泌尿生殖系统中最重要的一种,对其的治疗方法根据不同的年龄、分级和分期而不同。目前的治疗方法主要包括药物及物理去势(激素治疗)、手术摘除、放射治疗等。其中放射治疗由于其不开刀、痛苦小的优点而备受关注。但由于癌细胞本身的抗放射生物特性,使这一疗法一直没有达到预期的疗效。近来的研究显示自噬可能是癌细胞接受放射治疗后的一种重要的生物学反应。本课题通过干预细胞内自噬相关途径,借以改变抗放射前列腺癌细胞PC3的放射敏感性,从而从细胞水平、基因水平两个层次,对前列腺癌患者的临床放射治疗方法给予基础学支持。旨在提高放射治疗疗效,延长患者生存期和降低死亡率。 材料和方法: 1)应用GFP-LC3测定法分析前列腺PC3细胞内自噬途径的激活过程,观察在应用不同药物干预并结合放射治疗后,PC3细胞的自噬生物学特性随不同时间和不同剂量的变化规律。 2)对PC3细胞内LC3-II蛋白表达水平进行western blotting测定。 3)通过克隆存活形成实验和Annexin V assay 检测在Gamma射线放射并联合不同药物干预后,PC3 细胞放射敏感性的变化。 4)运用RT-PCR法检测PC3细胞内自噬相关基因表达谱。 结果: Gamma射线照射后,前列腺癌PC3 细胞可以表现出诱导性的自噬反应。应用特异性靶向自噬相关基因Atg7和/或Atg12的小干扰RNA阻断细胞内自噬途径可以显著的降低PC3细胞的放射敏感性。用于阻断自噬途径的氯喹不能明显影响接受射线干预后PC3细胞集落的形成,但它可以部分诱导I型程序性细胞死亡(20%)。被自噬诱导剂RAD001和Statin干预后的细胞,在接受gamma射线照射后表现出显著的细胞集落形成率的降低,表明这两种诱导剂可以增加PC3细胞的放射敏感性。Statin在干预后24-72小时,可以诱导PC3细胞自噬,但在这一时间段却没有明显的细胞凋亡。通过分析自噬相关基因表达谱,我们发现statin和gamma射线联合干预后,PC3细胞的所有84种相关基因中,有22种的表达量明显改变了,而且这22种基因中有9种是自噬机制不可或缺的。 结论: 前列腺癌细胞水平的研究证明,自噬可以被gamma射线诱导,并且不论联合应用自噬诱导剂/抑制剂还是siRNA内源调节自噬通路激活水平,都可以影响细胞的存活状态。进一步试验显示,细胞内自噬通路的激活可以提高细胞对gamma射线的放射敏感性,提示我们自噬诱导剂与放射治疗对于抑制肿瘤细胞生长,清除体内癌细胞有协同作用。具有潜在的临床应用前景。
Other AbstractPurpose: Treatment options vary depending on the age, stage and grade of the cancer. Current treatment for prostate cancer consists of extirpative, radiation or ablation based approaches for clinically for metastatic disease and androgen deprivation therapy for metastatic disease. Recent studies have indicated that autophagy may be one of the important pathways induced by ionizing radiation. In our study, changes in radiosensitivity of radioresistant human PC3 prostate cancer cells to radiotherapy by alteration of pathways related to autophagy were investigated. Methods and Materials: Activation of the autophagy pathway was analyzed using the GFP-LC3 assay and western blot to determine LC3-II expression. The radiosensitivity of PC3 cells was determined using the clonal survival assay and the Annexin V assay after exposure to gamma irradiation and/or various chemicals. The expression profiles of autophagy related genes were analyzed using a pathway specific real-time polymerase chain reaction array. Results: PC3 cells exhibited an induced autophagic response after exposure to gamma rays. Inhibition of the autophagic process using small interfering RNAs targeting Atg7 and/or Atg12 significantly reduced radiosensitivity of PC3 cells. The lysosomotropic agent chloroquine did not significantly affect the colony formation of the irradiated PC3 cells, but did induce apoptosis in a fraction of irradiated cells (20%). Both autophagy activators RAD001 and Statin sensitized the cells to gamma irradiation, showing significantly reduced colony forming efficiency in response to 6 Gy of gamma irradiation. Statin treatment of pc3 cells for 24h increased expression of GFP-LC3-II by more than 25% fold and keep expressing for more than 72h,whereas, apoptosis is not significantly induced within these moment. Analysis of autophagy and its regulatory gene profile showed that the expressions of 22 genes out of 84 genes assessed were significantly altered in the cells exposed to combined treatment with Statin and radiation, including nine genes encoding components of the autophagy machinery. Conclusions: The prostate cancer research on the cell level showed autophagy can be induced by gamma ray, and no matter combined with autophagy inducer/inhibitor or Atg specific siRNA to mediate activation of autophagy pathway, the cell survival status can be affected. The further experiment suggested that activate the cellular autophagy pathway can enhance the gamma ray radiosensitivit...
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/222524
Collection学院待认领
Affiliation临床医学院
Recommended Citation
GB/T 7714
贺振华. 细胞内自噬与前列腺癌细胞PC3放射敏感性关系的实验研究[D]. 兰州. 兰州大学,2011.
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