本文選題：NSCLC + EGFR基因突變　； 參考：《廣東藥科大學》2017年碩士論文

【摘要】：研究背景和目的非小細胞肺癌(non-small cell lung cancer,NSCLC)在肺癌中約占80%-85%[1],是死亡率最高的腫瘤之一[2]。放療是NSCLC的主要治療方法之一,不僅用于各個分期的NSCLC[3,4],也成為EGFR-TKIs獲得性耐藥局部進展或中樞神經(jīng)系統(tǒng)轉(zhuǎn)移的治療選擇之一[5-7]。但遺憾的是,目前臨床上缺乏放療敏感性預測因子。不管是臨床回顧性研究還是基礎(chǔ)研究均顯示,EGFR基因突變的NSCLC相對于EGFR野生型細胞對放射線敏感[8-15]。我們的一項前期臨床回顧性分析也顯示,EGFR突變型腦轉(zhuǎn)移NSCLC患者放療有效率更高[16,17],提示EGFR基因突變可能是潛在的特異性放療敏感性預測因子。但是,最近一項研究顯示EGFR突變狀態(tài)與NSCLC放射敏感性無相關(guān)性[18],提示EGFR基因突變與NSCLC放射敏感性的相關(guān)性尚需要進一步研究證實。對于EGFR-TKIs獲得性耐藥后局部進展或者中樞神經(jīng)系統(tǒng)轉(zhuǎn)移的患者,包括放療在內(nèi)的局部治療措施也是主要選擇之一。臨床研究中,EGFR-TKIs治療后局部進展或者中樞神經(jīng)系統(tǒng)轉(zhuǎn)移的患者在放療后繼續(xù)使用TKIs可以獲得較長的PFS以及OS[5,19,20]。既往研究發(fā)現(xiàn)EGFR-TKIs獲得性耐藥細胞株在放射線照射后都出現(xiàn)對gefitinib敏感性增高[21-24],并且發(fā)生間質(zhì)上皮轉(zhuǎn)換(Mesenchymal Epithelial Transition,MET)[25],但是其具體機制目前尚未明確�；谝陨涎芯勘尘�,我們擬對7株EGFR基因突變狀態(tài)不同的NSCLC細胞和2株EGFR-TKIs獲得性耐藥NSCLC細胞株進行放射敏感性檢測,并對其可能的機制進行研究。另外,初步研究了放射線對EGFR-TKIs獲得性耐藥細胞的藥物敏感性的作用及其可能的機制。本研究在既往研究背景以及前期臨床回顧性研究基礎(chǔ)上,尋找新的高特異性放療敏感性預測因子,優(yōu)化NSCLC患者個體化治療方案,對提高NSCLC患者治療有效率,延長NSCLC患者生存具有重要意義。方法1、EGFR基因突變與NSCLC放射敏感性的相關(guān)性:我們選用EGFR野生型肺鱗癌細胞H226、腺癌細胞A549、大細胞肺癌細胞H460,EGFR 19外顯子缺失突變型NSCLC細胞PC-9、HCC827,EGFR 21外顯子L858R突變型NSCLC細胞H1975、H3255,EGFR-TKIs獲得性耐藥細胞株P(guān)C-9/AB、PC-9/ZD。通過克隆成型實驗檢測各細胞株的放射敏感性,并擬合單擊多靶模型和線性平方和模型,得出各細胞株的放射生物學參數(shù)。2、EGFR基因突變對放射線敏感可能的機制:繪制生長曲線檢測各株細胞在放射線照射后群體增殖能力,流式細胞術(shù)檢測各株細胞在放射線照射后細胞周期分布及凋亡情況,免疫熒光法檢測各細胞株在放射線照射后DNA雙鏈斷裂(DNA double stranded breaks,DSBs)形成情況,western-blot實驗檢測各細胞株放射線照射后凋亡蛋白,抗凋亡蛋白及修復蛋白表達情況。3、研究放射線對EGFR-TKIs獲得性耐藥細胞gefitinib藥物敏感性作用及可能的機制:通過對EGFR-TKIs獲得性耐藥細胞PC-9/AB和PC-9/ZD進行4次劑量為6Gy的放射線照射后培育放射線照射細胞株P(guān)C-9/AB IR、PC-9/ZD IR。采用CCK8法檢測各細胞對gefitinib藥物敏感性,NGS(next-generation sequencing)法檢測放射線照射后基因突變、擴增情況,western-blot實驗檢測各株細胞E-cadherin、vimentin蛋白表達情況。4、統(tǒng)計學方法:應(yīng)用IBM SPSS Statistics 20.0進行統(tǒng)計學分析,各實驗重復3次,數(shù)據(jù)以均數(shù)±標準差表示,組間比較采用T檢驗,P㩳0.05時認為差異具有統(tǒng)計學意義。結(jié)果1、EGFR基因突變的NSCLC細胞對放射線敏感與EGFR野生型NSCLC細胞A549、H226對比,EGFR突變型細胞PC-9、HCC827、H1975、H3255和EGFR-TKIs繼發(fā)性耐藥細胞PC-9/AB、PC-9/ZD對放射線敏感,且EGFR突變型細胞與EGFR-TKIs繼發(fā)性耐藥細胞放射敏感性無明顯差異。EGFR野生型NSCLC細胞H460對比EGFR野生型NSCLC細胞A549、H226對放射線敏感,與EGFR突變型細胞、EGFR-TKIs繼發(fā)性耐藥細胞放射敏感性相近。放射生物學參數(shù)顯示,在代表37%殺傷的D0值和代表準閾殺傷的Dq值中,A549和H226細胞的D0值和Dq值明顯高于其他細胞。在代表修復能力的α/β值中,A549、H226、HCC827細胞明顯低于其他細胞,提示A549、H226、HCC827細胞的放射線照射后修復能力較其他細胞強。2、EGFR基因突變NSCLC細胞放射敏感性高的初步機制生長曲線顯示,EGFR突變型細胞在放射線照射后群體增殖能力低于H226細胞和A549細胞。流式細胞術(shù)檢測細胞周期分布及凋亡情況顯示,A549和H226細胞在放射線照射后沒有出現(xiàn)凋亡,其他細胞在放射線照射后24小時出現(xiàn)明顯凋亡。A549和H226細胞在放射線照射后出現(xiàn)細胞周期G0/G1、G2/M期增多,并持續(xù)增加至放射線照射后48小時,而其他細胞在放射線照射后僅出現(xiàn)G2/M期的增加,沒有出現(xiàn)G0/G1期的增加,且G2/M期增加在放射線照射后24小時達到峰值并開始下降。免疫熒光實驗顯示,A549和H226細胞放射線照射后照30min才出現(xiàn)明顯的γ-H2AX焦點形成,并在放射線照射6小時后γ-H2AX焦點形成消失。而其他細胞在放射線照射后15min就可出現(xiàn)明顯的γ-H2AX焦點形成,并持續(xù)到放射線照射后24小時。Western-blot實驗顯示,EGFR突變型細胞放射線照射后促凋亡蛋白Bax、凋亡蛋白Caspase-3表達,抗凋亡蛋白Bcl-2、修復蛋白DNA-PKcs不表達或表達減少。A549和H226細胞在放射線照射后表達Bcl-2、Caspase-3,不表達促Bax,DNA-PKcs表達至少持續(xù)至放射線照射后6h。H460細胞在放射線照射后Bax、Bcl-2、Caspase-3、DNA-PKcs均出現(xiàn)表達。3、放射線逆轉(zhuǎn)PC-9/AB、PC-9/ZD細胞對gefitinib獲得性耐藥及初步機制PC-9/AB IR和PC-9/ZD IR細胞形態(tài)發(fā)生改變,表現(xiàn)為細胞呈圓形,并且體積變小。CCK8法顯示PC-9/AB IR、PC-9/ZD IR細胞相對PC-9/AB、PC-9/ZD細胞,對gefitinib藥物敏感性恢復。NGS檢測并未發(fā)現(xiàn)放射線照射后出現(xiàn)基因突變、擴增或者融合,僅PC-9/ZD細胞在放射線照射后出現(xiàn)AKT1基因11外顯子F349L突變消失。Western-blot實驗顯示,PC-9/AB IR、PC-9/ZD IR細胞對比PC-9/AB、PC-9/ZD細胞E-cadherin表達增多、Vimenten表達降低,發(fā)生MET。結(jié)論EGFR突變與NSCLC放射敏感性相關(guān),修復能力下降、細胞周期分布、細胞增殖能力下降、更容易發(fā)生放射線引起的損傷是EGFR突變的細胞放射敏感性高可能的機制。EGFR-TKIs獲得性耐藥細胞未見到明顯的放射敏感性改變。放射線可以逆轉(zhuǎn)EGFR-TKIs獲得性耐藥細胞對gefitinib耐藥性,其機制可能是放射線照射后發(fā)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.

【學位授予單位】：廣東藥科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R734.2

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