DNA修复蛋白XPF影响肾癌细胞顺铂耐药的作用与机制研究

发布时间：2018-09-11 11:26

【摘要】：卫生部统计信息显示，我国肾癌发病率位居男性恶性肿瘤发病率前十位，且呈现逐年上升趋势。在泌尿生殖系统肿瘤中，肾癌发病率仅次于膀胱癌。早期或中期肾癌的治疗与其他肿瘤相同，均以外科手术治疗为主。晚期肾癌的治疗则明显区别于其他肿瘤。由于肾癌对顺铂等传统化疗药物普遍耐药，，所以晚期肾癌的治疗无法像其他肿瘤一样以化疗为主，只能以免疫治疗和靶向治疗为主，这也使得晚期肾癌治疗效果欠佳。与肾癌不同，睾丸癌的化疗效果非常好。以顺铂为基础的化疗方案对睾丸癌的治愈率可达90%以上。睾丸癌对顺铂的高度敏感性与其DNA修复蛋白XPF表达水平较低密切相关。XPF是一种重要的DNA修复蛋白，主要参与核苷酸切除修复途径。XPF表达水平降低能够导致细胞DNA修复能力降低，从而使其对DNA损伤类化疗药物的敏感性提高。低表达XPF的睾丸癌对顺铂高度敏感，该现象提示肾癌对顺铂等化疗药物耐药可能与其XPF表达水平较高有关。如果这种假设成立，那么可以通过降低肾癌XPF表达水平来提高其顺铂敏感性，使顺铂应用于肾癌治疗成为可能。运用免疫组织化学染色法检测癌组织及癌旁组织中XPF表达水平发现，在癌组织中，肾癌XPF阳性表达率最高，膀胱癌次之，睾丸癌最低；在癌旁组织中，肾癌癌旁组织XPF阳性表达率最高，膀胱癌癌旁次之，睾丸癌癌旁最低。利用蛋白印迹实验检测多种肾癌细胞和膀胱癌细胞中XPF表达水平发现，肾癌细胞的XPF表达水平高于膀胱癌细胞。采用克隆形成实验检测癌细胞的顺铂敏感性发现，肾癌细胞的顺铂敏感性低于膀胱癌细胞。通过shRNA干扰技术成功构建稳定低表达XPF的肾癌细胞株ACHN-shXPF，其顺铂敏感性显著提高。选用裸鼠皮下移植瘤模型检测ACHN-shXPF细胞在体顺铂敏感性发现，其在体顺铂敏感性提高。借助宿主反应实验、细胞周期检测实验和细胞凋亡检测实验分别对ACHN-shXPF细胞的DNA修复能力、顺铂诱导细胞周期阻滞情况和顺铂诱导凋亡情况进行检测，结果示ACHN-shXPF细胞DNA修复能力降低，顺铂诱导细胞周期阻滞异常，顺铂诱导凋亡增加。另外，细胞衰老染色实验发现，ACHN-shXPF衰老细胞比例增高。综合以上结果，肿瘤组织和肿瘤细胞的XPF表达水平越高，其顺铂敏感性越低，即XPF表达水平与顺铂敏感性负相关。体外实验和在体实验均证实降低XPF表达水平可显著提高肾癌细胞的顺铂敏感性。通过降低XPF表达水平使顺铂敏感性提高的分子机制包括细胞DNA修复能力降低、细胞周期在顺铂诱导下异常阻滞和顺铂诱导凋亡增加。衰老细胞比例增加可能也是造成顺铂敏感性升高的机制之一。本研究提示，通过设计小分子药物干扰肾癌细胞XPF表达可提高其顺铂敏感性，使顺铂成功应用于肾癌治疗。
[Abstract]:Statistics from the Ministry of Health show that the incidence of renal cell carcinoma in China ranks among the top ten male malignant tumors and is on the rise year by year.Among the tumors of the urogenital system, the incidence of renal cell carcinoma is second only to that of bladder cancer.The treatment of early or middle stage renal cell carcinoma is the same as that of other tumors, and surgical treatment is the main treatment.The treatment of advanced renal cell carcinoma is obvious. Different from other tumors, renal cell carcinoma is generally resistant to cisplatin and other traditional chemotherapy drugs, so the treatment of advanced renal cell carcinoma can not be as chemotherapy-based as other tumors, only immunotherapy and targeted therapy, which makes the treatment of advanced renal cell carcinoma poor.
Unlike renal cell carcinoma, testicular cancer is very effective in chemotherapy. The cure rate of testicular cancer can reach more than 90% with cisplatin-based chemotherapy. The high sensitivity of testicular cancer to cisplatin is closely related to the low expression of DNA repair protein XPF. XPF is an important DNA repair protein, mainly involved in nucleotide excision repair pathway. XPF table Low expression of XPF in testicular cancer is highly susceptible to cisplatin, suggesting that resistance to chemotherapy drugs such as cisplatin in renal cancer may be associated with a higher level of XPF expression. The expression level of XPF in renal cell carcinoma can enhance the sensitivity of cisplatin and make cisplatin possible for the treatment of renal cell carcinoma.
The expression of XPF was detected by immunohistochemical staining in cancer tissues and adjacent tissues. It was found that the expression of XPF was the highest in renal carcinoma, followed by bladder cancer and testicular cancer, and the highest in adjacent tissues, followed by bladder cancer and testicular cancer. The expression of XPF in renal cell carcinoma and bladder cancer cells was higher than that in bladder cancer cells. The cisplatin sensitivity of renal cell carcinoma cells was lower than that of bladder cancer cells by clone formation assay. The sensitivity of ACHN-shXPF cell line to cisplatin was significantly increased. The sensitivity of ACHN-shXPF cells to cisplatin in vivo was detected by subcutaneous transplantation tumor model in nude mice. Cell cycle arrest and cisplatin-induced apoptosis were detected. The results showed that the DNA repair ability of ACHN-shXPF cells decreased, the cell cycle arrest induced by cisplatin was abnormal, and the apoptosis induced by cisplatin was increased.
In conclusion, the higher the expression level of XPF in tumor tissue and tumor cells, the lower the sensitivity to cisplatin, that is, the negative correlation between the expression level of XPF and the sensitivity to cisplatin. Molecular mechanisms include decreased DNA repair, abnormal cell cycle arrest induced by cisplatin, and increased apoptosis induced by cisplatin. Increasing the proportion of senescent cells may also be one of the mechanisms responsible for the increased sensitivity to cisplatin. This study suggests that interfering with the expression of XPF in renal cancer cells by designing small molecule drugs may enhance the sensitivity to cisplatin. Platinum is successfully applied to the treatment of renal cell carcinoma.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.11

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