本文選題：非小細(xì)胞肺癌　切入點(diǎn)：EGFR突變L861Q　出處：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：研究背景非小細(xì)胞肺癌(NSCLC)由于其高發(fā)病率和高病死率成為世界性難題,近年來(lái)出現(xiàn)的靶向治療為NSCLC患者提供新的治療思路。表皮生長(zhǎng)因子受體(EGFR)突變是靶向治療是否有效的關(guān)鍵所在,酪氨酸激酶抑制劑(TKIs)可以延長(zhǎng)常見(jiàn)敏感突變患者(del-19, L858R)的非疾病進(jìn)展生存期(PFS)。然而EGFR罕見(jiàn)突變L861Q對(duì)靶向TKIs藥物治療敏感性尚不明確,需要進(jìn)一步研究。研究目的1 基礎(chǔ)實(shí)驗(yàn)方面,通過(guò)取得大量純度較高的EGFR野生型和各突變型目的蛋白,體外分析小分子TKIs藥物與EGFR突變蛋白的結(jié)合情況,探索EGFR突變蛋白的激酶活性以及小分子藥物抑制效果。2 臨床隨訪方面,探索L861Q突變患者臨床特點(diǎn)和預(yù)后情況,并分析該突變患者對(duì)TKIs藥物靶向治療的敏感性。研究方法1 基礎(chǔ)實(shí)驗(yàn)部分：通過(guò)NCBI檢索獲得野生型EGFR蛋白696-1022區(qū)域?qū)?yīng)的截短體基因,PCR擴(kuò)增后連接至載體質(zhì)粒。設(shè)計(jì)各突變蛋白基因引物,PCR獲得含有本研究目的基因的重組質(zhì)粒。之后利用Bac-to-Bac桿狀病毒表達(dá)系統(tǒng)將目的基因重組至昆蟲(chóng)真核細(xì)胞sf9中,大量傳代培養(yǎng)sf9細(xì)胞,之后破碎細(xì)胞獲得目的蛋白,通過(guò)鎳柱純化和分子篩層析純化,最終獲得大量純度高的野生型和各突變性EGFR目的蛋白。下一步,利用取得的目的蛋白進(jìn)行體外SPR實(shí)驗(yàn)初步分析小分子TKIs藥物與EGFR目的蛋白的親和力情況。之后通過(guò)蛋白激酶實(shí)驗(yàn)分析EGFR目的蛋白的激酶活性及TKIs藥物的抑制效果。2 臨床隨訪部分：回顧性收集全國(guó)范圍內(nèi)5125例在2009-2012年接受基因突變檢測(cè)的肺癌患者和北京協(xié)和醫(yī)院489例在2009-2014年接受基因突變檢測(cè)的肺癌患者,最終確定了6例含有L861Q突變的患者。針對(duì)6例患者收集臨床資料,包括性別、年齡、吸煙史、病理類(lèi)型、腫瘤分期、治療情況等,并隨訪生存期。最后分析L861Q突變患者的臨床特點(diǎn)和靶向治療敏感性。研究結(jié)果1 基礎(chǔ)實(shí)驗(yàn)部分：經(jīng)過(guò)提純濃縮獲得了500ul濃度2.5mg/ml的野生型EGFR蛋白、500ul濃度200ug/ml的EGFR-L858R突變蛋白、800ul濃度500ug/ml的EGFR-L861Q突變蛋白、500ul濃度315ug/ml的EGFR-L861Q/G719A聯(lián)合突變蛋白。之后的SPR實(shí)驗(yàn)未能擬合計(jì)算出親和力常數(shù)。激酶實(shí)驗(yàn)部分證實(shí)野生型和各突變型EGFR均具有激酶活性,且活性從高到低依次為：L858RL861Q/G719A L861QG719AWT。2 臨床隨訪部分：全國(guó)范圍的5125例肺癌患者和北京協(xié)和醫(yī)院隨訪的489例肺癌患者中,EGFR突變率分別為36.2%(1854/5125)和44.2%(216/489),其中聯(lián)合突變比例分別為7.2%(160/2208)和6.02%(13/216)。本研究的L861Q突變占總突變的比例分別為0.27%(5/1854)和0.46%(1/216),且所有L861Q突變均以聯(lián)合突變形式出現(xiàn),L861Q聯(lián)合突變比例為100%。其中最常見(jiàn)的聯(lián)合突變?yōu)長(zhǎng)861Q和G719A聯(lián)合,占66.7%(4/6)。L861Q患者初期使用吉非替尼治療有效,腫瘤穩(wěn)定無(wú)進(jìn)展,4-5個(gè)月出現(xiàn)耐藥,改用厄洛替尼無(wú)效。結(jié)論1 成功獲得大量純度高的EGFR野生型及研究的各突變型目的蛋白。2 本研究EGFR-L861Q突變蛋白具有激酶活性,L861Q突變?yōu)橹掳┩蛔�；各目的蛋白激酶活性從高到低為：L858RL861Q/G719AL861QG719AWT。3 EGFR-L861Q突變患者初期吉非替尼靶向治療有效,但很快出現(xiàn)耐藥(4-5個(gè)月),之后改用厄洛替尼無(wú)效。
[Abstract]:The research background of non-small cell lung cancer (NSCLC) due to its high incidence and high mortality rate has become a worldwide problem, in recent years the emergence of targeted therapy to provide new therapy of NSCLC patients. The epidermal growth factor receptor (EGFR) mutation is targeting key treatment is effective, tyrosine kinase inhibitors (TKIs) can extended common sensitive mutations (del-19, L858R) - progression free survival (PFS). However, EGFR L861Q of rare mutations targeting drug sensitivity of TKIs treatment is not clear, the need for further research. The purpose of the study of 1 basic experimental aspects, made by high purity EGFR wild type and the mutant protein. In vitro analysis combined with drugs and small molecule EGFR TKIs mutant protein, explore the EGFR mutant protein kinase activity and the inhibitory effect of.2 small molecule drugs clinical follow-up, to explore the mutation of L861Q patients The characteristics and prognosis, and to analyze the mutations of TKIs targeted drug sensitivity. Methods 1 basic experimental part: through NCBI retrieve the truncated gene corresponding to wild-type EGFR protein region 696-1022, amplified by PCR and connected to the plasmid. The design of each mutant protein gene primers containing the recombinant plasmid PCR objective to study the gene. After using Bac-to-Bac baculovirus expression system to recombinant eukaryotic cells to insect Sf9, a large number of cultured Sf9 cells were obtained after crushing protein cells, chromatography and molecular sieve purification by nickel column, finally get the wild type with high purity and the mutagenicity of EGFR protein. Step SPR in vitro experiment preliminary affinity analysis of small molecule drugs and EGFR TKIs protein using protein. The protein kinase. Experimental analysis of EGFR protein kinase activity and the inhibitory effect of.2 TKIs drug clinical follow-up part: retrospectively collected 5125 cases nationwide in 2009-2012 years to accept the detection of gene mutation in patients with lung cancer and 489 cases in Peking Union Medical College Hospital in 2009-2014 years for gene mutation assay in patients with lung cancer, and ultimately determine the 6 patients with the L861Q mutation. In 6 cases of patients with clinical data were collected, including gender, age, smoking history, pathological type, tumor staging, treatment, and survival analysis. Finally L861Q mutation of clinical characteristics and target patients to treatment sensitivity. Results of the 1 basic experimental part: after purification and concentration to obtain 500ul concentration of 2.5mg/ml wild type EGFR protein, 500ul concentration of 200ug/ml EGFR-L858R mutant protein, the concentration of 800ul 500ug/ml EGFR-L861Q mutant protein, the concentration of 500ul 315ug/ml EGFR-L861Q/G719A combined process Change of protein. SPR experiments failed to calculate the affinity constant after fitting. The experimental part confirmed that wild-type and kinase of the mutant EGFR have kinase activity, and the activity from high to low is: L858RL861Q/G719A L861QG719AWT.2: clinical follow-up of 489 lung cancer patients nationwide 5125 lung cancer patients and follow-up in Peking Union Medical College Hospital, EGFR mutation rates were 36.2% (1854/5125) and 44.2% (216/489), which combined mutation rates were 7.2% (160/2208) and 6.02% (13/216). The L861Q mutation of total mutation rates were 0.27% (5/1854) and 0.46% (1/216), and all the L861Q mutation was combined with mutation form, L861Q the mutation ratio of 100%. one of the most common mutation for L861Q and G719A, accounting for 66.7% (4/6).L861Q in patients with early use of gefitinib treatment is effective, stable tumor progression occurred in 4-5 months Resistance to erlotinib is invalid. Conclusion 1 successfully obtained a large number of wild type EGFR and study the high purity of the mutant protein the.2 mutant EGFR-L861Q protein with kinase activity, L861Q mutations for oncogenic mutations; the protein kinase activity from high to low: L858RL861Q/G719AL861QG719AWT.3 EGFR-L861Q mutation in patients with early gefitinib target to effective treatment, but soon the emergence of resistant (4-5 months), after the use of erlotinib is invalid.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R734.2

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本文編號(hào)：1699770