腫瘤干細胞的研究進展及臨床意義

岳冬麗，韓交玲,關方霞,張 毅#

1)鄭州大學第一附屬醫(yī)院生物細胞治療中心 鄭州 450052 2)鄭州大學第一附屬醫(yī)院腫瘤科 鄭州 450052 3)鄭州大學生物工程系 鄭州 450001

惡性腫瘤導致的死亡人數(shù)在發(fā)展中國家高居第二位，而在發(fā)達國家更是高居榜首，是發(fā)達國家的首要死亡原因[1]，嚴重威脅著人類的健康及生存。目前，惡性腫瘤的診斷與治療水平已有了極大提高，化療、放療、手術治療、生物治療等綜合治療手段的應用已經(jīng)極大改善了惡性腫瘤的治療效果，使腫瘤患者的生存率明顯提高。尤其是兒童白血病、霍奇金病和睪丸癌的治療取得了較大突破，已達治愈水平。由于早期診斷技術的進步以及腫瘤預防的開展，乳癌及前列腺癌患者的死亡率也急劇下降。但是大量腫瘤患者在確診時已是晚期，已發(fā)生遠處轉移，導致治療效果不佳，這成為腫瘤診斷中的一個嚴峻問題。因而提高惡性腫瘤的早期診斷水平并控制腫瘤的復發(fā)和轉移將是提高癌癥患者生存率與治愈率的關鍵。

近年來，腫瘤干細胞(cancer stem cell，CSC)學說備受關注。該學說認為腫瘤干細胞促進了腫瘤生長，是惡性腫瘤的起源；并且能夠耐受化療和放療等常用的腫瘤治療手段，是腫瘤轉移和復發(fā)的重要原因。腫瘤干細胞學說并不是近幾十年才提出的新概念，早在150年前已經(jīng)有人提出腫瘤干細胞的概念[2]。Reya等[3]明確提出腫瘤組織中存在少數(shù)具有干細胞性質的細胞亞群，具有自我更新和多向分化潛能，可以形成新腫瘤，并將這些細胞定義為腫瘤干細胞。近年來，大量研究證據(jù)證實了腫瘤干細胞的存在。作者主要從腫瘤干細胞學說的提出、腫瘤干細胞的生物學特性、表面標志物以及腫瘤干細胞學說對于惡性腫瘤診斷、治療及預后的意義等方面進行了綜述。

1 腫瘤干細胞的生物學特性

1867年Julius Cohnheim提出了腫瘤干細胞學說，但由于受到當時技術條件等的限制，未能分離出腫瘤干細胞，因而此學說在當時未獲得重視。隨著科學技術的進步，研究人員發(fā)現(xiàn)小部分腫瘤細胞具有不同程度的自我更新能力，即具備干細胞特性，證實了腫瘤干細胞的存在。近年來，隨著腫瘤干細胞研究的進展，腫瘤干細胞學說又被學術界所重視。

腫瘤干細胞的早期研究局限于細胞的體外擴增能力，并不能體現(xiàn)自我更新能力。1977年，Hamburger等[4]對來源于肺癌、卵巢癌和神經(jīng)母細胞瘤的腫瘤細胞進行體外培養(yǎng)，發(fā)現(xiàn)只有極少數(shù)的腫瘤細胞能夠形成克隆。他認為這些具有自我更新和多向分化潛能的極少數(shù)細胞可以形成新腫瘤，而這部分腫瘤細胞可能起源于遺傳突變的正常干細胞，進而明確提出“腫瘤干細胞學說”。1997年Bonnet等[5]分離出表型為CD34+C38-的白血病干細胞，首次在細胞水平上直觀證實了腫瘤干細胞的存在。近幾十年，隨著科學技術及相關研究的發(fā)展，腫瘤干細胞的研究進展迅速，人們已證實在多種實體腫瘤中存在腫瘤干細胞，如乳癌[6-7]、卵巢癌[8-9]、前列腺癌[10]、結直腸癌[11]和腦腫瘤[12]等。

腫瘤干細胞與正常組織干細胞具有相同特性，即均具備自我更新和多向分化潛能[13]。此外，腫瘤干細胞致瘤能力強，少量腫瘤干細胞(少至500個或 1 000 個[14])就能在裸鼠體內(nèi)形成腫瘤；對多種化療藥物(如阿霉素、氟尿嘧啶、環(huán)磷酰胺、依托泊苷及順鉑等)有耐藥性[15-16]；抗放療[17-18]；有特異性表面標記分子(如CD44+CD24-[6]，CD34+C38-[5]等)；Wnt/β-catein[19-20]、 Notch[21]、 Hedgehog[22]和TGF-β[23-24]等信號通路常異?；罨?圖1)。

根據(jù)腫瘤干細胞的特性，可通過不同方法富集腫瘤干細胞，例如誘導EMT(epithelial-to-mesenchymal transition)過程[25-26]、低黏附培養(yǎng)系統(tǒng)[27-28]、低氧培養(yǎng)[29]、化療藥物刺激[30]、側群分選[31]等方法，這為腫瘤干細胞的干細胞特性及分化能力的具體機制以及針對腫瘤干細胞的靶向治療等研究奠定了基礎。

2 腫瘤干細胞表面標志物

目前，有許多關于腫瘤干細胞表面標志物的研究，不同文獻報道的結果不盡相同，關于腫瘤干細胞表面標志物的研究尚無定論。但是，這些研究都將為腫瘤干細胞靶向治療提供理論依據(jù)。多種惡性腫瘤干細胞表面標志物見表1。

表1 腫瘤干細胞表面標志物

續(xù)表1

腫瘤類型腫瘤干細胞表面標志物腎癌CD105+[55]霍奇金淋巴瘤CD27+ALDH+[56]急性髓性白血病CD34+CD38-[5]ALDH+[57]CD44+[58]淋巴母細胞白血病CD90+CD110+[59]視網(wǎng)膜母細胞瘤Epcam+[60]非小細胞肺癌CD133+[61]惡性黑色素瘤CD44+CD133+CD24+[62]CD133+[63]CD271+[64]骨肉瘤CD133+[65]ALDH+[66]尤文肉瘤CD133[67]垂體腺瘤CD133[68]頭頸部腫瘤CD44[69]ALDH+[70]

圖1 腫瘤干細胞的生物學特性

3 腫瘤干細胞的臨床意義

3.1腫瘤干細胞在腫瘤診斷及預后中的意義腫瘤干細胞學說認為腫瘤干細胞是腫瘤的起源，因而檢測腫瘤干細胞表面標志物將會提高癌癥的早期診斷效率。急性髓性白血病的干細胞標志物CD34最早被發(fā)現(xiàn)，隨后很多實體腫瘤干細胞表面標志物陸續(xù)被鑒定出來，這在很大程度上提高了腫瘤診斷的特異性。Stenffensen等[71]對117例卵巢癌患者的CD44+細胞與無進展生存時間進行了分析，多數(shù)(57.1%)Ⅰ期患者的CD44+細胞數(shù)量較多(>20%)，而Ⅱ、Ⅲ和Ⅳ期患者的CD44+細胞數(shù)量較少；同時在Ⅰ期患者中，CD44+細胞數(shù)量多的患者(>20%)無進展生存時間縮短。另外，有研究[72]表明CD133+ALDH+卵巢癌患者的預后明顯較CD133-ALDH-患者差。大量研究表明腫瘤干細胞與多種腫瘤患者臨床預后相關，如乳癌[73]、卵巢癌[74]和結直腸癌[75]等。腫瘤細胞中腫瘤干細胞的比例有預后意義，比例越高，患者預后越差。這些結果提示腫瘤干細胞是影響腫瘤診斷及預后評價的重要指標。

3.2腫瘤干細胞在腫瘤治療中的意義目前抗癌藥物主要殺傷增殖期的腫瘤細胞，而對腫瘤干細胞的殺傷效果不佳。由于腫瘤干細胞主要處于靜止期，能夠通過改變細胞周期調(diào)控點、減弱凋亡途徑、增強DNA損傷修復能力以及高表達ABC轉運子把藥物轉運至胞外，降低細胞內(nèi)藥物積累[76]，導致治療失敗。如果腫瘤干細胞是腫瘤生長的根源，那么綜合應用腫瘤干細胞特異性或非特異性殺傷藥物將是腫瘤治療的重大突破。隨著分子生物學技術的發(fā)展，人們對腫瘤的細胞和分子水平上的發(fā)病機制有了進一步認識，腫瘤靶向治療的研究已經(jīng)進入一個全新時代。針對腫瘤干細胞靶向治療的研究也已取得重大進步，主要集中于以下幾個方面：調(diào)節(jié)腫瘤干細胞微環(huán)境，逆轉腫瘤干細胞抗放化療特性，靶向作用于腫瘤干細胞的特異性分子標志及信號通路，促進分化療法，腫瘤干細胞特異性免疫治療等。下面僅對逆轉腫瘤干細胞的抗放化療特性，靶向作用于腫瘤干細胞的特異性分子標志、信號通路及腫瘤干細胞特異性免疫治療方面做介紹。

3.2.1 逆轉腫瘤干細胞的抗放化療特性 腫瘤干細胞能夠耐受化療和放療[15-18]，從而導致惡性腫瘤復發(fā)和轉移，導致治療失敗。如果能夠扭轉腫瘤干細胞的這種特性，使其恢復對放化療的敏感性，將是腫瘤治療的重大突破。逆轉腫瘤干細胞抗放化療特性的示意圖見圖2。

圖2 逆轉腫瘤干細胞的抗放化療特性示意圖

Frank等[77]證實抑制多藥耐藥ABC轉運子(如ABCB5)活性能夠改善惡性黑色素瘤干細胞對化療的敏感性。Jang等[78]敲除乳癌細胞(MCF7,MDA-MB-231)ANT2(adenine nucleotide translocator-2)后，發(fā)現(xiàn)ABCG2表達下調(diào)而細胞對阿霉素的敏感性增強。Cheng等[79]證實通過抑制ABCB1和ABCG2介導的耐藥機制，能夠增強化療藥物殺傷腫瘤干細胞的能力。這些結果表明抑制ABC轉運子(如ABCB1、ABCB5、ABCG2)活性可逆轉腫瘤干細胞的耐藥性，因此ABC轉運子抑制劑可作為一類有效的腫瘤干細胞治療藥物。

放療是惡性腫瘤治療中不可或缺的手段，尤其是在惡性膠質瘤、鼻咽癌等腫瘤的治療中。放療對全部腫瘤細胞的損傷是一致的，但是腫瘤干細胞的損傷修復能力強、調(diào)控點基礎活性高，可以迅速對損傷做出反應，最終逃避細胞死亡。Eyler等[80]對體內(nèi)腫瘤生長及體外克隆形成等觀察后發(fā)現(xiàn)，抑制ChK1/2激酶活性能夠逆轉惡性膠質瘤干細胞的抗放療效應。

然而，腫瘤干細胞抗放化療的機制遠非上述幾種。有文獻報道[8，28，34]轉錄因子(如SOX2、NANOG、OCT4、C-MYC、LIN28、ALDH1A1等)也可能與腫瘤干細胞的生物學特性有關，某些信號通路活性的改變也是引起腫瘤干細胞上述特性的原因。目前，科研人員仍在對腫瘤干細胞抗放化療的機制進行深入研究。

3.2.2 靶向作用于腫瘤干細胞特異性分子標志及信號通路 糖蛋白CD44在上皮細胞、間質細胞或某些腫瘤細胞表面廣泛表達。CD44在惡性黑色素瘤、結直腸癌、前列腺癌、乳癌及白血病細胞中高表達，是一個潛在的治療靶點。Jin等[58]指出在慢性粒細胞性白血病及急性髓性白血病細胞中，應用CD44單克隆抗體阻斷CD44與腫瘤干細胞的干細胞穴的黏附，可誘導腫瘤干細胞迅速分化，達到清除白血病干細胞的目的。但是CD44在一些正常細胞表面上也存在，而且在某些正常細胞表面上的表達還很強，因而在臨床中是否能夠將CD44作為腫瘤干細胞的治療靶點還有待進一步的研究。ABC轉運蛋白ABCG2是腦腫瘤干細胞、黑色素瘤干細胞及腎癌干細胞等的表面標志物之一，因而可能成為腫瘤細胞治療的新靶點[81]。在多種惡性腫瘤中，如卵巢癌、乳癌、前列腺癌、結直腸癌、頭頸部腫瘤及胰腺癌細胞等，ALDH+細胞具有干細胞特性，是一個潛在的治療靶點。

2010年，Singh等[82]發(fā)現(xiàn)CXCL12-CXCR4信號通路在胰腺癌抗藥性中發(fā)揮重要作用，可能成為胰腺癌治療的新靶點。Wnt/β-catein[19-20]、Notch[21]、Hedgehog[22]和TGF-β[23-24]等信號通路在腫瘤干細胞中經(jīng)常發(fā)生異常調(diào)節(jié)，對腫瘤干細胞的干細胞特性保持起著重要作用。例如Wnt/β-catein信號通路能夠維持人胚胎干細胞、胃腸道干細胞、造血干細胞及皮膚干細胞的自我更新能力，抑制其分化[19]。在大腸癌干細胞中，Wnt信號通路活性增高，導致腫瘤細胞克隆形成能力增強、腸道分化標志表達下調(diào)[20]。故異常信號通路可作為促進腫瘤干細胞分化治療的特異性靶點。

3.2.3 腫瘤干細胞的特異性免疫治療 腫瘤特異性過繼細胞免疫治療，通過將體外大量擴增的腫瘤特異性效應細胞回輸給患者，使其直接殺傷腫瘤細胞，是目前療效最肯定的腫瘤生物治療手段。如果能夠獲得腫瘤干細胞特異性效應細胞，將可直接殺傷腫瘤干細胞，消除腫瘤復發(fā)轉移的根源。Visus等[83]發(fā)現(xiàn)ALDH1A1特異性CD8+T細胞能夠有效識別腫瘤細胞系和移植瘤中的ALDH+細胞，對荷瘤小鼠進行特異性CD8+T細胞過繼免疫治療，能夠清除體內(nèi)ALDH+細胞，抑制腫瘤生長及轉移，延長小鼠生存時間。

腫瘤干細胞疫苗，通過補體依賴性細胞毒性及細胞毒性T淋巴細胞直接靶向作用于腫瘤干細胞，誘導體液及細胞免疫反應，產(chǎn)生保護性抗腫瘤免疫。Ning等[84]證實腫瘤干細胞具有很強的免疫原性，能夠有效刺激機體產(chǎn)生保護性抗腫瘤免疫；腫瘤干細胞免疫小鼠的血清中含有高滴度抗體，能夠結合并裂解腫瘤干細胞；在體外實驗中，來自腫瘤干細胞免疫小鼠的外周血或者脾臟的細胞毒性T淋巴細胞能夠殺傷腫瘤干細胞。

以上結果顯示腫瘤干細胞特異性免疫治療能夠靶向作用于腫瘤干細胞。在臨床應用中，應密切考慮其與常規(guī)療法如化療和放療等的結合，以增強腫瘤的治療效果。腫瘤干細胞的免疫治療必將成為腫瘤治療的重要手段。

3.3腫瘤干細胞應用于臨床治療的雙面性腫瘤干細胞理論獲得了越來越多研究結果的支持，但目前還沒有一種腫瘤干細胞治療方案應用于臨床。腫瘤干細胞的臨床應用仍面臨著很多挑戰(zhàn)：首先，腫瘤干細胞與正常干細胞的生物學特性有很多相同之處，在腫瘤干細胞治療中，必須確保對腫瘤干細胞的特異性殺傷，避免對正常干細胞的攻擊。其次，腫瘤干細胞研究成果主要是在動物實驗中獲得的，由于人與動物間免疫系統(tǒng)存在較大差異，迄今尚無法將研究成果直接應用于人體。第三，腫瘤干細胞表面標志物研究迄今尚無統(tǒng)一標準，如Ricci-Vitiani等[50]認為CD133+細胞為結腸癌干細胞，但是Shmelkov等[85]卻發(fā)現(xiàn)CD133+和CD133-結腸癌細胞的致瘤性沒有太大差別。已報道的腫瘤干細胞表面標志物能否應用于腫瘤干細胞的分離，是否與腫瘤干細胞的功能有關，尚需更加廣泛而深入的研究證實。最后，由于腫瘤干細胞僅是一小部分細胞群體，被殺傷后并不會出現(xiàn)明顯的腫瘤縮小等直觀反映，因此，關于腫瘤干細胞治療的療效評價，需要確切的評價指標。

4 展望

近年來，大量腫瘤干細胞研究結果驗證了腫瘤干細胞學說，并已取得了很多鼓舞人心的成果。研究證實血液系統(tǒng)腫瘤和大多數(shù)實體瘤中存在腫瘤干細胞；CD133、CD44、ALDH和Epcam極有可能是腫瘤干細胞的表面標志物；SOX2、NANOG、OCT4、C-MYC、ABCG2、ALDH1A1等轉錄因子可能與腫瘤干細胞的生物學特性有關；并且在裸鼠實驗中證實了腫瘤干細胞具有很強的致瘤性。這些成果提示了腫瘤干細胞靶向治療的研究方向。

然而，在腫瘤干細胞的研究領域中還存在許多問題有待探索，如腫瘤干細胞維持干細胞特性及分化能力的機制等。總之，腫瘤干細胞的存在為腫瘤復發(fā)和轉移提供了一種新的理論解釋，也為腫瘤的診斷及預后評價開辟了一條新思路，同時腫瘤干細胞靶向治療藥物和普通腫瘤治療藥物的臨床結合應用，將提高腫瘤治愈率。相信通過研究人員的不懈努力，隨著腫瘤干細胞研究的不斷深入，一定可以找到腫瘤干細胞耐受放化療的具體機制，從而引發(fā)新一輪臨床腫瘤治療模式及靶向治療藥物研發(fā)的變革，推動腫瘤干細胞特異性治療的發(fā)展，為人類控制腫瘤、改善腫瘤預后增加希望。

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