NSCLC放射敏感性与EGFR基因突变的相关性及机制研究

发布时间：2018-05-02 06:47

本文选题：NSCLC + EGFR基因突变　；参考：《广东药科大学》2017年硕士论文

【摘要】：研究背景和目的非小细胞肺癌(non-small cell lung cancer,NSCLC)在肺癌中约占80%-85%[1],是死亡率最高的肿瘤之一[2]。放疗是NSCLC的主要治疗方法之一,不仅用于各个分期的NSCLC[3,4],也成为EGFR-TKIs获得性耐药局部进展或中枢神经系统转移的治疗选择之一[5-7]。但遗憾的是,目前临床上缺乏放疗敏感性预测因子。不管是临床回顾性研究还是基础研究均显示,EGFR基因突变的NSCLC相对于EGFR野生型细胞对放射线敏感[8-15]。我们的一项前期临床回顾性分析也显示,EGFR突变型脑转移NSCLC患者放疗有效率更高[16,17],提示EGFR基因突变可能是潜在的特异性放疗敏感性预测因子。但是,最近一项研究显示EGFR突变状态与NSCLC放射敏感性无相关性[18],提示EGFR基因突变与NSCLC放射敏感性的相关性尚需要进一步研究证实。对于EGFR-TKIs获得性耐药后局部进展或者中枢神经系统转移的患者,包括放疗在内的局部治疗措施也是主要选择之一。临床研究中,EGFR-TKIs治疗后局部进展或者中枢神经系统转移的患者在放疗后继续使用TKIs可以获得较长的PFS以及OS[5,19,20]。既往研究发现EGFR-TKIs获得性耐药细胞株在放射线照射后都出现对gefitinib敏感性增高[21-24],并且发生间质上皮转换(Mesenchymal Epithelial Transition,MET)[25],但是其具体机制目前尚未明确。基于以上研究背景,我们拟对7株EGFR基因突变状态不同的NSCLC细胞和2株EGFR-TKIs获得性耐药NSCLC细胞株进行放射敏感性检测,并对其可能的机制进行研究。另外,初步研究了放射线对EGFR-TKIs获得性耐药细胞的药物敏感性的作用及其可能的机制。本研究在既往研究背景以及前期临床回顾性研究基础上,寻找新的高特异性放疗敏感性预测因子,优化NSCLC患者个体化治疗方案,对提高NSCLC患者治疗有效率,延长NSCLC患者生存具有重要意义。方法1、EGFR基因突变与NSCLC放射敏感性的相关性:我们选用EGFR野生型肺鳞癌细胞H226、腺癌细胞A549、大细胞肺癌细胞H460,EGFR 19外显子缺失突变型NSCLC细胞PC-9、HCC827,EGFR 21外显子L858R突变型NSCLC细胞H1975、H3255,EGFR-TKIs获得性耐药细胞株PC-9/AB、PC-9/ZD。通过克隆成型实验检测各细胞株的放射敏感性,并拟合单击多靶模型和线性平方和模型,得出各细胞株的放射生物学参数。2、EGFR基因突变对放射线敏感可能的机制:绘制生长曲线检测各株细胞在放射线照射后群体增殖能力,流式细胞术检测各株细胞在放射线照射后细胞周期分布及凋亡情况,免疫荧光法检测各细胞株在放射线照射后DNA双链断裂(DNA double stranded breaks,DSBs)形成情况,western-blot实验检测各细胞株放射线照射后凋亡蛋白,抗凋亡蛋白及修复蛋白表达情况。3、研究放射线对EGFR-TKIs获得性耐药细胞gefitinib药物敏感性作用及可能的机制:通过对EGFR-TKIs获得性耐药细胞PC-9/AB和PC-9/ZD进行4次剂量为6Gy的放射线照射后培育放射线照射细胞株PC-9/AB IR、PC-9/ZD IR。采用CCK8法检测各细胞对gefitinib药物敏感性,NGS(next-generation sequencing)法检测放射线照射后基因突变、扩增情况,western-blot实验检测各株细胞E-cadherin、vimentin蛋白表达情况。4、统计学方法:应用IBM SPSS Statistics 20.0进行统计学分析,各实验重复3次,数据以均数±标准差表示,组间比较采用T检验,P㩳0.05时认为差异具有统计学意义。结果1、EGFR基因突变的NSCLC细胞对放射线敏感与EGFR野生型NSCLC细胞A549、H226对比,EGFR突变型细胞PC-9、HCC827、H1975、H3255和EGFR-TKIs继发性耐药细胞PC-9/AB、PC-9/ZD对放射线敏感,且EGFR突变型细胞与EGFR-TKIs继发性耐药细胞放射敏感性无明显差异。EGFR野生型NSCLC细胞H460对比EGFR野生型NSCLC细胞A549、H226对放射线敏感,与EGFR突变型细胞、EGFR-TKIs继发性耐药细胞放射敏感性相近。放射生物学参数显示,在代表37%杀伤的D0值和代表准阈杀伤的Dq值中,A549和H226细胞的D0值和Dq值明显高于其他细胞。在代表修复能力的α/β值中,A549、H226、HCC827细胞明显低于其他细胞,提示A549、H226、HCC827细胞的放射线照射后修复能力较其他细胞强。2、EGFR基因突变NSCLC细胞放射敏感性高的初步机制生长曲线显示,EGFR突变型细胞在放射线照射后群体增殖能力低于H226细胞和A549细胞。流式细胞术检测细胞周期分布及凋亡情况显示,A549和H226细胞在放射线照射后没有出现凋亡,其他细胞在放射线照射后24小时出现明显凋亡。A549和H226细胞在放射线照射后出现细胞周期G0/G1、G2/M期增多,并持续增加至放射线照射后48小时,而其他细胞在放射线照射后仅出现G2/M期的增加,没有出现G0/G1期的增加,且G2/M期增加在放射线照射后24小时达到峰值并开始下降。免疫荧光实验显示,A549和H226细胞放射线照射后照30min才出现明显的γ-H2AX焦点形成,并在放射线照射6小时后γ-H2AX焦点形成消失。而其他细胞在放射线照射后15min就可出现明显的γ-H2AX焦点形成,并持续到放射线照射后24小时。Western-blot实验显示,EGFR突变型细胞放射线照射后促凋亡蛋白Bax、凋亡蛋白Caspase-3表达,抗凋亡蛋白Bcl-2、修复蛋白DNA-PKcs不表达或表达减少。A549和H226细胞在放射线照射后表达Bcl-2、Caspase-3,不表达促Bax,DNA-PKcs表达至少持续至放射线照射后6h。H460细胞在放射线照射后Bax、Bcl-2、Caspase-3、DNA-PKcs均出现表达。3、放射线逆转PC-9/AB、PC-9/ZD细胞对gefitinib获得性耐药及初步机制PC-9/AB IR和PC-9/ZD IR细胞形态发生改变,表现为细胞呈圆形,并且体积变小。CCK8法显示PC-9/AB IR、PC-9/ZD IR细胞相对PC-9/AB、PC-9/ZD细胞,对gefitinib药物敏感性恢复。NGS检测并未发现放射线照射后出现基因突变、扩增或者融合,仅PC-9/ZD细胞在放射线照射后出现AKT1基因11外显子F349L突变消失。Western-blot实验显示,PC-9/AB IR、PC-9/ZD IR细胞对比PC-9/AB、PC-9/ZD细胞E-cadherin表达增多、Vimenten表达降低,发生MET。结论EGFR突变与NSCLC放射敏感性相关,修复能力下降、细胞周期分布、细胞增殖能力下降、更容易发生放射线引起的损伤是EGFR突变的细胞放射敏感性高可能的机制。EGFR-TKIs获得性耐药细胞未见到明显的放射敏感性改变。放射线可以逆转EGFR-TKIs获得性耐药细胞对gefitinib耐药性,其机制可能是放射线照射后发生MET。
[Abstract]:Background and objective non small cell lung cancer (non-small cell lung cancer, NSCLC) is about 80%-85%[1] in lung cancer, and is one of the most fatal tumors. [2]. radiotherapy is one of the main treatments for NSCLC. It is not only used in each stage of NSCLC[3,4], but also as a cure for local progression of EGFR-TKIs acquired resistance or metastasis of central nervous system. One of the choice of [5-7]. but regrets that there is a lack of predictors of radiosensitivity in the clinic. Both clinical review and basic research have shown that the NSCLC mutation of the EGFR gene is relative to the EGFR wild type cells for the radiation sensitive [8-15].. Our preliminary clinical review also showed that the EGFR mutated brain metastases NSCL The effective rate of radiotherapy in C patients is higher [16,17], suggesting that EGFR gene mutation may be a potential predictor of specific radiation sensitivity. However, a recent study showed that there was no correlation between EGFR mutation and NSCLC radiosensitivity, suggesting that the correlation between the EGFR gene mutation and the radiosensitivity of NSCLC needs further study. Local treatment, including radiotherapy, is also one of the major options in patients with TKIs acquired resistance to local progress or central nervous system metastasis. In clinical studies, local progress after EGFR-TKIs treatment or the continued use of TKIs after radiotherapy for patients with central nervous system metastases can obtain longer PFS and OS[5,19,20]. in the past. The study found that EGFR-TKIs acquired resistance cell lines were sensitive to gefitinib sensitivity [21-24] and Mesenchymal Epithelial Transition (MET) [25], but its specific mechanism was not clear at present. Based on the above study, we propose to 7 strains of EGFR gene mutation in NSC. LC cells and 2 EGFR-TKIs acquired resistant NSCLC cell lines were tested for radiosensitivity, and their possible mechanisms were studied. In addition, the effects and possible mechanisms of radiation on drug sensitivity of EGFR-TKIs acquired resistant cells were preliminarily studied. At the same time, looking for new high specificity radiotherapy sensitivity predictors and optimizing the individualized treatment of NSCLC patients is of great significance for improving the efficiency of NSCLC patients and prolonging the survival of NSCLC patients. Method 1, the correlation between EGFR mutation and NSCLC radiosensitivity: we choose EGFR wild type lung squamous cell carcinoma cell H226, adenocarcinoma cell A549, Large cell lung cancer cells H460, EGFR 19 exon deletion mutant NSCLC cells PC-9, HCC827, EGFR 21 exon L858R process NSCLC cells H1975, H3255, EGFR-TKIs acquired resistance cell strain PC-9/AB. The radibiologic parameters of each cell line,.2, and EGFR gene mutation, were likely to be sensitive to the radiating line. The growth curve was drawn to detect the proliferation ability of each cell after radiation exposure. Flow cytometry was used to detect the cell cycle distribution and apoptosis of each cell after irradiation, and the immunofluorescence method was used to detect the radiation of each cell line. The formation of DNA double strand breaks (DNA double stranded breaks, DSBs) after line irradiation. The Western-blot test detected the apoptotic protein, anti apoptotic protein and the expression of repair protein after radiation of each cell line.3, and studied the effect of radiation on gefitinib drug sensitivity of EGFR-TKIs acquired drug-resistant cells and the possible mechanism: through EGFR-TKI. S acquired drug-resistant cells PC-9/AB and PC-9/ZD were irradiated with radioactive rays at 4 doses of 6Gy to cultivate radiation irradiated cell line PC-9/AB IR, PC-9/ZD IR. was used to detect drug sensitivity to gefitinib by CCK8 method, and NGS (next-generation) method was used to detect gene mutation and amplification. The expression of E-cadherin and vimentin protein in each cell was.4, statistical method: statistical analysis was carried out with IBM SPSS Statistics 20, each experiment was repeated for 3 times, the data were expressed with mean number of standard deviation, T test was used in the group, and P? 0.05 thought the difference was statistically significant. Results 1, EGFR gene mutation of NSCLC cells was sensitive to the radiating line. Compared with EGFR wild type NSCLC cells, A549, H226, EGFR mutant cells PC-9, HCC827, H1975, H3255 and EGFR-TKIs secondary drug-resistant cells are PC-9/AB, PC-9/ZD is sensitive to radiation, and there is no significant difference in radiosensitivity from secondary resistant cells. 9, H226 is sensitive to radiosensitivity, and is similar to the radiosensitivity of EGFR mutants and EGFR-TKIs secondary resistant cells. Radibiologic parameters show that D0 and Dq values of A549 and H226 cells are significantly higher in A549 and H226 cells than in other cells in the D0 values that represent the killing of the cells and the Dq values representing the quasi threshold killing. In the alpha / beta values representing the restoration ability, A549, H226, HCC827 The cells were significantly lower than other cells, suggesting that the radiation of A549, H226 and HCC827 cells was stronger than that of other cells after irradiation, and the initial mechanism growth curve of EGFR gene mutation NSCLC cells with high radiosensitivity showed that the proliferation ability of EGFR mutated cells was lower than that of H226 and A549 cells after radiation exposure. Flow cytometry was performed. The cell cycle distribution and apoptosis showed that there was no apoptosis in A549 and H226 cells after radiation. Other cells appeared obviously apoptotic.A549 and H226 cells at 24 hours after irradiation. The cell cycle was G0/G1, G2/M period increased after radiation and increased to 48 hours after radiation, while other cells were in the radiation. The increase of G2/M stage only appeared after radiation, and no increase in G0/G1 stage, and the increase of G2/M period reached its peak value at 24 hours after irradiation. The immunofluorescence experiment showed that the A549 and H226 cells irradiated with 30min only to appear the obvious gamma ray -H2AX focus form, and the gamma -H2AX focus after irradiation of 6 hours after radiation. The formation of 15min was formed in other cells after radiation, and the formation of a significant gamma -H2AX focal point could occur after radiation of radiation, and 24 hours after radiation irradiation, the.Western-blot experiment showed that the apoptotic protein Bax, the expression of apoptotic protein Caspase-3, the anti apoptotic protein Bcl-2, the non expression of protein DNA-PKcs, or the expression of the protein DNA-PKcs were not expressed or expressed in the EGFR mutated cells after radiation. To reduce.A549 and H226 cells to express Bcl-2, Caspase-3, and no expression of Bax, and at least the expression of DNA-PKcs in 6h.H460 cells after radiation exposure to Bax, Bcl-2, Caspase-3, DNA-PKcs after irradiation. The morphologic changes of IR and PC-9/ZD IR cells showed that the cells were round, and the volume smaller.CCK8 method showed PC-9/AB IR, PC-9/ZD IR cells were relative PC-9/AB, PC-9/ZD cells, and gefitinib drug sensitivity recovery.NGS detection did not find gene mutation, amplification or fusion after radiation exposure, only irradiated cells were irradiated by radiation. After AKT1 gene 11 exon F349L mutation disappeared.Western-blot experiment showed that PC-9/AB IR, PC-9/ZD IR cells compared PC-9/AB, PC-9/ZD cell E-cadherin expression increased, Vimenten expression decreased, occurrence of MET. conclusion mutation was related to radiosensitivity, repair ability descended, cell cycle distribution, cell proliferation ability decreased, easier The damage caused by radiation is the mechanism of high radiosensitivity of EGFR mutant cells..EGFR-TKIs acquired resistance cells do not see significant radiosensitivity changes. Radiation can reverse the resistance of EGFR-TKIs acquired resistant cells to gefitinib, and the mechanism may be MET. after radiation radiation.

【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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