張先鋒，黃遠見，肖 娟，張志偉，黃衛(wèi)國

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·論著·

miR-150促進鼻咽癌細胞增殖和侵襲的機制研究

張先鋒，黃遠見，肖 娟，張志偉，黃衛(wèi)國

目的探討miR-150是否通過靶向調(diào)控程序性細胞死亡因子4(PDCD4)促進鼻咽癌細胞增殖和侵襲，進一步揭示miR-150在鼻咽癌中的癌基因作用。方法2014年3—6月，培養(yǎng)鼻咽癌細胞株CNE-2，取生長良好的CNE-2用于實驗。設(shè)計合成PDCD4野生型引物序列以及突變型引物序列，連接到含有熒光素酶載體質(zhì)粒上，檢測熒光素酶活性。分別瞬時轉(zhuǎn)染Vector、pcDNA3.1(+)-PDCD4、miR-ctr、miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4于CNE-2，48 h后采用Western blotting法檢測PDCD4表達水平。分別瞬時轉(zhuǎn)染Vector、pcDNA3.1(+)-PDCD4、miR-150 inhibitors、miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4于CNE-2，分別于培養(yǎng)24、48、72、96 h后測定其吸光度(OD)值，反映其增殖能力。分別瞬時轉(zhuǎn)染Vector、pcDNA3.1(+)-PDCD4、miR-150 inhibitors、miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4于CNE-2，采用Transwell侵襲實驗檢測CNE-2侵襲能力。結(jié)果無miR-150轉(zhuǎn)染的PDCD4野生型細胞株熒光素酶活性為(0.975±0.112)，miR-150轉(zhuǎn)染的PDCD4野生型細胞株熒光素酶活性為(0.588±0.042)，差異有統(tǒng)計學意義(t=7.853,P=0.018)。無miR-150轉(zhuǎn)染的PDCD4突變型細胞株熒光素酶活性為(0.992±0.135)，miR-150轉(zhuǎn)染的PDCD4突變型細胞株熒光素酶活性為(0.875±0.095)，差異無統(tǒng)計學意義(t=1.461,P=0.281)。轉(zhuǎn)染Vector、pcDNA3.1(+)-PDCD4、miR-ctr、miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4的CNE-2 PDCD4表達水平分別為(0.655±0.058)、(1.147±0.152)、(0.704±0.068)、(0.313±0.036)，差異有統(tǒng)計學意義(F=43.410，P<0.001)；其中，轉(zhuǎn)染Vector、miR-ctr、miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4的CNE-2 PDCD4表達水平均低于轉(zhuǎn)染pcDNA3.1(+)-PDCD4的CNE-2，差異有統(tǒng)計學意義(P<0.05)。轉(zhuǎn)染物和時間對CNE-2增殖能力存在交互作用，轉(zhuǎn)染物和時間對CNE-2增殖能力均存在主效應(yīng)(P<0.001)。轉(zhuǎn)染Vector、pcDNA3.1(+)-PDCD4、miR-150 inhibitors、miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4的CNE-2穿膜細胞數(shù)分別為(56.6±7.5)、(26.5±3.7)、(30.5±4.7)、(55.2±6.9)個，差異有統(tǒng)計學意義(F=18.550，P=0.014)；其中，轉(zhuǎn)染pcDNA3.1(+)-PDCD4、miR-150 inhibitors的CNE-2穿膜細胞數(shù)少于轉(zhuǎn)染Vector、miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4的CNE-2，差異有統(tǒng)計學意義(P<0.05)。結(jié)論miR-150通過抑制PDCD4的表達，繼而增強鼻咽癌細胞的增殖能力和侵襲能力。

鼻咽腫瘤；miR-150；程序性細胞死亡因子4；細胞增殖；侵襲

張先鋒，黃遠見，肖娟，等.miR-150促進鼻咽癌細胞增殖和侵襲的機制研究[J].中國全科醫(yī)學，2016，19(21)：2513-2517.[www.chinagp.net]

ZHANG X F,HUANG Y J，XIAO J,et al.Mechanism research of miR-150 promoting the proliferation and invasion of nasopharyngeal carcinoma[J].Chinese General Practice，2016，19(21)：2513-2517.

miRNA是長21～25 nt具有調(diào)控功能的單鏈非編碼小RNA，在進化過程中高度保守，是近年來生物醫(yī)學領(lǐng)域研究的熱點[1]。miRNA通過與靶基因mRNA的3′非編碼區(qū)(3′UTR)完全或不完全堿基配對，致使靶基因mRNA降解或其蛋白翻譯受抑制，從而負向調(diào)控轉(zhuǎn)錄水平。研究表明，miR-150在胰腺癌、食管癌、結(jié)腸癌以及肝癌細胞中表達下調(diào)[2-5]，而在胃癌、乳腺癌和非小細胞肺癌細胞中表達上調(diào)[6-8]。對比正常鼻咽上皮細胞和鼻咽癌細胞miRNA表達譜發(fā)現(xiàn)，miR-150在鼻咽癌細胞株CNE-1、CNE-2中上調(diào)最為明顯，因此miR-150極有可能參與了鼻咽癌的發(fā)生發(fā)展過程[9]。程序性細胞死亡因子4(PDCD4)是與細胞凋亡相關(guān)的基因，在多種實體瘤中表達下調(diào)。PDCD4基因低表達能夠促使腫瘤細胞增殖、侵襲和轉(zhuǎn)移。同時，低表達的PDCD4常提示腫瘤患者的不良預(yù)后，提示PDCD4基因可能成為腫瘤生物治療新靶點[10]。本研究運用分子生物學技術(shù)，證實miR-150可通過靶向調(diào)控PDCD4促進鼻咽癌細胞增殖和侵襲能力，進一步揭示了miR-150在鼻咽癌中的促癌分子機制。

1　材料與方法

1.1主要材料低分化鼻咽癌細胞株CNE-2購于上海細胞研究所，由南華大學腫瘤研究所保存。熒光素酶活性檢測試劑盒購自美國Promega公司；miR-150 mimics、miR-150 inhibitors購自上海拜力生物科技有限公司；PDCD4單克隆抗體、β-actin抗體購自美國Epigentek公司。

1.2細胞培養(yǎng)2014年3—6月，CNE-2培養(yǎng)于含10%新生胎牛血清的RPMI 1640培養(yǎng)基中，37 ℃,5% CO2飽和濕度條件下傳代培養(yǎng)，每1～2 d更換1次培養(yǎng)基，取生長良好的CNE-2用于實驗。

1.3熒光素酶活性檢測根據(jù)PDCD4基因3′UTR序列與miR-150結(jié)合位點設(shè)計合成PDCD4野生型引物序列以及突變型引物序列，PDCD4野生型上游引物序列：5′-GGTCTGACCTCCCCGACC-3′，下游引物序列：5′-CATCTGATTGTTAGGCTT-3′；PDCD4突變型上游引物序列：5′-ATACCGTCAAGCTCTTGGCA-3′，下游引物序列：5′-TTCTCTGGGCCGTTTTACCA-3′。引物序列分別引入限制性內(nèi)切酶Spe Ⅰ和Hind Ⅲ識別位點。取上、下游引物序列各10 μmol/L，加單蒸水(dH2O)至20 μl，94 ℃持續(xù)5 min，50 ℃持續(xù)10 min，將其連接到含有熒光素酶報告載體質(zhì)粒上，移入CNE-2，并經(jīng)miR-150轉(zhuǎn)染。吸去培養(yǎng)基，磷酸鹽緩沖液(PBS)洗1次；每孔加入100 μl 裂解緩沖液，置搖床上劇烈振蕩15 min。振蕩后將每孔中的液體移入1.5 ml離心管中，10 000 r/min離心1 min(離心半徑為10 cm)。取20 μl上清液加入96孔板中，采用多功能酶標儀檢測熒光素酶活性，熒光素酶活性以Rellina活性正態(tài)化后得到的相對熒光素值表示，實驗數(shù)據(jù)取3個重復(fù)孔平均值，實驗重復(fù)3次。

1.4Western blotting法檢測PDCD4表達水平采用脂質(zhì)體2000分別瞬時轉(zhuǎn)染Vector、pcDNA3.1(+)-PDCD4、miR-ctr、miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4于CNE-2。轉(zhuǎn)染48 h后，采用胰蛋白酶消化，以5 000個/孔接種在6孔板中。收集蛋白，加入RIPA細胞裂解液以及蛋白酶抑制劑，提取細胞總蛋白，采用BCA法測定總蛋白水平。取不同轉(zhuǎn)染細胞等量蛋白，10%十二烷基硫酸鈉-聚丙烯酰胺凝膠(SDS-PAGE)電泳，轉(zhuǎn)移至聚偏氟乙烯(PVDF)膜上，在含5%脫脂牛奶的三羥甲基氨基甲烷(Tris)緩沖溶液中封閉，加入β-actin抗體以及PDCD4抗體，4 ℃過夜，洗膜，加入辣根過氧化物酶(HRP)二抗，室溫孵育1 h，TBST洗膜30 min，加入發(fā)光劑，X片曝光、顯影、定影。

1.5增殖實驗采用脂質(zhì)體2000分別瞬時轉(zhuǎn)染Vector、pcDNA3.1(+)-PDCD4、miR-150 inhibitors、miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4于CNE-2。轉(zhuǎn)染48 h后，采用胰蛋白酶進行消化，以3 000個/200 μl細胞懸液接種在96孔板內(nèi)，每種細胞設(shè)置4個復(fù)孔，置于37 ℃，5% CO2培養(yǎng)箱中培養(yǎng)；每孔加入5 mg/ml MTT試劑20 μl，置于37 ℃，5% CO2培養(yǎng)箱中繼續(xù)培養(yǎng)4 h，小心吸去每孔中含有MTT的培養(yǎng)基，分別加入150 μl二甲基亞砜(DMSO)，將96孔板置于搖床上避光低速振蕩10 min，以使活細胞內(nèi)形成的結(jié)晶物充分溶解。使用多功能酶標儀，分別于細胞培養(yǎng)24、48、72、96 h后在570 nm波長處測定其吸光度(OD)值，同時設(shè)置490 nm波長背景對照。實驗重復(fù)3次。

1.6Transwell侵襲實驗采用脂質(zhì)體2000分別瞬時轉(zhuǎn)染Vector、pcDNA3.1(+)-PDCD4、miR-150 inhibitors、miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4于CNE-2。采用不含胎牛血清(FBS)的DMEM培養(yǎng)基重懸細胞至5×104個/ml，將鋪有基質(zhì)膠的Transwell小室預(yù)熱至37 ℃，在24孔板內(nèi)小室下層外孔加入含10% FBS的DMEM培養(yǎng)基，37 ℃孵箱培養(yǎng)。48 h后，取出小室，吸除小室底部Matrigel培養(yǎng)基，以PBS洗2次；用小棉簽小心擦拭小室上層底部的細胞和Matrigel培養(yǎng)基；PBS洗1次，蘇木精復(fù)染10～15 min，自來水洗去多余藍色。顯微鏡觀察5個視野，拍照，取圖，記錄穿膜細胞數(shù)。

2　結(jié)果

2.1熒光素酶活性無miR-150轉(zhuǎn)染的PDCD4野生型細胞株熒光素酶活性為(0.975±0.112)，miR-150轉(zhuǎn)染的PDCD4野生型細胞株熒光素酶活性為(0.588±0.042)，差異有統(tǒng)計學意義(t=7.853,P=0.018)。無miR-150轉(zhuǎn)染的PDCD4突變型細胞株熒光素酶活性為(0.992±0.135)，miR-150轉(zhuǎn)染的PDCD4突變型細胞株熒光素酶活性為(0.875±0.095)，差異無統(tǒng)計學意義(t=1.461,P=0.281)。

2.2PDCD4表達水平轉(zhuǎn)染Vector、pcDNA3.1(+)-PDCD4、miR-ctr、miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4的CNE-2 PDCD4表達水平分別為(0.655±0.058)、(1.147±0.152)、(0.704±0.068)、(0.313±0.036)，差異有統(tǒng)計學意義(F=43.410，P<0.001)；其中，轉(zhuǎn)染Vector、miR-ctr、miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4的CNE-2 PDCD4表達水平均低于轉(zhuǎn)染pcDNA3.1(+)-PDCD4的CNE-2，差異有統(tǒng)計學意義(P<0.05)。

2.3增殖能力轉(zhuǎn)染物和時間對CNE-2增殖能力存在交互作用，轉(zhuǎn)染物和時間對CNE-2增殖能力均存在主效應(yīng)(P<0.001)。48 h時，轉(zhuǎn)染pcDNA3.1(+)-PDCD4、miR-150 inhibitors的CNE-2增殖能力低于轉(zhuǎn)染Vector的CNE-2，轉(zhuǎn)染miR-150 inhibitors、miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4的CNE-2增殖能力高于轉(zhuǎn)染pcDNA3.1(+)-PDCD4的CNE-2，轉(zhuǎn)染miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4的CNE-2增殖能力高于轉(zhuǎn)染miR-150 inhibitors的CNE-2，差異均有統(tǒng)計學意義(P<0.05)；72 h時，轉(zhuǎn)染pcDNA3.1(+)-PDCD4、miR-150 inhibitors、miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4的CNE-2增殖能力低于轉(zhuǎn)染Vector的CNE-2，轉(zhuǎn)染miR-150 inhibitors、miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4的CNE-2增殖能力高于轉(zhuǎn)染pcDNA3.1(+)-PDCD4，轉(zhuǎn)染miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4的CNE-2增殖能力高于轉(zhuǎn)染miR-150 inhibitors的CNE-2，差異均有統(tǒng)計學意義(P<0.05)；96 h時，轉(zhuǎn)染pcDNA3.1(+)-PDCD4、miR-150 inhibitors、miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4的CNE-2增殖能力低于轉(zhuǎn)染Vector的CNE-2，轉(zhuǎn)染miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4的CNE-2增殖能力高于轉(zhuǎn)染pcDNA3.1(+)-PDCD4、miR-150 inhibitors的CNE-2，差異均有統(tǒng)計學意義(P<0.05，見表1)。

2.4侵襲能力轉(zhuǎn)染Vector、pcDNA3.1(+)-PDCD4、miR-150 inhibitors、miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4的CNE-2穿膜細胞數(shù)分別為(56.6±7.5)、(26.5±3.7)、(30.5±4.7)、(55.2±6.9)個，差異有統(tǒng)計學意義(F=18.550，P=0.014)；其中，轉(zhuǎn)染pcDNA3.1(+)-PDCD4、miR-150 inhibitors的CNE-2穿膜細胞數(shù)少于轉(zhuǎn)染轉(zhuǎn)染Vector、miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4的CNE-2，差異有統(tǒng)計學意義(P<0.05，見圖1，本文彩圖見本刊官網(wǎng)www.chinagp.net電子期刊相應(yīng)文章附件)。

3　討論

鼻咽癌是我國高發(fā)腫瘤之一，研究發(fā)現(xiàn)，鼻咽癌是一個多階段、多途徑改變的病理發(fā)展過程，其發(fā)生與遺傳、EB病毒感染、環(huán)境等因素有關(guān)，侵襲和轉(zhuǎn)移是鼻咽癌患者死亡的主要原因[11]。miRNA作為近年來腫瘤研究領(lǐng)域的熱點，其參與調(diào)控細胞生命活動中眾多信號轉(zhuǎn)導途徑，與腫瘤的發(fā)生發(fā)展密不可分。隨著對miRNA研究深入，miRNA可為早期腫瘤的監(jiān)測、診斷、治療、預(yù)后及解決放化療抵抗提供新的應(yīng)用價值[3]。miR-150是近年發(fā)現(xiàn)的miRNAs家族中的重要分子之一，其在不同腫瘤組織中的表達存在明顯差異，與腫瘤患者的臨床特征、生物學行為以及預(yù)后相關(guān)[3]。miR-150在不同腫瘤中的功能可能完全不同，目前miR-150在鼻咽癌中的功能尚不明確[7]。

SHIBAHARA等[12]運用差異顯示法在小鼠體內(nèi)發(fā)現(xiàn)了PDCD4基因，隨后又有研究在大鼠以及人類中發(fā)現(xiàn)了相同的基因[13-14]。PDCD4作為凋亡相關(guān)基因，其抑制腫瘤的作用引起廣泛關(guān)注。PDCD4不僅影響細胞程序性死亡，并且通過抑制蛋白翻譯的起始從而抑制腫瘤的發(fā)生發(fā)展。有研究證實，PDCD4的高頻缺失與多種實體瘤相關(guān)，如大腸癌、腎癌、乳腺癌、卵巢癌、膠質(zhì)瘤等[15-19]，而PDCD4在鼻咽癌中的作用尚未見到相關(guān)文獻報道。

本研究通過熒光素酶活性檢測發(fā)現(xiàn)，miR-150轉(zhuǎn)染和無miR-150轉(zhuǎn)染的PDCD4野生型細胞株熒光素酶活性存在差異，而miR-150轉(zhuǎn)染和無miR-150轉(zhuǎn)染的PDCD4突變型細胞株熒光素酶活性無差異，提示miR-150能夠直接靶向結(jié)合PDCD4 mRNA 3′UTR。進一步通過Western blotting法發(fā)現(xiàn)，轉(zhuǎn)染Vector、miR-ctr、miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4的CNE-2 PDCD4表達水平均低于轉(zhuǎn)染pcDNA3.1(+)-PDCD4的CNE-2，證實miR-150對內(nèi)源性PDCD4的表達具有抑制作用；Transwell侵襲實驗發(fā)現(xiàn)，48、72、96 h轉(zhuǎn)染miR-150 inhibitors的CNE-2增殖能力均低于轉(zhuǎn)染Vectors的CEN-2,轉(zhuǎn)染miR-150 inhibitors的CNE-2穿膜細胞數(shù)少于轉(zhuǎn)染Vector的CNE-2，提示miR-150能夠通過調(diào)節(jié)PDCD4影響鼻咽癌CNE-2的增殖能力與侵襲能力。其具體機制尚未見有相關(guān)報道，而LEI等[20]對膀胱癌細胞研究發(fā)現(xiàn)，miR-150可通過靶向調(diào)節(jié)PCDC4抑制膀胱癌細胞的侵襲能力，增加對順鉑化療的敏感性。

表1　不同轉(zhuǎn)染物對CNE-2增殖能力影響的比較±s,n=3)

注：與Vector比較，aP<0.05；與pcDNA3.1(+)-PDCD4比較，bP<0.05；與miR-150 inhibitors比較，cP<0.05

注：圖A～D分別為轉(zhuǎn)染Vector、pcDNA3.1(+)-PDCD4、miR-150 inhibitors、miR-150 mimics聯(lián)合pcDNA3.1(+)-PDCD4的CNE-2

圖1不同轉(zhuǎn)染物對CNE-2侵襲能力的影響(結(jié)晶紫染色，×100)

Figure 1Influence of various transfected objects in the invasion capacity of CNE-2

綜上所述，miR-150通過抑制PDCD4的表達，繼而增強鼻咽癌細胞的增殖能力和侵襲能力，該結(jié)論為鼻咽癌的診斷、治療及預(yù)后預(yù)測提供新的依據(jù)。

作者貢獻：張先鋒、黃遠見、肖娟、張志偉、黃衛(wèi)國進行實驗設(shè)計與實施、資料收集整理、撰寫論文、成文并對文章負責；張志偉進行質(zhì)量控制及審校。

本文無利益沖突。

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(本文編輯：吳立波)

Mechanism Research of miR-150 Promoting the Proliferation and Invasion of Nasopharyngeal Carcinoma

ZHANGXian-feng,HUANGYuan-jian,XIAOJuan,etal.

DepartmentofOtorhinolaryngology,theSecondHospital,UniversityofSouthChina,Hengyang421001,China

ObjectiveTo investigate whether miR-150 promotes cell proliferation and invasion by target regulating of programmed cell death 4(PDCD4) or not,and further reveal the oncogeme function of miR-150 in nasopharyngeal carcinoma.MethodsFrom March to June 2014,we cultured the nasopharyngeal carcinoma CNE-2,and selected the well-grown ones in the experiment.We designed and synthesized PDCD4 of wild type primer sequence and mutant primer sequence,which would be linked to the carrier vector with luciferase reporter gene，and luciferase activity was detected.Vector,pcDNA3.1(+)-PDCD4,miR-ctr,and miR-150 mimics combined with pcDNA3.1(+)-PDCD4 were transient transfected into CNE-2 respectively,and the expression level of PDCD4 was detected after 48 h by Western blotting method.Vector,pcDNA3.1(+)-PDCD4,miR-150 inhibitors and miR-150 mimics combined with pcDNA3.1(+)-PDCD4 were transient tranfected into CNE-2 respectively,and respectively cultured for 24,48,72,and 96 h to detect the absorbance(OD) value to reflect its ascending ability.Vector,pcDNA3.1(+)-PDCD4,miR-150 inhibitors and miR-150 mimics combined with pcDNA3.1(+)-PDCD4 were transient transfected into CNE-2 respectively,and the invasion ability of CNE-2 was reflected by Transwell invasion experiment.ResultsThe luciferase activity of PDCD4 of wild type cell lines that without miR-150 transfection was(0.975±0.112),and luciferase activity of PDCD4 of wild type cell lines that with miR-150 transfection was(0.588±0.042),which showed significant differences(t=7.853,P=0.018).The luciferase activity of PDCD4 of mutant type cell lines that without miR-150 transfection was(0.992±0.135),mutant type cell lines that with miR-150 transfection was(0.875±0.095),which showed no significant differences(t=1.461,P=0.281).The expression levels of PDCD4 that were transient transfected with Vector,pcDNA3.1(+)-PDCD4,miR-ctr,miR-150 mimics combined with pcDNA3.1(+)-PDCD4 were(0.665±0.058),(1.147±0.152),(0.074±0.068),and(0.313±0.036) respectively,which showed significant differences(F=43.410,P<0.011);the expression level of CNE-2 PDCD4 that had been transfected with Vector,miR-ctr,miR-150 mimics combined with pcDNA3.1(+)-PDCD4 was lower than that of the CNE-2 with transfection of pcDNA3.1(+)-PCDC4,which showed significant differences(P<0.05).The transfected objects and time played an interactive role in the proliferation capacity of CNE-2,and both of them had a main effect in the proliferation capacity of CNE-2(P<0.001).The numbers of transmembrane cells of CNE-2 that were transient transfected with Vector,pcDNA3.1(+)-PDCD4,miR-150 inhibitors and miR-150 mimics combined with pcDNA3.1(+)-PDCD4 were(56.6±7.5),(26.5±3.7),(30.5±4.7),and(55.2±6.9),which showed significant differences(F=18.550,P=0.014);the number of transmembrane cells of CNE-2 that were transfected with pcDNA3.1(+)-PDCD4,and miR-150inhibitors was less than that of CNE-2 that were transfected with Vector,miR-150mimics combined with pcDNA3.1(+)-PDCD4,which showed significant differences(P<0.05).ConclusionmiR-150 enhances the proliferation and invasion capacity of nasopharyngeal carcinoma cells by inhibiting PDCD4 expression.

Nasopharyngeal neoplasms;miR-150;PDCD4;Cell proliferation;Invasion

國家自然科學基金青年基金資助項目(81100568)；湖南省科技計劃項目(2014SK3081)

421001湖南省衡陽市，南華大學附屬第二醫(yī)院耳鼻咽喉科(張先鋒，黃遠見，肖娟)；南華大學腫瘤研究所(張志偉，黃衛(wèi)國)

張志偉，421001湖南省衡陽市，南華大學腫瘤研究所；E-mail：nhdxzzw@qq.com

R 739.63

A

10.3969/j.issn.1007-9572.2016.21.005

2015-12-14；

2016-06-06)