人肺腺癌辐射耐受细胞株的建立及其放射抵抗机制探讨

Establishment of Human Lung Adenocarcinoma Radioresistant Cell Lines and the Mechanism of Radioresistance

背景与目的 放疗是肺癌最常见的治疗方法之一,40%-50%的患者在放疗后会出现局部肿瘤未控或复发,放射抵抗是导致肺癌局部控制失败的重要原因。然而,体外放疗抵抗模型的缺乏是阻碍其机制研究的主要因素。因此,本研究旨在通过建立人肺腺癌H1975和H1299辐射耐受细胞株,为未来开展肺癌放射抵抗相关性研究创建体外模型,并初步探索其放射抵抗机制。方法 对H1975和H1299细胞进行等剂量的X射线分次照射,构建辐射耐受细胞株H1975DR和H1299DR;采用克隆形成实验比较H1975和H1975DR细胞、H1299和H1299DR细胞的克隆形成能力,线性二次模型拟合细胞存活分数曲线;DNA损伤修复能力的检测采用彗星实验,并计算DNA尾部百分比(Tail DNA%);利用光学显微镜、CCK-8、FACS、细胞划痕和侵袭等方法比较细胞形态、细胞增殖能力、细胞凋亡水平和周期分布、细胞迁移和侵袭能力等生物学特性;通过Western blot免疫印迹分析DNA-PKcs、53BP1、RAD51、p-ATM等DNA损伤修复因子的表达。结果 历经5个月的照射及持续培养获得稳定的辐射耐受细胞株H1975DR和H1299DR。X射线照射下,两株辐射耐受株的细胞增殖活性、克隆形成能力、DNA损伤修复能力均显著提高;细胞周期G2期/M期比例均显著下调,G0期/G1期比例上调;细胞迁移及侵袭能力显著增强。非同源末端连接(nonhomologous end-joining, NHEJ)修复通路的p-DNA-PKcs(Ser2056)、53BP1及同源重组(homologous recombination, HR)修复通路的p-ATM(Ser1981)、D51相对蛋白表达量较H1975及H1299均有不同程度的增高。结论 通过等剂量分次照射可诱导H1975和H1299细胞株分化为肺腺癌辐射耐受细胞株H1975DR和H1299DR,为肺癌患者放疗抵抗的机制研究提供了体外细胞学模型。

Background and objective Radiotherapy is one of the most common treatments for lung cancer,and about 40%-50% of patients after radiotherapy will appear uncontrolled or recurrence in the case of local tumors.Radioresistance is the predominant cause of local therapeutic failure.Nevertheless,the lack of in vitro radioresistance models is an influential factor obstructing the study of its mechanism.Therefore,the establishment of radioresistant cell lines,H1975DR and H1299DR,was beneficial to explore the mechanism of radioresistance in lung adenocarcinoma.Methods The radioresistant cell lines of H1975DR and H1299DR were obtained from H1975 and H1299 cells irradiated with equal doses of X-rays;Clonogenic assays were performed to compare the clone-forming ability of H1975 vs H1975DR cells,H1299 vs H1299DR cells,then fitting cell survival curve by linear quadratic model;The comet assay was employed to examine DNA damage repair and calculate the percentage of DNA tails;The optical microscopy,CCK-8,flow cytometry,Transwell invasion assays were used to compare biological characteristics such as cell morphology,cell proliferation,apoptosis level,cycle distribution,cell migration and invasion;Western blot was carried out to measure the protein expression of DNA damage repair factors,such as DNAPKcs,53BP1,RAD51,and p-ATM.Results After five months of continuous irradiation and stable culture,radioresistant cell lines H1975DR and H1299DR were obtained.The cell proliferation activity,clone formation ability and DNA damage repair ability of the two radioresistant cell lines were significantly improved under X-ray irradiation.The proportion of the G2/M phase was markedly decreased and the proportion of the G0/G1phase was increased.Cell migration and invasion ability were significantly enhanced.Relative expression levels of p-DNA-PKcs(Ser2056),53BP1 in the nonhomologous end-joining(NHEJ) repair pathway and p-ATM(Ser1981),RAD51 in the homologous recombination(HR) repair pathway were higher than those in H1975 and H1299.Conclusion H1975 and H1299 cell lines can be able to differentiate into lung adenocarcinoma radioresistant cell lines H1975DR and H1299DR by equal dose fractional irradiation,which provided an in vitro cytological model for the study of radiotherapy resistance mechanism of lung cancer patients.