崔青松，樸紅梅，崔晶剛，王富佳，安昌善

·論著·

肝細(xì)胞生長(zhǎng)因子誘導(dǎo)敏感非小細(xì)胞肺癌細(xì)胞對(duì)厄洛替尼耐藥及機(jī)制研究

崔青松，樸紅梅，崔晶剛，王富佳，安昌善*

目的 研究肝細(xì)胞生長(zhǎng)因子(HGF)誘導(dǎo)敏感非小細(xì)胞肺癌(NSCLC)細(xì)胞對(duì)厄洛替尼耐藥的機(jī)制，觀察c-Met及其下游信號(hào)通道蛋白是否參與HGF誘導(dǎo)不同基因型NSCLC細(xì)胞對(duì)厄洛替尼耐藥。方法 2014年1月—2015年1月，選擇人NSCLC細(xì)胞株P(guān)C-9〔表皮生長(zhǎng)因子受體(EGFR)突變型，敏感株〕、H292(EGFR野生型，敏感株)及人胚肺成纖維細(xì)胞MRC-5細(xì)胞，通過(guò)ELISA法檢測(cè)PC-9、H292、MRC-5細(xì)胞培養(yǎng)上清液中HGF水平。用MRC-5細(xì)胞培養(yǎng)上清液誘導(dǎo)PC-9、H292細(xì)胞，采用Western blotting法檢測(cè)c-Met及其下游通道蛋白表達(dá)情況。將56只雌性、SPF級(jí)BALB/c裸鼠隨機(jī)分為8組，每組7只。在PC-9細(xì)胞誘導(dǎo)模型中，對(duì)照組(C組)和厄洛替尼處理組(E組)裸鼠皮下接種PC-9細(xì)胞懸液，MRC-5誘導(dǎo)組(H組)、MRC-5和厄洛替尼處理組(HE組)裸鼠皮下接種PC-9+MRC-5細(xì)胞懸液；當(dāng)移植瘤直徑達(dá)到4 mm時(shí)，C組和H組采用0.9%氯化鈉溶液灌胃，E組和HE組采用厄洛替尼灌胃。在H292細(xì)胞誘導(dǎo)模型中，C組、E組裸鼠皮下接種H292細(xì)胞懸液，H組、HE組裸鼠皮下接種H292+MRC-5細(xì)胞懸液；模型建立后灌胃方式同PC-9細(xì)胞誘導(dǎo)模型。給藥結(jié)束后處死裸鼠，比較PC-9、H292細(xì)胞誘導(dǎo)模型中各組移植瘤重量。采用免疫組化法檢測(cè)裸鼠移植瘤組織中c-Met及其下游通道蛋白表達(dá)水平。結(jié)果 PC-9、H292細(xì)胞培養(yǎng)上清液中均未檢測(cè)到HGF，MRC-5細(xì)胞培養(yǎng)上清液中HGF水平為(1 262±90)pg/ml。Western blotting法結(jié)果顯示，MRC-5細(xì)胞培養(yǎng)上清液中HGF能活化PC-9、H292細(xì)胞中p-Met、p-Akt、p-Stat3、磷酸化細(xì)胞外調(diào)節(jié)蛋白激酶1/2(p-Erk1/2)活性。PC-9細(xì)胞誘導(dǎo)模型中：E組移植瘤重量小于C組(P<0.05)；HE組移植瘤重量小于H組，大于E組(P<0.05)。H292細(xì)胞誘導(dǎo)模型中：E組移植瘤重量小于C組(P<0.05)；HE組移植瘤重量小于H組，大于E組(P<0.05)。c-Met、p-Met分別定位于細(xì)胞膜和細(xì)胞質(zhì)。在PC-9、H292細(xì)胞誘導(dǎo)模型中：C組、H組、E組、HE組c-Met表達(dá)水平比較，差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；H組、HE組p-Met表達(dá)水平分別高于C組、E組(P<0.05)。Stat3定位于細(xì)胞質(zhì)，p-Stat3定位于細(xì)胞核。在PC-9、H292細(xì)胞誘導(dǎo)模型中：C組、H組、E組、HE組Stat3表達(dá)水平比較，差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；H組、HE組p-Stat3表達(dá)水平分別高于C組、E組(P<0.05)。Akt、p-Akt均定位于細(xì)胞質(zhì)。在PC-9、H292細(xì)胞誘導(dǎo)模型中：C組、H組、E組、HE組Akt表達(dá)水平比較，差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；H組、HE組p-Akt表達(dá)水平分別高于C組、E組(P<0.05)。Erk1/2定位于細(xì)胞質(zhì)，p-Erk1/2定位于細(xì)胞核。在PC-9、H292細(xì)胞誘導(dǎo)模型中：C組、H組、E組、HE組Erk1/2表達(dá)水平比較，差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；H組、HE組p-Erk1/2表達(dá)水平分別高于C組、E組(P<0.05)。結(jié)論 MRC-5細(xì)胞分泌的HGF能夠在裸鼠體內(nèi)誘導(dǎo)敏感NSCLC細(xì)胞PC-9、H292對(duì)厄洛替尼耐藥，HGF通過(guò)激活c-Met及其下游通道蛋白的磷酸化可能是不同基因型NSCLC細(xì)胞對(duì)厄洛替尼耐藥的重要機(jī)制。

癌，非小細(xì)胞肺；肝細(xì)胞生長(zhǎng)因子；抗藥性，腫瘤

崔青松，樸紅梅，崔晶剛，等.肝細(xì)胞生長(zhǎng)因子誘導(dǎo)敏感非小細(xì)胞肺癌細(xì)胞對(duì)厄洛替尼耐藥及機(jī)制研究[J].中國(guó)全科醫(yī)學(xué)，2017，20(9)：1076-1083.[www.chinagp.net]

CUI Q S,PIAO H M,CUI J G,et al.Mechanism of erlotinib resistance induced by hepatocyte growth factor in sensitive non-small-cell lung cancer cells in vivo[J].Chinese General Practice，2017，20(9)：1076-1083.

厄洛替尼是表皮生長(zhǎng)因子受體酪氨酸激酶抑制劑(epidermal growth factor receptor-tyrosine kinase inhibitor，EGFR-TKI)，是目前治療表皮生長(zhǎng)因子受體(epidermal growth factor receptor，EGFR)敏感突變的非小細(xì)胞肺癌(non-small cell lung cancer，NSCLC)最常用的分子靶向藥物，是治療NSCLC的一線藥物[1-3]。臨床研究顯示，厄洛替尼對(duì)部分EGFR野生狀態(tài)NSCLC細(xì)胞亦有一定的抑制作用[4-5]。由于厄洛替尼存在原發(fā)性耐藥或獲得性耐藥，僅對(duì)部分有特殊基因突變或腫瘤標(biāo)志物表達(dá)的NSCLC有效[6]。c-Met是肝細(xì)胞生長(zhǎng)因子(hepatocyte growth factor，HGF)的受體，在腫瘤增殖、侵襲過(guò)程中起重要作用。有研究顯示，在體外HGF誘導(dǎo)不同基因型NSCLC細(xì)胞對(duì)EGFR-TKI耐藥，可能與其誘導(dǎo)c-Met磷酸化活化有關(guān)[7-8]。在肺癌組織細(xì)胞微環(huán)境中，成纖維細(xì)胞普遍存在，可表達(dá)、分泌大量與腫瘤細(xì)胞增殖、存活相關(guān)的細(xì)胞因子及基質(zhì)成分。本研究選擇EGFR不同基因型人NSCLC敏感細(xì)胞株(EGFR突變型和EGFR野生型)，并采用人胚肺成纖維細(xì)胞MRC-5細(xì)胞建立HGF誘導(dǎo)，采用Western blotting法檢測(cè)c-Met及其下游通道蛋白的表達(dá)情況，將MRC-5細(xì)胞分別與PC-9、H292細(xì)胞混合種植于裸鼠右側(cè)肋下皮下，建立PC-9、H292細(xì)胞的裸鼠移植瘤模型，觀察腫瘤生長(zhǎng)情況，并檢測(cè)裸鼠移植瘤組織中c-Met及其下游通道蛋白的表達(dá)情況，旨在探究體內(nèi)HGF誘導(dǎo)不同基因型NSCLC細(xì)胞對(duì)厄洛替尼耐藥機(jī)制。

本研究背景：

本課題組前期研究表明,在體外肝細(xì)胞生長(zhǎng)因子(HGF)誘導(dǎo)敏感非小細(xì)胞肺癌(NSCLC)細(xì)胞對(duì)表皮生長(zhǎng)因子受體酪氨酸激酶抑制劑(EGFR-TKI)厄洛替尼耐藥，HGF刺激c-Met及其下游通道蛋白的磷酸化可能是敏感NSCLC細(xì)胞對(duì)EGFR-TKI耐藥的重要機(jī)制。

1 材料與方法

1.1 實(shí)驗(yàn)材料 人NSCLC細(xì)胞株P(guān)C-9(EGFR突變型，敏感株)、H292(EGFR野生型，敏感株)由同濟(jì)大學(xué)附屬上海肺科醫(yī)院中心實(shí)驗(yàn)室提供；人胚肺成纖維細(xì)胞MRC-5細(xì)胞購(gòu)自上海生命科學(xué)院細(xì)胞庫(kù)；56只雌性、4周齡、SPF級(jí)BALB/c裸鼠購(gòu)自上海市斯萊克實(shí)驗(yàn)動(dòng)物有限公司，許可證號(hào)SCXK(滬)2003-0003，放在標(biāo)準(zhǔn)塑料盒中由專職飼養(yǎng)員負(fù)責(zé)飼養(yǎng)；鹽酸厄洛替尼原料購(gòu)自大連美侖生物技術(shù)有限公司；Human HGF ELISA試劑盒購(gòu)自美國(guó)R&D Systems公司，兔抗人磷酸化c-Met(p-Met)(Tyr1234/1235,145 kDa)、c-Met(140/170 kDa)、磷酸化Akt(p-Akt)(Ser473，60 kDa)、Akt(60 kDa)、磷酸化細(xì)胞外調(diào)節(jié)蛋白激酶1/2(p-Erk1/2)(Tyr202/Y204,42/44 kDa)、Erk1/2(42/44 kDa)購(gòu)自CST公司，磷酸化Stat3(p-Stat3)(S727,98 kDa)、Stat3(92 kDa)購(gòu)自Abcam公司。

1.2 細(xì)胞培養(yǎng)及藥物配制 2014年1月—2015年1月，將人NSCLC細(xì)胞株P(guān)C-9、H292的液氮凍存管迅速放入37 ℃恒溫水浴箱中快速融化，PBS洗滌后，常規(guī)培養(yǎng)于含10%胎牛血清的DMEM培養(yǎng)液中，置于37 ℃、5% CO2恒溫細(xì)胞培養(yǎng)箱中孵育，3～4 d換液傳代1次。厄洛替尼原料采用DMSO溶解稀釋成濃度為50 μmol/ml的母液，用藥時(shí)DMSO終質(zhì)量濃度應(yīng)小于0.1%，稱取150 mg厄洛替尼加入45 ml 0.9%氯化鈉溶液中，再加入5 ml Tween-80,避光、4 ℃保存，使用前充分?jǐn)嚢琛?/p>

1.3 ELISA法檢測(cè)細(xì)胞上清液中HGF水平 采用胰酶消化細(xì)胞，分別將PC-9(2×105個(gè))、H292(2×105個(gè))、MRC-5(2×105個(gè))細(xì)胞接種于6孔板，放入細(xì)胞培養(yǎng)箱孵育48 h，取細(xì)胞培養(yǎng)上清液。在酶標(biāo)板的包被中加入150 μl試劑稀釋液后，每孔加入50 μl標(biāo)準(zhǔn)品、細(xì)胞培養(yǎng)上清液，輕晃混勻，用封板膜封板后室溫孵育2 h。棄去液體，甩干，沖洗4次，每孔加入200 μl酶標(biāo)試劑，室溫孵育1.75 h。再次棄去液體，甩干，用沖洗液沖洗4次，每孔加入200 μl顯色劑，室溫孵育30 min。每孔加入50 μl終止液，用酶標(biāo)儀測(cè)量波長(zhǎng)450 nm處光密度(OD)值；以標(biāo)準(zhǔn)物的濃度為橫坐標(biāo)，OD值為縱坐標(biāo)繪制標(biāo)準(zhǔn)曲線，根據(jù)細(xì)胞培養(yǎng)上清液的OD值由標(biāo)準(zhǔn)曲線查出相應(yīng)的濃度，再乘稀釋倍數(shù)即為細(xì)胞培養(yǎng)上清液中HGF水平。實(shí)驗(yàn)獨(dú)立重復(fù)3次。

1.4 Western blotting法檢測(cè)c-Met及其下游信號(hào)通道蛋白表達(dá)情況 取對(duì)數(shù)生長(zhǎng)期的PC-9(5×105個(gè))、H292(5×105個(gè))細(xì)胞接種于6孔板，細(xì)胞饑餓過(guò)夜后加入MRC-5(2×105個(gè))細(xì)胞培養(yǎng)48 h，收集上清液1 ml，立即冰上裂解細(xì)胞，4 ℃下14 800×g離心30 min，收集蛋白裂解液。采用BCA蛋白定量法進(jìn)行蛋白定量。取30～40 μg蛋白經(jīng)8%～10%十二烷基硫酸鈉-聚丙烯酰氨凝膠電泳(SDS-PAGE)后，轉(zhuǎn)印至硝酸纖維素(NC)膜上，用5%脫脂奶粉封閉1 h，一抗孵育4 ℃過(guò)夜，TBST洗膜(10 min)3次后二抗室溫?fù)u床孵育1 h，TBST洗膜(5 min)5次后ECL化學(xué)發(fā)光試劑顯色、曝光成像。

1.5 裸鼠移植瘤模型的建立及藥物抑瘤分析 將PC-9細(xì)胞、H292細(xì)胞和MRC-5細(xì)胞分別制成終密度為5×106個(gè)/ml、5×106個(gè)/ml、5×106個(gè)/ml的細(xì)胞懸液，而后將PC-9細(xì)胞、H292細(xì)胞分別與MRC-5細(xì)胞混合，制成混懸液PC-9(5×106個(gè)/ml)+MRC-5(5×106個(gè)/ml)、H292(5×106個(gè)/ml)+MRC-5(5×106個(gè)/ml)；當(dāng)裸鼠飼養(yǎng)到6周齡時(shí)，隨機(jī)分為8組，每組7只。

在PC-9細(xì)胞誘導(dǎo)模型中，對(duì)照組(C組)和厄洛替尼處理組(E組)裸鼠于右側(cè)肋下皮下接種PC-9細(xì)胞懸液100 μl，MRC-5誘導(dǎo)組(H組)、MRC-5和厄洛替尼處理組(HE組)裸鼠右側(cè)肋下皮下接種PC-9+MRC-5細(xì)胞懸液100 μl；當(dāng)移植瘤直徑達(dá)到4 mm時(shí)，C組和H組采用含1%Tween-80的0.9%氯化鈉溶液灌胃，E組和HE組采用已配制好的厄洛替尼25 mg·kg-1·d-1灌胃，每周給藥5 d，連續(xù)4周。

按照上述實(shí)驗(yàn)步驟及方法，在H292細(xì)胞誘導(dǎo)模型中，C組、E組裸鼠于右側(cè)肋下皮下接種H292細(xì)胞懸液100 μl，H組、HE組裸鼠右側(cè)肋下皮下接種H292+MRC-5細(xì)胞懸液100 μl；模型建立后灌胃方式亦同上。測(cè)量8組裸鼠第4、7、11、14、18、21、25天移植瘤體積(移植瘤體積=移植瘤長(zhǎng)徑×移植瘤短徑2/2)，并繪制移植瘤生長(zhǎng)曲線。給藥結(jié)束后處死裸鼠，剝離移植瘤組織后稱重。

1.6 免疫組化法檢測(cè)移植瘤組織中c-Met及其下游信號(hào)通道蛋白表達(dá)水平 將剝離的移植瘤組織固定、包埋，制成石蠟切片，脫蠟、水化，PBS洗滌3次，3 min/次。用含有3%過(guò)氧化氫的PBS孵育切片30 min，PBS洗滌3次，3 min/次。用微波法進(jìn)行抗原修復(fù)，PBS洗滌3次，3 min/次，將切片取出，加入5%羊血清，室溫孵育30 min。棄去切片上的羊血清，用PBS洗滌3次，5 min/次，加入已稀釋的一抗后，放入濕盒中室溫靜置1 h，然后4 ℃過(guò)夜。將一抗棄去并用PBS洗滌5次，5 min/次；加入二抗后室溫孵育2 h，PBS洗滌5次，5 min/次；快速加入DAB，孵育約5 min；PBS洗滌3次，3 min/次；加上蘇木素染液，再用PBS返藍(lán)5 min。系列梯度乙醇脫水，透明，中性樹(shù)膠封固。將所有切片照相，每組選取10張照片，所有照片用IPP 6.0圖像分析軟件進(jìn)行定量分析。

2 結(jié)果

2.1 ELISA法檢測(cè)細(xì)胞上清液中HGF水平 PC-9、H292細(xì)胞培養(yǎng)上清液中均未檢測(cè)到HGF，MRC-5細(xì)胞培養(yǎng)上清液中HGF水平為(1 262±90) pg/ml。

2.2 Western blotting法檢測(cè)c-Met及其下游信號(hào)通道蛋白表達(dá)情況 MRC-5細(xì)胞培養(yǎng)上清液中HGF能活化PC-9、H292細(xì)胞中p-Met、p-Stat3、p-Akt、p-Erk1/2活性(見(jiàn)圖1)。

注：p-Met=磷酸化c-Met，p-Stat3=磷酸化Stat3，p-Akt=磷酸化Akt，p-Erk1/2=磷酸化細(xì)胞外調(diào)節(jié)蛋白激酶1/2

圖1 Western blotting法檢測(cè)c-Met及其下游信號(hào)通道蛋白表達(dá)情況

Figure 1 Expressions of c-Met and its downstream signaling pathway proteins examined by Western blotting

2.3 PC-9、H292細(xì)胞誘導(dǎo)模型中各組不同時(shí)間點(diǎn)移植瘤體積比較 PC-9細(xì)胞誘導(dǎo)模型中：4組第4、7天移植瘤體積比較，差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；4組第11、14、18、21、25天移植瘤體積比較，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；其中E組第11、14、18、21、25天移植瘤體積小于C組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；HE組第18、21、25天移植瘤體積小于H組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；HE組第11、14、18、21、25天移植瘤體積大于E組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見(jiàn)表1)。

H292細(xì)胞誘導(dǎo)模型中：4組第4、7天移植瘤體積比較，差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；4組第11、14、18、21、25天移植瘤體積比較，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；其中E組第11、14、18、21、25天移植瘤體積小于C組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；HE組第11、14、18、21、25天移植瘤體積小于H組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；HE組第11、14、18、21、25天移植瘤體積大于E組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見(jiàn)表2)。

2.4 給藥結(jié)束后PC-9、H292細(xì)胞誘導(dǎo)模型中各組移植瘤重量比較 PC-9細(xì)胞誘導(dǎo)模型中：C組、H組、E組、HE組移植瘤重量分別為(680±373)、(805±492)、(109±84)、(361±172)mg；4組移植瘤重量比較，差異有統(tǒng)計(jì)學(xué)意義(F=6.628，P=0.002)；其中E組移植瘤重量小于C組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；HE組移植瘤重量小于H組，大于E組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。H292細(xì)胞誘導(dǎo)模型中：C組、H組、E組、HE組移植瘤重量分別為(920±122)、(979±176)、(389±109)、(564±176)mg；4組移植瘤重量比較，差異有統(tǒng)計(jì)學(xué)意義(F=25.454，P<0.001)；其中E組移植瘤重量小于C組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；HE組移植瘤重量小于H組，大于E組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。

2.5 免疫組化法檢測(cè)移植瘤組織中c-Met及其下游信號(hào)通道蛋白表達(dá)水平 c-Met、p-Met分別定位于細(xì)胞膜和細(xì)胞質(zhì)(見(jiàn)圖2～3，本文圖2～3彩圖見(jiàn)本刊官網(wǎng)www.chinagp.net電子期刊相應(yīng)文章附件)。在PC-9、H292細(xì)胞誘導(dǎo)模型中：各組c-Met表達(dá)水平比較，差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；各組p-Met表達(dá)水平比較，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；其中H組、HE組p-Met表達(dá)水平分別高于C組、E組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見(jiàn)表3～4)。

Stat3定位于細(xì)胞質(zhì)，p-Stat3定位于細(xì)胞核(見(jiàn)圖2～3)。在PC-9、H292細(xì)胞誘導(dǎo)模型中：各組Stat3

達(dá)水平比較，差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；各組p-Stat3表達(dá)水平比較，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；其中H組、HE組p-Stat3表達(dá)水平分別高于C組、E組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見(jiàn)表3～4)。

Akt、p-Akt均定位于細(xì)胞質(zhì)(見(jiàn)圖2～3)。在PC-9、H292細(xì)胞誘導(dǎo)模型中：各組Akt表達(dá)水平比較，差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。各組p-Akt表達(dá)水平比較，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)；其中H組、HE組p-Akt表達(dá)水平分別高于C組、E組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見(jiàn)表3～4)。

Erk1/2定位于細(xì)胞質(zhì)，p-Erk1/2定位于細(xì)胞核(見(jiàn)圖2～3)。在PC-9、H292細(xì)胞誘導(dǎo)模型中：各組Erk1/2表達(dá)水平比較，差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；各組p-Erk1/2表達(dá)水平比較，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)；其中H組、HE組p-Erk1/2表達(dá)水平分別高于C組、E組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見(jiàn)表3～4)。

表1 PC-9細(xì)胞誘導(dǎo)模型中各組不同時(shí)間點(diǎn)移植瘤體積比較

注：PC-9細(xì)胞誘導(dǎo)模型中，C組=對(duì)照組，H組=MRC-5誘導(dǎo)組，E組=厄洛替尼處理組，HE組=MRC-5和厄洛替尼處理組；與C組比較，aP<0.05；與H組比較，bP<0.05；與E組比較，cP<0.05

表2 H292細(xì)胞誘導(dǎo)模型中各組不同時(shí)間點(diǎn)移植瘤體積比較

注：H292細(xì)胞誘導(dǎo)模型中，C組=對(duì)照組，H組=MRC-5誘導(dǎo)組，E組=厄洛替尼處理組，HE組=MRC-5和厄洛替尼處理組；與C組比較，aP<0.05；與H組比較，bP<0.05；與E組比較，cP<0.05

Table 3 Comparison of expressions of c-Met and its downstream signaling pathway proteins in transplanted tumor tissues in groups of C,H,E and HE in PC-9 cell-induced model

組別只數(shù)p-Metc-Metp-Stat3Stat3p-AktAktp-Erk1/2Erk1/2C組70.167±0.0320.139±0.0220.100±0.0420.132±0.0320.157±0.0100.101±0.0240.121±0.0110.113±0.015H組70.225±0.012a0.150±0.0240.233±0.025a0.140±0.0100.225±0.004a0.102±0.0280.216±0.040a0.122±0.017E組70.154±0.0190.153±0.0180.096±0.0190.141±0.0300.151±0.0040.100±0.0110.101±0.0080.114±0.005HE組70.218±0.017b0.147±0.0420.252±0.022b0.140±0.0170.228±0.017b0.110±0.0290.210±0.041b0.116±0.012F值27.8620.46187.3590.290166.4570.56146.9650.841P值<0.0010.711<0.0010.832<0.0010.644<0.0010.480

注：p-Met=磷酸化c-Met，p-Stat3=磷酸化Stat3，p-Akt=磷酸化Akt，p-Erk1/2=磷酸化細(xì)胞外調(diào)節(jié)蛋白激酶1/2；與C組比較，aP<0.05；與E組比較，bP<0.05

Table 4 Comparison of expressions of c-Met and its downstream signaling pathway proteins in transplanted tumor tissues in groups of C,H,E and HE in H292 cell-induced model

組別只數(shù)p-Metc-Metp-Stat3Stat3p-AktAktp-Erk1/2Erk1/2C組70.137±0.0280.149±0.0230.122±0.0350.128±0.0230.131±0.0380.145±0.0410.164±0.0040.104±0.055H組70.205±0.017a0.148±0.0250.263±0.051a0.121±0.0200.260±0.011a0.123±0.0570.287±0.011a0.102±0.049E組70.150±0.0680.139±0.0210.131±0.0190.125±0.0290.120±0.0420.143±0.0250.171±0.0070.094±0.033HE組70.201±0.027b0.125±0.0120.255±0.061b0.140±0.0190.243±0.015b0.130±0.0190.282±0.020b0.103±0.043F值27.1842.84329.9201.24959.2861.940307.1740.110P值<0.0010.052<0.0010.306<0.0010.141<0.0010.954

注：與C組比較，aP<0.05；與E組比較，bP<0.05

注：PC-9細(xì)胞誘導(dǎo)模型中，C組=對(duì)照組，H組=MRC-5誘導(dǎo)組，E組=厄洛替尼處理組，HE組=MRC-5和厄洛替尼處理組

圖2 PC-9細(xì)胞誘導(dǎo)模型中各組移植瘤組織中c-Met及其下游信號(hào)通道蛋白表達(dá)(免疫組化染色，×200)

Figure 2 Expressions of c-Met and its downstream signaling pathway proteins in transplanted tumor tissues in groups of C,H,E and HE in PC-9 cell-induced model stained by immunohistochemistry

3 討論

肺癌已經(jīng)成為發(fā)病率和病死率最高的癌癥。由于肺癌的臨床癥狀隱蔽，早期不易篩查出來(lái)，約75%的NSCLC患者發(fā)現(xiàn)時(shí)已處于疾病晚期，而常規(guī)化療后肺癌患者的中位生存期僅為8～10個(gè)月[9]。EGFR-TKI在改善晚期NSCLC患者的近、遠(yuǎn)期療效與生活質(zhì)量中的作用顯著，已成為晚期NSCLC的有效治療手段，但臨床治療時(shí)仍存在EGFR-TKI原發(fā)、繼發(fā)耐藥問(wèn)題。EGFR突變與EGFR-TKI治療有效率間存在較高相關(guān)性[10]。目前EGFR-TKI獲得性耐藥公認(rèn)的兩大主要機(jī)制為EGFR二次突變(T790M)和MET基因擴(kuò)增，其他可能的機(jī)制有胰島素樣生長(zhǎng)因子1受體(IGF-1R)過(guò)表達(dá)[11]、蛋白酪氨酸磷酸酶基因(phosphatase and tensinhomolog deleted on chromosome ten,PTEN)缺失[12]、BIM下調(diào)[13]、整合素β1上調(diào)[14]、HGF高表達(dá)[15]等。2007年，ENGELMAN等[16]首次發(fā)現(xiàn)c-Met基因擴(kuò)增引起了EGFR-TKI耐藥。SENGUTA等[17]研究發(fā)現(xiàn)，部分肺癌患者血清中HGF水平明顯升高，而這些患者均屬于高侵襲型肺癌。HGF也被稱為分散因子(scatter facter，SF)，是成纖維細(xì)胞的衍生因子，由間質(zhì)細(xì)胞(如成纖維細(xì)胞、巨噬細(xì)胞)產(chǎn)生，具有多種生物學(xué)功能的生物信號(hào)分子，HGF與其特異性受體c-Met結(jié)合，使c-Met磷酸化為p-Met，激活下游通道，包括MAPKs、PI13K/Akt、c-Src/FAK、STAT信號(hào)通路，這些信號(hào)通路中部分蛋白與EGFR下游通道蛋白重疊，因此，雖然EGFR-TKI有效阻斷了EGFR及其下游通道，但是當(dāng)患者血清中HGF水平升高，并激活c-Met及其下游通道蛋白，便可以繞過(guò)EGFR通路，仍然能夠引起腫瘤細(xì)胞增殖、血管生成并轉(zhuǎn)移。

注：H292細(xì)胞誘導(dǎo)模型中，C組=對(duì)照組，H組=MRC-5誘導(dǎo)組，E組=厄洛替尼處理組，HE組=MRC-5和厄洛替尼處理組

圖3 H292細(xì)胞誘導(dǎo)模型中各組移植瘤組織中c-Met及其下游信號(hào)通道蛋白表達(dá)(免疫組化染色，×200)

Figure 3 Expressions of c-Met and its downstream signaling pathway proteins in transplanted tumor tissues in groups of C,H,E and HE in H292 cell-induced model stained by immunohistochemistry

本課題組前期研究測(cè)得厄洛替尼在體外抑制PC-9、H292細(xì)胞增殖的作用均呈濃度依賴性，HGF誘導(dǎo)后IC50值顯著升高，說(shuō)明HGF增加了細(xì)胞耐藥性，其耐藥機(jī)制與活化的c-Met相關(guān)[7]。

腫瘤微環(huán)境由腫瘤細(xì)胞和宿主基質(zhì)細(xì)胞組成，可促進(jìn)腫瘤細(xì)胞的生長(zhǎng)和轉(zhuǎn)移，并影響腫瘤細(xì)胞對(duì)抗腫瘤藥物的敏感性[18-20]。有研究結(jié)果顯示，在腫瘤微環(huán)境中，由基質(zhì)成纖維細(xì)胞分泌的HGF可降低多種腫瘤對(duì)靶向藥物的敏感性，如PLX4720(RAF抑制劑)[21]、Lapatinib(EGFR及EGFR-2抑制劑)[22]等。MRC-5細(xì)胞是人胚肺成纖維細(xì)胞，有研究表明其能夠分泌HGF[23]，本研究中通過(guò)ELISA法檢測(cè)到PC-9、H292細(xì)胞不產(chǎn)生HGF，而MRC-5細(xì)胞分泌HGF，其水平為(1 262±90)pg/ml。本研究選取PC-9細(xì)胞(EGFR突變型，敏感株)和H292細(xì)胞(EGFR野生型，敏感株)，將PC-9、H292細(xì)胞與MRC-5細(xì)胞培養(yǎng)上清液混合培養(yǎng)后，MRC-5細(xì)胞培養(yǎng)上清液中的HGF能夠活化PC-9、H292細(xì)胞中p-Met、p-Stat3、p-Akt、p-Erk1/2的活性；表明MRC-5細(xì)胞能夠分泌高水平HGF，并且其分泌的HGF能使PC-9、H292細(xì)胞中c-Met及其下游蛋白磷酸化。因此，本研究將MRC-5細(xì)胞分別與PC-9、H292細(xì)胞混合種植于裸鼠右側(cè)肋下皮下，建立HGF誘導(dǎo)PC-9、H292細(xì)胞耐藥模型，使PC-9、H292細(xì)胞在裸鼠體內(nèi)能夠持續(xù)受到HGF影響。通過(guò)對(duì)移植瘤生長(zhǎng)的觀察以及重量的稱取，發(fā)現(xiàn)厄洛替尼能夠抑制PC-9、H292細(xì)胞誘導(dǎo)模型E組、HE組腫瘤的生長(zhǎng)，但是厄洛替尼對(duì)PC-9、H292細(xì)胞誘導(dǎo)模型E組腫瘤的抑制作用更加明顯，說(shuō)明MRC-5細(xì)胞分泌的HGF能夠降低厄洛替尼對(duì)PC-9、H292細(xì)胞的抑制作用。免疫組化法檢測(cè)結(jié)果顯示，PC-9、H292細(xì)胞誘導(dǎo)模型中H組、HE組中p-Met、p-Stat3、p-Akt、p-Erk1/2表達(dá)水平均增高，與體外實(shí)驗(yàn)一致[8]。

綜上所述，本研究進(jìn)一步在裸鼠體內(nèi)證實(shí)HGF通過(guò)使PC-9、H292細(xì)胞中c-Met及其下游蛋白磷酸化，誘導(dǎo)PC-9、H292細(xì)胞對(duì)厄洛替尼耐藥，并且得出其耐藥機(jī)制與活化的c-Met及其下游信號(hào)通道蛋白相關(guān)。

作者貢獻(xiàn)：崔青松進(jìn)行實(shí)驗(yàn)設(shè)計(jì)與實(shí)施、資料收集整理、撰寫論文并對(duì)文章負(fù)責(zé)；樸紅梅、崔晶剛、王福佳進(jìn)行實(shí)驗(yàn)實(shí)施、評(píng)估、資料收集；安昌善進(jìn)行質(zhì)量控制及審校。

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(本文編輯：毛亞敏)

Mechanism of Erlotinib Resistance Induced by Hepatocyte Growth Factor in Sensitive Non-small-cell Lung Cancer Cells in Vivo

CUIQing-song,PIAOHong-mei,CUIJing-gang,WANGFu-jia，ANChang-shan*

DepartmentofRespiratoryMedicine,YanbianUniversityHospital,Yanji133000,China

*Correspondingauthor:ANChang-shan，Chiefphysician；E-mail：cs_an2003@aliyun.com

Objective To study the mechanism of erlotinib resistance induced by hepatocyte growth factor(HGF) in non-small-cell lung cancer(NSCLC) cells and to investigate whether c-Met and its downstream signaling pathway proteins participate in the HGF-induced erlotinib resistance of NSCLC cells with different genetypes in vivo.Methods This study was conducted from January 2014 to January 2015.We selected NSCLC cell lines with different EGFR genes(PC-9:EGFR-mutation type,sensitive;H292:EGFR-wild type,sensitive) and human embryonic lung fibroblasts MRC-5.The HGF level in cell culture supernatants secreted by PC-9,H292 and MRC-5 cells was quantified by ELISA.PC-9 and H292 cells were induced by MRC-5 cell culture supernatant;the expressions of c-Met and its downstream signaling pathway proteins were examined by Western blotting.Fifty-six female and SPF BALB/c nude mice were randomly divided into 8 groups,with each group containing 7.In building the PC-9 cell-induced model,the control group(group C) and erlotinib treatment group(group E) were subcutaneously inoculated PC-9 cell suspension and MRC-5 induced group(group H),MRC-5 and erlotinib treatment group(group HE) were inoculated PC-9+MRC-5 cell suspension subcutaneously.In establishing the H292 cell-induced model,group C and group E were inoculated H292 cell suspension subcutaneously while group H and group HE were inoculated H292+MRC-5 cell suspension subcutaneously.When the tumor diameter reached 4 mm in all groups,group C and group H were lavaged with 0.9% sodium chloride solution while group E and group HE were lavaged with erlotinib,respectively,in both built models.Mice were euthanized at after dosing.Comparisons of the weight of transplanted tumor were made between all groups in models induced by PC-9 and H292 cells.The expressions of c-Met and its downstream signaling pathway proteins in transplanted tumor tissues in nude mice were determined via immunohistochemistry.Results The concentration of HGF was not detected in PC-9 and H292 cell culture supernatants,while that in the supernatant of MRC-5 cells was(1 262 ±90) pg/ml.Western blotting results showed that HGF in the MRC-5 cell culture supernatant could stimulate the expression of p-Met,p-Akt,p-Stat3 and phosphorylated extracellular regulated protein kinase 1/2(p-Erk1/2) in PC-9 and H292 cells.The weight of transplanted tumor in group E was less than that in group C(P<0.05) in PC-9 cell-induced model;the weight of transplanted tumor in group HE was less than that in group H,but was greater than that in group E(P<0.05) in PC-9 cell-induced model.The weight of transplanted tumor in group E was less than in group C(P<0.05) in H292 cell-induced model;the weight of transplanted tumor in group HE was less than that in group H,whereas it was greater than that in group E(P<0.05) in H292 cell-induced model.c-Met and p-Met were localized in the cell membrane and cytoplasm,respectively.In both models,expressions of c-Met did not differ significantly between groups of C,H,E,and HE(P0.05);group H had higher expressions of p-Met than group C(P<0.05),and group HE had higher expressions of p-Met than group E(P<0.05).Stat3 was localized in cytoplasm and p-Stat3 in nucleus.In both models,no distinct differences in the expressions of Stat3 were noted between groups of C,H,E and HE(P0.05);group H had higher expressions of Stat3 than group C(P<0.05),and group HE showed higher expressions of Stat3 than group E(P<0.05).Akt and p-Akt were located in cytoplasm.In both models,the differences in the expressions of Akt were not prominent between groups of C,H,E and HE(P0.05);expressions of p-Akt in group H were higher than those in group C(P<0.05);expressions of p-Akt were found to be higher in group HE than in group E(P<0.05).Erk1/2 was localized in cytoplasm and p-Erk1/2 was localized in nucleus.In both models,expressions of Erk1/2 demonstrated no significant differences between groups of C,H,E and HE(P0.05);the expressions of p-Erk1/2 in H group were higher than those in group C(P<0.05);expressions of p-Erk1/2 in group HE were higher than those in group E(P<0.05).Conclusion HGF secreted by MRC-5 cell induces Erlotinib resistance in PC-9 and H292 cells in sensitive NSCLC cells.The HGF-induced activation of c-Met and its downstream signaling pathway proteins phosphorylation may be an important mechanism of Erlotinib resistance in sensitive NSCLC cells with different genetypes.

Carcinoma,non-small-cell lung；Hepatocyte growth factor;Drug resistance,neoplasm

國(guó)家自然科學(xué)基金資助項(xiàng)目(81160291)

R 730.26

A

10.3969/j.issn.1007-9572.2017.09.011

2016-10-21；

2017-01-27)

133000吉林省延吉市，延邊大學(xué)附屬醫(yī)院呼吸內(nèi)科

*通信作者：安昌善，主任醫(yī)師；E-mail：cs_an2003@aliyun.com