【摘要】：目的:肺癌是世界上發(fā)病率死亡率均居于首位的惡性腫瘤,其中非小細(xì)胞肺癌占所有肺癌的80-85%。異質(zhì)性強(qiáng)、生物學(xué)行為復(fù)雜易于侵襲轉(zhuǎn)移是其主要特點(diǎn),而易于遠(yuǎn)處轉(zhuǎn)移進(jìn)入晚期又是導(dǎo)致臨床治療失敗及患者死亡的最主要原因之一。近年,以表皮生長因子受體酪氨酸激酶抑制劑(epidermal growth factor receptor-tyrosine kinase inhibitors,EGFR-TKIs)為代表的高效、低毒的分子靶向藥物在治療EGFR突變的非小細(xì)胞肺癌領(lǐng)域取得了突破性進(jìn)展,并被美國國立綜合癌癥網(wǎng)絡(luò)(National Comprehensive Cancer Network,NCCN)推薦作為表皮生長因子受體(epidermal growth factor receptor,EGFR)突變晚期非小細(xì)胞肺癌治療的首選藥物。EGFR-TKI的主要作用機(jī)制是通過干預(yù)腫瘤細(xì)胞EGFR胞內(nèi)區(qū)的酪氨酸激酶結(jié)構(gòu)域,阻止其異�；罨�,進(jìn)而阻斷下游信號(hào)轉(zhuǎn)導(dǎo),發(fā)揮抑制腫瘤細(xì)胞增殖、促進(jìn)腫瘤細(xì)胞凋亡等作用。然而臨床效果觀察發(fā)現(xiàn),部分服用EGFR-TKI的患者不僅腫瘤生長受到抑制,其整體轉(zhuǎn)移趨勢也受到影響。這就提示EGFR-TKI可能不僅作用于腫瘤細(xì)胞本身,很可能還通過某種潛在途徑調(diào)節(jié)腫瘤微環(huán)境,進(jìn)而對(duì)腫瘤轉(zhuǎn)移產(chǎn)生影響,但具體機(jī)制目前還尚不明了。黏附分子CD44是腫瘤微環(huán)境中黏附分子家族重要成員之一,其可介導(dǎo)細(xì)胞與細(xì)胞之間、細(xì)胞與基質(zhì)之間的黏附作用,并通過參與一系列信號(hào)轉(zhuǎn)導(dǎo)為腫瘤轉(zhuǎn)移提供足夠動(dòng)力。從頭頸部腫瘤、乳腺癌等的研究結(jié)果中發(fā)現(xiàn),EGFR信號(hào)通路與CD44之間可能存在著某種交叉協(xié)同關(guān)系,并在腫瘤轉(zhuǎn)移中發(fā)揮重要作用,但二者在非小細(xì)胞肺癌(non-small cell lung cancer,NSCLC)中是否也存在這種交互作用并對(duì)轉(zhuǎn)移產(chǎn)生影響以及具體機(jī)制目前還不清楚。遂本研究的目的即通過多種體外實(shí)驗(yàn)方法探討在EGFR突變的NSCLC細(xì)胞系中,EGFR-TKI抑制腫瘤轉(zhuǎn)移的機(jī)制是否與下調(diào)黏附分子CD44表達(dá)有關(guān),并初步探索連接二者信號(hào)通路的關(guān)鍵信號(hào)分子。此研究不僅有利于我們對(duì)信號(hào)通路產(chǎn)生更深入的認(rèn)識(shí),更為未來尋找更多抑制轉(zhuǎn)移的新藥尋求靶點(diǎn)。方法:1細(xì)胞培養(yǎng)使用含15%胎牛血清、100U/ml青霉素及100μg/ml鏈霉素的RPMI-1640培養(yǎng)基培養(yǎng)HCC827細(xì)胞(EGFR19外顯子突變肺腺癌細(xì)胞)和A549細(xì)胞(EGFR野生型肺腺癌細(xì)胞),置于37℃、5%CO2的恒溫培養(yǎng)箱中,隔天換液,細(xì)胞長至80%以上處于對(duì)數(shù)生長期時(shí)進(jìn)行實(shí)驗(yàn)。2采用實(shí)時(shí)無標(biāo)記細(xì)胞增殖實(shí)驗(yàn)方法分別檢測HCC827細(xì)胞(EGFR突變型)和A549細(xì)胞(EGFR野生型)對(duì)EGFR-TKI代表藥物厄洛替尼的敏感性,進(jìn)一步確定實(shí)驗(yàn)用細(xì)胞。3四甲基偶氮唑藍(lán)(MTT)法檢測不同濃度厄洛替尼對(duì)HCC827的增殖抑制作用。在加入相應(yīng)濃度藥物的同時(shí)并加入合適濃度(50ng/ml)的EGF刺激因子,以模擬人體內(nèi)環(huán)境,藥物作用48h后計(jì)算細(xì)胞增殖抑制率及半數(shù)抑制濃度(IC50),做出增殖抑制率曲線。4采用Transwell小室侵襲實(shí)驗(yàn)及劃痕實(shí)驗(yàn)分別觀察四組(control、EGF(50ng/ml)刺激組、厄洛替尼(0.3μM)+EGF(50ng/ml)處理組、CD44中和抗體(20μg/ml)+EGF(50ng/ml)處理組)腫瘤細(xì)胞侵襲及遷移能力的變化情況。5采用流式細(xì)胞術(shù)檢測三組(control、EGF(50ng/ml)刺激組、厄洛替尼(0.3μM)+EGF(50ng/ml)處理組)腫瘤細(xì)胞表面CD44表達(dá)情況。6采用Western-blot實(shí)驗(yàn)方法檢測三組(control、EGF(50ng/ml)刺激組、厄洛替尼(0.3μM)+EGF(50ng/ml)處理組)腫瘤細(xì)胞CD44蛋白表達(dá)水平變化。7采用q RT-PCR實(shí)驗(yàn)技術(shù)檢測三組(control、EGF(50ng/ml)刺激組、厄洛替尼(0.3μM)+EGF(50ng/ml)處理組)腫瘤細(xì)胞CD44m RNA的表達(dá)水平變化。8采用Western-blot實(shí)驗(yàn)方法檢測四組(control、EGF(50ng/ml)刺激組、厄洛替尼(0.3μM)+EGF(50ng/ml)處理組、STAT3阻斷劑(S3I-20150μM)+EGF(50ng/ml)處理組)細(xì)胞CD44、STAT3、磷酸化STAT3蛋白表達(dá)水平。9統(tǒng)計(jì)學(xué)方法:采用SPSS 21.0統(tǒng)計(jì)軟件進(jìn)行數(shù)據(jù)處理,計(jì)量數(shù)據(jù)以(?)±s或M±QR表示,組間比較以單因素方差分析或秩和檢驗(yàn)進(jìn)行統(tǒng)計(jì)分析,LSD法進(jìn)行各組間兩兩比較,檢驗(yàn)水準(zhǔn)α=0.05,以P0.05為差異具有統(tǒng)計(jì)學(xué)意義。結(jié)果:1實(shí)時(shí)無標(biāo)記細(xì)胞增殖實(shí)驗(yàn)結(jié)果如Fig.1所示:EGFR-TKI代表藥物厄洛替尼(0.3μM)對(duì)EGFR突變的非小細(xì)胞肺癌細(xì)胞系HCC827具有較強(qiáng)的增殖抑制作用,并呈現(xiàn)時(shí)間依賴性;而對(duì)EGFR野生型的肺腺癌細(xì)胞系A(chǔ)549幾乎不產(chǎn)生作用;遂選定HCC827進(jìn)行后續(xù)實(shí)驗(yàn)。2 MTT法檢測不同濃度厄洛替尼對(duì)HCC827細(xì)胞的增殖抑制作用,結(jié)果顯示:隨著藥物濃度增大(0.001、0.01、0.1、0.5、1、10μM),厄洛替尼作用HCC827細(xì)胞48h后的增殖抑制率也逐漸增高,且呈劑量依賴性(Fig.2、Table1,P0.05)。結(jié)合細(xì)胞增殖抑制率,采用直線回歸方法得出IC50為0.323μM;3 Transwell侵襲實(shí)驗(yàn)結(jié)果如Fig.3、Fig.4和Table2所示:control、EGF刺激組、厄洛替尼+EGF處理組、CD44中和抗體+EGF處理組穿膜細(xì)胞數(shù)分別為(64.07±1.51)個(gè)、(129.53±4.20)個(gè)、(21.0±1.06)個(gè)、(23.87±1.70)個(gè);實(shí)驗(yàn)組與對(duì)照組比較差異具有統(tǒng)計(jì)學(xué)意義(P0.05);劃痕實(shí)驗(yàn)結(jié)果如Fig.5、Fig.6和Table3所示:各組細(xì)胞遷移距離分別為(78.65±3.19)μm、(119.98±1.62)μm、(51.73±4.23)μm、(53.18±6.71)μm;實(shí)驗(yàn)組與對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義(P0.05);結(jié)果證明厄洛替尼可抑制HCC827細(xì)胞的侵襲遷移能力。4流式細(xì)胞術(shù)檢測三組(control、EGF(50ng/ml)刺激組、厄洛替尼(0.3μM)+EGF(50ng/ml)處理組)HCC827細(xì)胞表面CD44表達(dá)水平分別為(25.87±3.46)%、(48.37±2.21)%、(15.50±1.11)%;結(jié)果顯示加入厄洛替尼后細(xì)胞表面CD44表達(dá)顯著低于前兩組,差異有統(tǒng)計(jì)學(xué)意義(Fig.7、Table4,P0.05);5 Western-blot方法檢測三組(同4)腫瘤細(xì)胞CD44蛋白半定量結(jié)果分別為(0.72±0.03)、(0.83±0.04)、(0.21±0.03),加入厄洛替尼組CD44蛋白表達(dá)顯著低于對(duì)照組和EGF刺激組,差異有統(tǒng)計(jì)學(xué)意義(Fig.8、Table5,P0.05);結(jié)果從蛋白水平進(jìn)一步證明厄洛替尼可下調(diào)CD44表達(dá)。6 qRT-PCR法檢測三組(同4)腫瘤細(xì)胞CD44 m RNA表達(dá)變化:設(shè)對(duì)照組為1,EGF刺激組及厄洛替尼+EGF處理組CD44 m RNA表達(dá)量分別是對(duì)照組的(2.22±0.17)倍和(0.50±0.04)倍;差異有統(tǒng)計(jì)學(xué)意義(Fig.9、Table6,P0.05);實(shí)驗(yàn)結(jié)果從基因水平上再次證明厄洛替尼可下調(diào)CD44表達(dá)。7 Western-blot方法檢測四組(control、EGF(50ng/ml)刺激組、厄洛替尼(0.3μM)+EGF(50ng/ml)處理組、STAT3阻斷劑(S3I-201 50μM)+EGF(50ng/ml)處理組)腫瘤細(xì)胞CD44、STAT3、磷酸化STAT3蛋白表達(dá)水平,結(jié)果如Fig.10、Fig.11、Fig.12和Table7所示:與對(duì)照組比較,厄洛替尼組CD44和p-STAT3蛋白表達(dá)水平均被顯著下調(diào)(P0.05),而在使用STAT3特異性阻斷劑阻斷STAT3信號(hào)通路后CD44蛋白表達(dá)仍被下調(diào)(P0.05);綜合以上結(jié)果初步得出,厄洛替尼阻斷EGFR信號(hào)通路的同時(shí),其間接下調(diào)黏附分子CD44表達(dá)很可能是通過EGFR/STAT3信號(hào)通路進(jìn)行的,但其深入機(jī)制還需進(jìn)一步探索。結(jié)論:1 HCC827細(xì)胞對(duì)EGFR-TKI代表藥物厄洛替尼高度敏感,適合作為本實(shí)驗(yàn)用細(xì)胞。2 EGFR-TKI藥物不僅可顯著抑制EGFR突變NSCLC細(xì)胞株HCC827細(xì)胞的增殖,還對(duì)其侵襲轉(zhuǎn)移能力產(chǎn)生明顯影響,并可能與CD44被下調(diào)有關(guān)。3 EGFR-TKI藥物可明顯下調(diào)黏附分子CD44表達(dá),且可能與EGFR/STAT3信號(hào)通路有關(guān)。
[Abstract]:Objective: Lung cancer is one of the most common malignant tumors in the world, in which non-small cell lung cancer accounts for 80-85% of all lung cancers. In recent years, low-toxicity molecular targeted drugs, represented by epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), have made breakthroughs in the treatment of EGFR-mutated non-small cell lung cancer (NSCLC), and have been used by the National Compreh Cancer Network (NCN). The main mechanism of EGFR-TKI is to block the abnormal activation of tyrosine kinase domains in the intracellular domain of EGFR, thereby blocking the downstream signal. However, some patients taking EGFR-TKI not only inhibited tumor growth, but also affected the overall metastasis trend. This suggests that EGFR-TKI may not only affect tumor cells themselves, but also possibly regulate them through some potential pathways. Adhesion molecule CD44 is one of the important members of the adhesion molecule family in tumor microenvironment, which mediates cell-to-cell and cell-to-matrix adhesion, and provides sufficient information for tumor metastasis by participating in a series of signal transduction pathways. Dynamics. In the study of head and neck tumors, breast cancer and so on, we found that there may be some cross-synergistic relationship between EGFR signaling pathway and CD44, which plays an important role in tumor metastasis, but whether EGFR signaling pathway and CD44 signaling pathway also have this interaction in non-small cell lung cancer (NSCLC) and affect metastasis. The purpose of this study is to investigate whether the mechanism of EGFR-TKI inhibiting tumor metastasis is related to the down-regulation of CD44 expression in NSCLC cell lines with EGFR mutation and to explore the key signaling molecules linking the two signaling pathways. Methods: 1. HCC 827 cells (EGFR19 exon mutant lung adenocarcinoma cells) and A549 cells (EGFR wild type lung adenocarcinoma cells) were cultured in RPMI-1640 medium containing 15% fetal bovine serum, 100U/ml penicillin and 100ug/ml streptomycin. HCC 827 cells (EGFR mutant) and A549 cells (EGFR wild type) were tested for their sensitivity to erlotinib, a representative of EGFR-TKI, by real-time labeled cell proliferation assay. Cell proliferation inhibitory effect of erlotinib on HCC 827 was detected by MTT assay. The proliferation inhibitory rate and half inhibitory concentration (IC50) were calculated 48 hours after the drug was added to the corresponding concentration of erlotinib and the appropriate concentration of EGF stimulator (50ng/ml) was added to simulate the human body environment. The invasion and migration of tumor cells were observed by Transwell chamber invasion test and scratch test respectively in four groups (control, EGF (50ng/ml), erlotinib (0.3ugM) + EGF (50ng/ml) and CD44 neutralizing antibody (20ug/ml) + EGF (50ng/ml). Flow cytometry was used to detect the invasion and migration of tumor cells in three groups (control, EGF (50n/ml). The expression of CD44 on the surface of tumor cells was detected by Western blot. The expression of CD44 on tumor cells was detected by Q RT-PCR in three groups (control, EGF (50ng/ml) and erlotinib (0.3 mu M) + EGF (50ng/ml) respectively. The expression of CD44m RNA in tumor cells of EGF (50ng/ml) stimulation group, Erlotinib (0.3ugM) + EGF (50ng/ml) treatment group and Western blot assay were used to detect CD44, STAT3, phosphoric acid in four groups (control, EGF (50ng/ml) stimulation group, Erlotinib (0.3ugM) + EGF (50ng/ml) treatment group, STAT3 blocker (S3I-20150ugM) + EGF (50ng/ml) treatment group. Statistical methods: SPSS 21.0 statistical software was used to process the data. The measurement data were expressed as (?) + s or M + QR. Single factor analysis of variance or rank sum test were used to analyze the statistical data. LSD method was used to compare the expression of STAT3 protein between groups. The test level was alpha = 0.05, and the difference was statistically significant with P 0.05. Real-time label-free cell proliferation experiment showed that the EGFR-TKI representative drug Erlotinib (0.3 mu M) inhibited the proliferation of non-small cell lung cancer cell line HCC827 with EGFR mutation in a time-dependent manner, but had little effect on wild-type lung adenocarcinoma cell line A549 with EGFR. HCC827 was selected for follow-up study. 2 MTT assay was used to detect the inhibitory effect of different concentrations of erlotinib on the proliferation of HCC 827 cells. The results showed that with the increase of the concentration of erlotinib (0.001, 0.01, 0.1, 0.5, 1, 10 mu M), the inhibitory rate of proliferation of HCC 827 cells increased gradually after 48 hours and was dose-dependent (Fig.2, Table1, P 0.05). Combined with the inhibitory rate of cell proliferation, the inhibitory rate of erlotinib was linear. Regression analysis showed that IC50 was 0.323 mu M; 3 Transwell invasion test results were shown as Fig.3, Fig.4 and Table2: control, EGF stimulation group, erlotinib + EGF treatment group, CD44 neutralizing antibody + EGF treatment group, the number of penetrating membrane cells were (64.07 [1.51], (129.53 [4.20], (21.0 [1.06], (23.87 [1.70], respectively; the experimental group compared with the control group. Scratch test showed that the migration distances of HCC 827 cells in each group were (78.65 (+ 3.19) micron, (119.98 (+ 1.62) micron, (51.73 (+ 4.23) micron, (53.18 (+ 6.71) micron), respectively. There was significant difference between the experimental group and the control group (P 0.05); the results showed that erlotinib could inhibit the invasion and migration of HCC 827 cells. The expression of CD44 on the surface of HCC 827 cells in three groups (control, EGF (50ng/ml) and erlotinib (0.3 mu M) + EGF (50ng/ml) was detected by flow cytometry. The results showed that the expression of CD44 on the surface of HCC 827 cells in the control, EGF (50 ng/ml) and erlotinib (0.3 mu) + EGF (50 ng/ml) groups was significantly lower than that in the former two groups (Fig) respectively. The semi-quantitative results of CD44 protein in tumor cells of the three groups (the same 4) by Western-blot were (0.72.03), (0.83.04), (0.21.03), respectively. The expression of CD44 protein in erlotinib group was significantly lower than that in control group and EGF stimulation group (Fig.8, Table5, P 0.05). Lotinib could down-regulate the expression of CD44. 6 qRT-PCR assay was used to detect the expression of CD44 m RNA in tumor cells of three groups (the same 4). The expression of CD44 m RNA in EGF-stimulated group and Erlotinib+EGF-treated group was (2.22+0.17) and (0.50+0.04) times higher than that in control group (Fig.9, Table6, P 0.05), respectively. Western-blot method was used to detect the expression of CD44, STAT3 and phosphorylated STAT3 protein in tumor cells of four groups (control, EGF (50ng/ml) stimulation group, erlotinib (0.3ugm) + EGF (50ng/ml) treatment group, STAT3 blocker (S3I-201 50ugM) + EGF (50ng/ml) treatment group). Compared with the control group, the expression of CD44 and p-STAT3 protein in the erlotinib group were significantly down-regulated (P 0.05), while the expression of CD44 protein was still down-regulated after STAT3 specific blocker was used to block the STAT3 signaling pathway (P 0.05); based on the above results, it was preliminarily concluded that erlotinib blocked the EGFR signaling pathway and indirectly down-regulated the adhesion molecule C. The expression of D44 may be mediated by EGFR/STAT3 signaling pathway, but its mechanism still needs further exploration. Conclusion: 1. HCC827 cells are highly sensitive to erlotinib, which is the representative drug of EGFR-TKI. 2 EGFR-TKI drugs can not only inhibit the proliferation of EGFR mutant NSCLC cell line HCC827, but also inhibit its invasion. The ability of metastasis may be related to the down-regulation of CD44. 3 EGFR-TKI can down-regulate the expression of CD44 and may be related to the EGFR/STAT3 signaling pathway.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R734.2

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