楊麗華,沈星凱,李靜秋,楊杰,樂燕萍,龔朝輝

1. 寧波大學(xué)醫(yī)學(xué)院生物化學(xué)與分子生物學(xué)研究所,寧波 315211;

2. 浙江省病理生理學(xué)技術(shù)研究重點(diǎn)實(shí)驗(yàn)室,寧波 315211

據(jù)估計(jì),2013年僅美國就有580350人死于癌癥,平均每天約1600人,癌癥已成為僅次于心臟疾病的第二大死因[1]。在我國,根據(jù)中國衛(wèi)生統(tǒng)計(jì)年鑒(2012 版)統(tǒng)計(jì),1973～1975、1990～1992、2004～2005年中國惡性腫瘤死亡抽樣回顧調(diào)查結(jié)果顯示,惡性腫瘤死亡率呈顯著上升趨勢,分別為 83.65/100000、108.26/100000和134.80/100000。癌癥致人死亡的原因近 90%是癌癥發(fā)生了轉(zhuǎn)移。轉(zhuǎn)移是惡性腫瘤最顯著的特征之一,它是一個(gè)多步驟的過程[2]。腫瘤細(xì)胞發(fā)生轉(zhuǎn)移的初始會(huì)發(fā)生上皮間質(zhì)轉(zhuǎn)化(Epithelialmesenchymal transition,EMT),EMT過程又受到多種因素調(diào)控,如轉(zhuǎn)錄因子、微RNA(MicroRNA,miRNA)等。miRNA是一類內(nèi)源性非編碼小分子RNA,與細(xì)胞生長、凋亡和信號轉(zhuǎn)導(dǎo)密切相關(guān)。同時(shí),miRNA可以調(diào)控EMT過程,在腫瘤轉(zhuǎn)移發(fā)面發(fā)揮了重要作用。

1 EMT和腫瘤轉(zhuǎn)移

EMT是胚胎正常發(fā)育、傷口愈合和惡性上皮腫瘤發(fā)生過程中的一個(gè)極其重要的基本過程[3],它可以使上皮細(xì)胞發(fā)生形態(tài)改變,轉(zhuǎn)換成有運(yùn)動(dòng)能力的間充質(zhì)細(xì)胞[4]。這些改變會(huì)使細(xì)胞-細(xì)胞之間和細(xì)胞-細(xì)胞外基質(zhì)之間的連接受到破壞,因而細(xì)胞可以遷移到身體的其他部位參與組織重建和修復(fù)[5]。EMT在腫瘤細(xì)胞的遷移/侵襲以及多種癌癥進(jìn)程方面也起到非常重要的作用,是腫瘤細(xì)胞轉(zhuǎn)移中的一個(gè)關(guān)鍵步驟,也是一個(gè)特定的信號通路[6,7]。EMT的特點(diǎn)是細(xì)胞粘附能力喪失,E-鈣粘蛋白(E-cadherin,CDH1)表達(dá)下降,間葉標(biāo)志物(包括 N-鈣粘蛋白(N-cadherin)、波形蛋白(Vimentin)和纖維連接蛋白(Fibronectin)表達(dá)上升,細(xì)胞運(yùn)動(dòng)能力和侵襲能力增強(qiáng)[8]。原發(fā)部位的腫瘤細(xì)胞可以發(fā)生EMT來獲得運(yùn)動(dòng)能力和侵襲能力,而間質(zhì)上皮轉(zhuǎn)化(Mesenchymalepithelial transition,MET)在腫瘤轉(zhuǎn)移的最后一步起到重要作用,即從血管或淋巴管中滲出的腫瘤細(xì)胞會(huì)恢復(fù)到上皮細(xì)胞狀態(tài),然后增殖形成巨大的甚至肉眼可見的繼發(fā)性腫瘤[9]。腫瘤EMT過程受內(nèi)在因素和外在因素或者是二者共同作用的影響(圖1),其中內(nèi)在因素包括腫瘤發(fā)生過程中產(chǎn)生的基因突變。突變的K-RAS途徑即是一個(gè)典型的例子[10],它可以單獨(dú)誘導(dǎo) EMT,也可以聯(lián)合 EMT誘導(dǎo)因子如轉(zhuǎn)化生長因子-β(Transforming growth factor-β,TGF-β)或者缺氧誘導(dǎo)因子 -2α(Hypoxia-inducible factor-2α,HIF-2α)共同作用。在某種程度上,由煙草(Nicotiana tabacum L.)引起的缺氧或者炎性腫瘤微環(huán)境,通過細(xì)胞外信號通路的失調(diào),創(chuàng)造一個(gè)促進(jìn)EMT發(fā)生的環(huán)境,特別是 TGF-β和環(huán)氧化酶/前列腺素 E2途徑在腫瘤微環(huán)境中具有重要作用[11]。在肺癌中,缺氧腫瘤微環(huán)境誘導(dǎo)EMT主要是上調(diào)了堿性螺旋-環(huán)-螺旋類(Basic helix-loop helix,bHLH)轉(zhuǎn)錄因子和HIF的表達(dá)。在EMT的細(xì)胞內(nèi)外信號通路中,EMT誘導(dǎo)轉(zhuǎn)錄因子和 miRNA起主要作用。在多發(fā)性腫瘤中,EMT被認(rèn)為是轉(zhuǎn)移的標(biāo)志,與許多轉(zhuǎn)錄因子緊密相關(guān)。已有研究表明,轉(zhuǎn)錄因子能夠促進(jìn) EMT和腫瘤轉(zhuǎn)移[12～16]。轉(zhuǎn)移過程中的多個(gè)步驟都受到這些轉(zhuǎn)錄因子調(diào)節(jié),而這些因子本身的表達(dá)同時(shí)受到miRNA的精細(xì)調(diào)控。

圖1 癌變過程中多種因素共同誘導(dǎo)EMT

2 影響EMT過程的轉(zhuǎn)錄因子

影響 EMT的轉(zhuǎn)錄抑制因子包括 Snail1[12]、Snail2[13]、Twist1[14]、Zeb1[15]、Zeb2[16]、Goosecoid[17]、FOXC2[18]、FoxQ1[19]、KLF8[20]、Prrx1[21]等。本文主要總結(jié)了 Snail、Twist和 Zeb轉(zhuǎn)錄因子家族及其在EMT和腫瘤轉(zhuǎn)移方面的網(wǎng)絡(luò)調(diào)控作用。

2.1 Snail鋅指轉(zhuǎn)錄抑制因子家族

鋅指簇可以與靶基因調(diào)控區(qū)的 E-box結(jié)合進(jìn)行調(diào)控,Snail家族是EMT轉(zhuǎn)錄抑制因子中研究最多的。諸多研究表明,很多信號通路會(huì)誘導(dǎo) Snail1和Snail2 的表達(dá),包括 TGF-β[22]、Notch[23]和 Wnt[24]信號通路,活性氧[25]和缺氧[26]。其中,TGF-β信號通路是最重要的EMT誘導(dǎo)細(xì)胞因子之一,它可以激活包括Snail1/2在內(nèi)的一系列EMT轉(zhuǎn)錄因子[27,28]。肝纖維化模型在缺氧條件下,基質(zhì)細(xì)胞會(huì)分泌大量的TGF-β,隨后腫瘤細(xì)胞受到 TGF-β的刺激后,促進(jìn)Snail1表達(dá),誘發(fā)EMT[29],這表明EMT誘導(dǎo)的各種信號通路之間互有關(guān)聯(lián)。

2.2 Twist bHLH轉(zhuǎn)錄因子家族

參與EMT調(diào)控的bHLH轉(zhuǎn)錄因子包括E12和E47(E2A基因選擇性剪切產(chǎn)物)、Twist1和2、Id1-4[30],均可以直接負(fù)調(diào)控CDH1的表達(dá)。Twist1和Twist2表達(dá)異常誘發(fā) EMT的發(fā)生[14,31]。這是由于 Twist1可以結(jié)合Snail2啟動(dòng)子區(qū),從而促進(jìn)Snail2基因轉(zhuǎn)錄,過表達(dá)的 Snail2蛋白介導(dǎo)乳腺上皮腫瘤細(xì)胞發(fā)生EMT[32]。同時(shí),Twist1亦可通過直接與血小板衍生生長因子受體α(Platelet-derived growth factor receptor-alpha,PDGFRα)啟動(dòng)子區(qū)結(jié)合,促進(jìn) PDGFα及其下游基因的表達(dá),促進(jìn)侵襲性偽足的形成,引起腫瘤細(xì)胞的侵襲和轉(zhuǎn)移[33]。在干細(xì)胞和癌癥中經(jīng)常呈現(xiàn)高表達(dá)的多梳抑制復(fù)合體蛋白 Bmi1同Twist1形成復(fù)合體,進(jìn)而抑制CDH1和p16INK4a基因表達(dá)[34]。Twist亦可誘導(dǎo)miR-10b的表達(dá),miR- 10b通過靶向抑制Homebox D10的翻譯,造成促轉(zhuǎn)移基因RHOC的表達(dá)上調(diào),最終影響乳腺癌轉(zhuǎn)移[35]。一些癌基因可以誘導(dǎo)細(xì)胞異常增殖和異常轉(zhuǎn)化,但細(xì)胞衰老會(huì)抑制腫瘤發(fā)生EMT和轉(zhuǎn)移[36]。Twist2亦可下調(diào)CDH1的表達(dá),參與TGF-β和p21誘導(dǎo)結(jié)腸癌細(xì)胞發(fā)生EMT這一過程[37]。在轉(zhuǎn)錄水平,Twist1主要受缺氧信號通路的調(diào)控[38]。在轉(zhuǎn)錄后水平,幾種微RNA可以直接靶向Twist1 mRNA[39]。此外,通過激酶作用,Twist1蛋白在 Ser68處發(fā)生磷酸化,使Twist1更穩(wěn)定,并且促進(jìn)乳腺癌細(xì)胞的侵襲[40]。以上研究表明,Twist bHLH家族通過EMT影響腫瘤轉(zhuǎn)移。

2.3 Zeb鋅指轉(zhuǎn)錄因子家族

Zeb家族包括鋅指/同源結(jié)構(gòu)域蛋白-ZEB1和ZEB2,在物種間高度保守,和其他轉(zhuǎn)錄因子共同作用調(diào)節(jié)EMT,并且在這一過程中受SUMO化、乙?；土姿峄恼{(diào)控。這些蛋白均可通過抑制上皮型標(biāo)志物和激活間質(zhì)型標(biāo)志物的表達(dá)來誘發(fā) EMT過程。多種因子和信號通路可直接激活Zeb家族的表達(dá),如生長激素和類固醇激素、缺氧條件下的HIF-1α、炎性細(xì)胞因子、配體(成纖維細(xì)胞生長因子、胰島素樣生長因子-1、血小板衍生生長因子等受體的配體)、Ras-ERK2-Fra1、NF-κB 和 JAK/STAT3 等腫瘤中經(jīng)常處于激活狀態(tài)的下游通路、Wnt和Notch信號通路。Zeb家族也受miR-200的抑制,它們之間存在一個(gè)負(fù)反饋環(huán)路,最終誘發(fā)多種腫瘤的EMT[41,42]。

3 miRNA通過EMT調(diào)節(jié)腫瘤轉(zhuǎn)移

miRNA是一類長約 22 nt的小分子RNA,可在轉(zhuǎn)錄后水平通過結(jié)合靶 mRNA的 3′UTR阻止靶mRNA的翻譯或者促進(jìn)靶mRNA的降解,實(shí)現(xiàn)對其靶基因的負(fù)調(diào)控[43]。據(jù)估計(jì),miRNA可以調(diào)控人類30%左右蛋白的表達(dá)[44]。miRNA通過調(diào)節(jié)腫瘤細(xì)胞EMT相關(guān)靶蛋白表達(dá),進(jìn)而抑制或促進(jìn)腫瘤轉(zhuǎn)移(表 1)。

3.1 miRNA通過 CDH1、波形蛋白和轉(zhuǎn)錄因子調(diào)節(jié)EMT

CDH1是 EMT/MET中非常重要的調(diào)控基因,miRNA可以通過多種機(jī)制對其進(jìn)行調(diào)節(jié)。miR-9通過直接靶向CDH1 mRNA引發(fā)EMT,促進(jìn)乳腺癌轉(zhuǎn)移。和未發(fā)生轉(zhuǎn)移的乳腺癌患者相比,發(fā)生轉(zhuǎn)移患者的原位癌組織中miR-9的表達(dá)升高。進(jìn)一步研究發(fā)現(xiàn),miR-9增加細(xì)胞的運(yùn)動(dòng)和侵襲能力,因而使非轉(zhuǎn)移的乳腺癌細(xì)胞形成微轉(zhuǎn)移[46]。miR-23a也直接靶向CDH1調(diào)控肺癌發(fā)生EMT[49]。Xu等[45]研究表明,miR-25在食管鱗狀細(xì)胞癌中表達(dá)上調(diào),其表達(dá)和淋巴結(jié)轉(zhuǎn)移及腫瘤分期相關(guān),這是由于miR-25直接負(fù)調(diào)控CDH1,進(jìn)而促進(jìn)細(xì)胞侵襲和遷移。

表1 參與腫瘤EMT的miRNA

miRNA除了直接靶向CDH1外,亦可間接調(diào)控CDH1。miR-138表達(dá)下調(diào)會(huì)促進(jìn) EMT的發(fā)生,其直接靶點(diǎn)是波形蛋白[47]。除了波形蛋白,miR-138還靶向EMT相關(guān)的其他靶基因,如ZEB2和靶向zeste基因增強(qiáng)子人類同源物2 (Enhancer of zeste homologue 2,EZH2)。由于EZH2可抑制組蛋白去乙?；?/2(Histone deacetylases 1/2,HDAC1/2)和Snail的表達(dá),間接降低 CDH1的表達(dá),最終誘發(fā)鼻咽癌發(fā)生EMT[55]。

miRNA還可通過直接調(diào)控相關(guān)轉(zhuǎn)錄因子進(jìn)而作用于 CDH1。據(jù)報(bào)道,幾種誘發(fā) EMT的因子,如ZEB1、ZEB2、Twist1、Snai1、Snai2、TGF-β 和 E47均可通過與 CDH1啟動(dòng)子區(qū)的 E-box結(jié)合抑制CDH1轉(zhuǎn)錄,促進(jìn)腫瘤侵襲和轉(zhuǎn)移[56～58]。

在EMT中,miR-200家族是研究最多的miRNA[59]。miR-200家族包括 miR-200a、-200b、-200c、-141和-429。miR-200家族直接結(jié)合ZEB1和ZEB2 mRNA 3′UTR,進(jìn)而抑制其表達(dá)[48],間接上調(diào) CDH1表達(dá),最終抑制EMT過程。同時(shí),ZEB1和ZEB2反過來又可結(jié)合miR-200家族成員啟動(dòng)子中的E-box,從而抑制miR-200家族成員的表達(dá),這表明 miR-200家族和ZEB因子相互負(fù)調(diào)控,它們之間形成了一個(gè)負(fù)反饋環(huán)路[42]。Gibbons等[60]對 40株人非小細(xì)胞肺癌(Non-small cell lung cancer,NSCLC)細(xì)胞進(jìn)行了分析,發(fā)現(xiàn)miR-200家族的表達(dá)和EMT標(biāo)志物具有相關(guān)性。此后,他們又將小鼠K-RAS和p53基因突變獲得肺腺癌模型后,用 TGF-β處理小鼠誘導(dǎo)其發(fā)生EMT,使得 miR-200家族低表達(dá),而上調(diào) miR-200家族的表達(dá)會(huì)阻礙EMT的發(fā)生,這表明NSCLC發(fā)生 EMT這一過程取決于 miR-200家族的表達(dá)。在miR-200家族上游調(diào)控方面,Yang等[61]發(fā)現(xiàn)具有轉(zhuǎn)移傾向的鼠肺腺癌細(xì)胞能高表達(dá)Notch和Notch配體,其中Notch配體Jagged2能上調(diào)GATA結(jié)合因子的表達(dá),而GATA結(jié)合因子可以抑制EMT轉(zhuǎn)錄抑制因子 miR-200家族的表達(dá),從而誘導(dǎo) EMT,同時(shí)miR-200又可調(diào)節(jié)Gata3的表達(dá),他們互相調(diào)控。這些結(jié)果表明,miR-200家族有可能成為肺癌治療的潛在靶點(diǎn)。

最近,Kumarswamy等[39]通過生物信息學(xué)方法分析發(fā)現(xiàn),在NSCLC細(xì)胞株中,Snail的上游調(diào)控基因 miR-30a與 CDH1和 N-鈣粘蛋白的表達(dá)相關(guān),miR-30a可靶向CDH1的轉(zhuǎn)錄抑制因子Snai1來抑制EMT,而且與相應(yīng)的正常組織比較,miR-30a在NSCLC中表達(dá)下調(diào)。在非肺癌組織和細(xì)胞中,有研究證實(shí)miR-30a可以靶向Snai1阻斷TGF-β誘導(dǎo)的EMT過程[62]。肺癌發(fā)生 EMT,除了 miR-30a,miR-34[50]也可作用于CDH1轉(zhuǎn)錄抑制物Snai1。miR-10b是Twist1的直接靶點(diǎn),但是單獨(dú)改變miR-10b的表達(dá)并不能誘導(dǎo)人類乳腺癌細(xì)胞發(fā)生EMT[35]。這表明,miRNA在 EMT過程中的調(diào)控作用并非唯一,有其他基因的參與,甚至是一個(gè)網(wǎng)絡(luò)。

3.2 miRNA直接靶向DICER調(diào)節(jié)EMT

Dicer是一種核糖核酸內(nèi)切酶,屬于 RNase III家族中特異識別雙鏈 RNA的一員。Bernstein等[63]首次在小干擾RNA(Small interfering RNA,siRNA)形成過程中明確了其作用,在胞質(zhì)中其可對 premiRNA進(jìn)行剪切形成成熟的miRNA[64,65]。在腫瘤的基因組中經(jīng)常會(huì)出現(xiàn) Dicer1的缺失突變,上調(diào)Dicer1的表達(dá)則會(huì)抑制腫瘤的形成[66],反之亦然[67]。在間質(zhì)型或發(fā)生遠(yuǎn)處骨轉(zhuǎn)移的乳腺癌細(xì)胞中,Dicer1呈現(xiàn)低表達(dá)[68]。這些研究表明,Dicer1在腫瘤中具有雙面作用：它是腫瘤細(xì)胞增殖和細(xì)胞存活所不可或缺的,同時(shí)又是腫瘤向遠(yuǎn)處轉(zhuǎn)移的一道屏障。

Dicer1是 miR-103/107的靶基因,其表達(dá)高低和腫瘤轉(zhuǎn)移及乳腺癌患者的預(yù)后緊密相關(guān)。在穩(wěn)定表達(dá) miR-103/107的MDA-MB-231細(xì)胞中,Dicer1和成熟 miRNA的表達(dá)均下降。在臨床上,高表達(dá)miR-103/107的乳腺癌患者發(fā)生轉(zhuǎn)移的機(jī)率更高。同時(shí),miR-103/107高表達(dá)會(huì)誘發(fā) EMT,這是由于miR-107可通過miR-200通路調(diào)節(jié)ZEB1/ZEB2的表達(dá)[51]。總之,miR-103/miR-107可以調(diào)節(jié)Dicer1的表達(dá),進(jìn)而減少成熟miRNA的生物合成,最終增強(qiáng)細(xì)胞的遷移能力及體內(nèi)腫瘤的轉(zhuǎn)移。

Su等[69]研究表明,TAp63可調(diào)控Dicer1的表達(dá),Dicer低表達(dá)會(huì)增加細(xì)胞的侵襲能力,同時(shí)TAp63可以反式激活miR-130b的表達(dá),miR-130b與轉(zhuǎn)移密切相關(guān)并且其加工成熟需Dicer1的參與。在TAp63缺失表達(dá)的細(xì)胞中上調(diào) Dicer1和 miR-130b的表達(dá)顯著影響其轉(zhuǎn)移能力。通過調(diào)控 Dicer1和 miR-130b的表達(dá),TAp63可抑制腫瘤的形成和轉(zhuǎn)移。

miR-18a作為癌基因miR-17-92簇的一員,在鼻咽癌組織及細(xì)胞中呈現(xiàn)高表達(dá),并且與中晚期鼻咽癌發(fā)展密切相關(guān)。研究表明,miR-18a可與Dicer1基因 3′UTR結(jié)合抑制 Dicer1翻譯,因而包括 miR-200家族和miR-143在內(nèi)的miRNA合成受到影響,最終誘發(fā)EMT,促進(jìn)腫瘤轉(zhuǎn)移[52]。因此,miRNA可通過調(diào)控Dicer1的表達(dá)影響EMT。

3.3 miRNA通過鋅指蛋白調(diào)節(jié)EMT

鋅指蛋白(Tristetraprolin,TTP)是一種可降解靶基因mRNA 3′UTR中富含AU序列的蛋白。在Ras信號通路的作用下,下調(diào)TTP可以引起EMT和癌癥轉(zhuǎn)移。miR-29a通過結(jié)合3′UTR的2個(gè)位點(diǎn)調(diào)節(jié)TTP的表達(dá),因而miR-29a可通過TTP參與EMT及乳腺癌的轉(zhuǎn)移[53]。

3.4 miRNA通過NF-κB調(diào)節(jié)EMT

NF-κB 是一個(gè)蛋白復(fù)合體,在 DNA 轉(zhuǎn)錄成mRNA過程中起調(diào)控作用。在多種腫瘤中,NF-κB出現(xiàn)異常(包括表達(dá)改變和位置變化),并且和轉(zhuǎn)移有關(guān)[70～73]。在化療反應(yīng)中,NF-κB和miR-448之間可形成正反饋環(huán)路,通過EMT調(diào)節(jié)腫瘤轉(zhuǎn)移[54]。

4 miRNA參與腫瘤轉(zhuǎn)移其他步驟的調(diào)控

腫瘤轉(zhuǎn)移是一個(gè)多步驟的過程,包括腫瘤細(xì)胞從原發(fā)部位擴(kuò)散,入侵組織,滲入血管或淋巴管,隨血液循環(huán)系統(tǒng)或淋巴循環(huán)系統(tǒng)在體內(nèi)播散,遷出血管或淋巴管以及在新的地點(diǎn)定居和增殖[2]。除EMT外,miRNA也參與腫瘤轉(zhuǎn)移所必須經(jīng)歷的其他過程(表2),如細(xì)胞外基質(zhì)降解和血管生成等方面。眾所周知,腫瘤細(xì)胞在轉(zhuǎn)移過程中需要將細(xì)胞外基質(zhì)蛋白降解才可出入自由,參與這一過程的miRNA包括 miR-335[74]、miR-373和 miR-520c[75]。腫瘤在發(fā)生微轉(zhuǎn)移后,血管生成是其生長存活所必須的,miR-9在乳腺癌中高表達(dá),其在轉(zhuǎn)移中具有雙重作用,不但引發(fā)細(xì)胞 EMT,而且刺激血管的形成[46]。miR-9介導(dǎo)的CDH1下降會(huì)激活β-catenin信號通路,使血管內(nèi)皮生長因子表達(dá)上升,最終導(dǎo)致血管生成增加。有研究表明,miR-126[76,77]和miR-205[78]也調(diào)節(jié)血管生成。因此,miRNA可調(diào)控ECM降解和血管形成繼而影響腫瘤轉(zhuǎn)移。

表2 在腫瘤轉(zhuǎn)移中起作用的其他 miRNA(不包括調(diào)控EMT/MET的miRNA)

5 結(jié) 語

miRNAs可以作為潛在的癌基因或者抑制基因在EMT過程中起作用,可作為腫瘤惡化或者逆轉(zhuǎn)的生物標(biāo)志物。今后的研究無疑是要將注意力集中在不同腫瘤 EMT中發(fā)生特異性改變的 miRNA,以及這些 miRNA下游和上游調(diào)控相關(guān)的一些尚未被發(fā)現(xiàn)的新的調(diào)控因子上。EMT是一個(gè)多步驟過程,而這個(gè)過程涉及到許多信號分子、通路和轉(zhuǎn)錄因子。更為重要的是,EMT也和轉(zhuǎn)移、耐藥以及腫瘤干細(xì)胞的形成密切相關(guān)。越來越多的證據(jù)表明,促進(jìn)EMT和腫瘤干細(xì)胞形成的治療性藥物[83]以及分子靶向治療也可促進(jìn)轉(zhuǎn)移[84]。鑒于EMT在腫瘤發(fā)展中的重要作用,針對參與EMT的蛋白和miRNA可以提供一個(gè)具有特異性的治療策略,以防止腫瘤的轉(zhuǎn)移、耐藥和復(fù)發(fā)[85]。這些新的治療策略將開辟癌癥個(gè)性化治療的新方向,將提高人們對腫瘤EMT和轉(zhuǎn)移的認(rèn)識,最終在腫瘤綜合診治的方案及策略選擇上有所突破。

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