腫瘤干細(xì)胞的研究進(jìn)展

董 敏,畢經(jīng)旺

(濟(jì)南軍區(qū)總醫(yī)院腫瘤科,山東濟(jì)南,250000)

早期理論認(rèn)為,腫瘤的發(fā)生和發(fā)展主要來源于正常組織細(xì)胞的基因突變,使細(xì)胞增殖能力增強(qiáng),分化度下降,細(xì)胞凋亡減少,細(xì)胞持續(xù)處于高增殖狀態(tài)。按照該理論,這種低分化細(xì)胞持續(xù)增殖會導(dǎo)致更多的細(xì)胞出現(xiàn)更進(jìn)一步的致癌突變,而使同一腫瘤中存在多種腫瘤細(xì)胞,這和大多數(shù)臨床結(jié)果是不相符的。近年來越來越多的學(xué)者[1]提出,干細(xì)胞不僅在多細(xì)胞有機(jī)體形成中具有重要的作用,同時(shí)在腫瘤的發(fā)生過程中同樣起到不可忽視的推動作用,即“腫瘤干細(xì)胞假說”學(xué)說。許多研究[2-5]表明,在腫瘤組織中存在一類特殊的細(xì)胞群,具有自我更新能力并能產(chǎn)生異質(zhì)性腫瘤細(xì)胞,促進(jìn)腫瘤形成和增殖,即腫瘤干細(xì)胞,也稱癌干細(xì)胞(CSCs)。由于這類細(xì)胞的存在,使得腫瘤組織具有自我更新、多向分化和無限增殖的能力,是腫瘤的轉(zhuǎn)移和復(fù)發(fā)過程中的關(guān)鍵因素。因此能否徹底清除腫瘤干細(xì)胞成了腫瘤能否緩解甚至治愈的重要前提。

雖然腫瘤干細(xì)胞理論提出較早,但直到20世紀(jì)80年代單克隆抗體和流式細(xì)胞術(shù)廣泛應(yīng)用后,才使該理論的證實(shí)成為了可能。利用表面標(biāo)記物的差別,多種腫瘤干細(xì)胞被分離得到。腫瘤干細(xì)胞表現(xiàn)出失控的自我復(fù)制和分化過程,其在分化過程中能夠產(chǎn)生處于不同分化階段和不同類型的腫瘤細(xì)胞,并由大量子細(xì)胞聚集構(gòu)成腫瘤,但在腫瘤中只有腫瘤干細(xì)胞具有自我更新能力。部分學(xué)者認(rèn)為腫瘤干細(xì)胞可能來自于正常干細(xì)胞的基因突變,也有人認(rèn)為其來自于祖細(xì)胞突變[6-7],是祖細(xì)胞突變過程中保留了自我更新能力的部分細(xì)胞。

1 造血系統(tǒng)腫瘤干細(xì)胞

白血病干細(xì)胞是最早分離出的腫瘤干細(xì)胞。John Dick等[8]將分離得到的CD34+CD38-人白血病干細(xì)胞群移植入非肥胖糖尿病/重度聯(lián)合免疫缺陷(NOD/SCID)小鼠體內(nèi),發(fā)現(xiàn)在其體內(nèi)形成白血病移植瘤細(xì)胞群。Holyoake等[9]在慢性髓細(xì)胞樣白血病患者體內(nèi)提取到了腫瘤干細(xì)胞。Cox等[10]研究發(fā)現(xiàn),急性淋巴細(xì)胞白血病患者骨髓樣本內(nèi)同樣存在類似的腫瘤干細(xì)胞。Jordan等[11]發(fā)現(xiàn)急性髓細(xì)胞樣白血病干細(xì)胞中存在IL-3受體表面標(biāo)記物,其他研究者也發(fā)現(xiàn)CD33同樣存在于多種白血病干細(xì)胞中[12-13],同時(shí)還有公認(rèn)的 CD96、CD123、CLL-1等特異的表面標(biāo)記物,均為白血病干細(xì)胞的靶向清除提供了重要依據(jù)。

2 肺癌腫瘤干細(xì)胞

Kim等[14]于2005年采用流式細(xì)胞儀,以熒光激活細(xì)胞分選(FACS)的方法在支氣管-肺泡導(dǎo)管分界區(qū)(BADJ)分離得到了表面標(biāo)記特征為Sca-1+CD45Pecam-CD34+的細(xì)胞,并將其命名為支氣管肺泡干細(xì)胞(BASCs)。該細(xì)胞在正常時(shí)處于靜止?fàn)顟B(tài),在機(jī)體損傷時(shí)則表現(xiàn)出自我更新和多向分化的能力等干細(xì)胞特性。Ho等[15]應(yīng)用Hoechst33342染料染色和FACS方法,從肺癌細(xì)胞系(H460,H23,HTB-58,A549,H441,H2170)中分離出可自我更新、分化的側(cè)群細(xì)胞(SPCs),該細(xì)胞有非常高的體外侵襲力和體內(nèi)成瘤特征,同時(shí)可表達(dá)ABCG2等ABC跨膜轉(zhuǎn)運(yùn)蛋白,且正常狀態(tài)下處于靜止期,對化療藥物有很高的耐藥性。Eramo等[16]研究發(fā)現(xiàn),在小細(xì)胞肺癌和非小細(xì)胞肺癌組織中存在極少量(0.32%～1.13%)表達(dá)CD133+表面標(biāo)記的細(xì)胞群,將該細(xì)胞群接種至NOD/SCID小鼠體內(nèi),可迅速產(chǎn)生與原發(fā)瘤相同的移植瘤。Maria等[17]也發(fā)現(xiàn),在部分非小細(xì)胞肺癌患者臨床標(biāo)本中存在CD133+和 nestin+的細(xì)胞。這些研究也證實(shí)CD133可作為肺癌干細(xì)胞的特異性表面標(biāo)記物。

3 腦腫瘤干細(xì)胞

Ignatova等[18]最早報(bào)道了腦腫瘤干細(xì)胞的存在,他們發(fā)現(xiàn)人多形性膠質(zhì)母細(xì)胞瘤具有形成神經(jīng)球的能力,并從中分離出可克隆擴(kuò)增形成神經(jīng)球的前體細(xì)胞群,同時(shí)將克隆神經(jīng)球分析法用于分離膠質(zhì)母細(xì)胞瘤。Singh等[19]用神經(jīng)干細(xì)胞分離法從14例兒童腦腫瘤患者樣本中分離培養(yǎng)出CD133+的細(xì)胞群落,發(fā)現(xiàn)該細(xì)胞群落具有體外增殖和自我更新能力,并可向腦腫瘤細(xì)胞分化。將分離得到的CD133+細(xì)胞接種至NOD/SCID小鼠額葉中,結(jié)果僅少量該細(xì)胞即可引起接種小鼠腦腫瘤生長[20]。

4 肝癌干細(xì)胞

Haraguchi等[21]于2006年首次利用側(cè)群細(xì)胞分選技術(shù)在Huh7、Hep3B肝癌細(xì)胞系中分離出側(cè)群細(xì)胞,并發(fā)現(xiàn)Huh7細(xì)胞系中的側(cè)群細(xì)胞具有自我更新和多向分化能力,且絕大多數(shù)處于靜止期,對多種化療藥具有很強(qiáng)的耐藥性。Chiba等[22]從Huh7和PLC/PRE/5人肝癌細(xì)胞系中分別篩選出0.25%和0.8%的側(cè)群細(xì)胞,該細(xì)胞具有很強(qiáng)的增殖和抗凋亡能力,并且接種至NOD/SCID小鼠體內(nèi)后表現(xiàn)出很高的致瘤能力。Suetsugu等[23]首次利用CD133對肝癌細(xì)胞系Huh7細(xì)胞進(jìn)行分選,發(fā)現(xiàn)CD133+細(xì)胞較CD133-細(xì)胞具有更高的增殖能力,成熟度較低,且甲胎蛋白的mRNA表達(dá)水平更高。通過接種NOD/SCID小鼠發(fā)現(xiàn)前者具有高致瘤性。Yang等[24]從肝癌細(xì)胞 系 MIHA、HepG2、Hep3B、PLC、Huh7、MHCC97L和MHCC97H中分離出CD90+細(xì)胞,并進(jìn)行NOD/SCID裸鼠接種,發(fā)現(xiàn)CD90+細(xì)胞具有明顯的致瘤性,而CD90-細(xì)胞則沒有,并且91.6%的肝癌患者血液樣本中存在CD90+CD45-細(xì)胞群,相對于CD133具有更高的特異性。同時(shí)該研究還發(fā)現(xiàn)CD90+CD44+細(xì)胞較CD90+CD44-細(xì)胞具有更強(qiáng)的致瘤性和更高的轉(zhuǎn)移瘤風(fēng)險(xiǎn)。Zhu等[25]研究則發(fā)現(xiàn)CD133+CD44+肝癌細(xì)胞具有自我更新、增殖和分化的特性,并且具有較強(qiáng)的抗藥性和更高的致瘤性。據(jù)此可以看出,目前對于肝癌干細(xì)胞檢測并沒有特異的表面標(biāo)記物,但多種標(biāo)記物聯(lián)合檢測的特異性明顯高于單一標(biāo)記物檢測。

5 乳腺癌干細(xì)胞

Al-Hajj等[4]于2003年首次從人原發(fā)性乳腺癌和乳腺癌轉(zhuǎn)移性胸水滲出液中分離出具有ESA+CD44+CD24-/low Lin-標(biāo)記的細(xì)胞群,該細(xì)胞具有自我更新和多向分化能力,且將其接種于NOD/SCID小鼠乳腺脂肪墊中可形成具有親代異質(zhì)性的移植瘤,而超過20 000種的其他表型細(xì)胞群均不能致瘤。但在Al-Hajj的研究中,6只小鼠中僅有1只出現(xiàn)了移植瘤,Giatromanolaki和Sheridan的研究也同樣發(fā)現(xiàn)[26-27],并非所有CD44+CD24-細(xì)胞都具有致瘤性,且CD44+CD24-表型細(xì)胞在basal型乳腺癌中較多,而在luminal型乳腺癌中卻非常少。因此越來越多的學(xué)者提出,對于乳腺癌干細(xì)胞的檢測,應(yīng)該使用多種表面標(biāo)記聯(lián)合的方法才能提高其特異性和檢出率。

6 結(jié)腸癌干細(xì)胞

O′Brien等[28]于 2007年首次報(bào)道了從7例原發(fā)性結(jié)腸癌和10例結(jié)腸外轉(zhuǎn)移灶中分離出具有自我更新、增殖和分化能力CD133+的細(xì)胞群,并且明確該細(xì)胞群具有明顯的致瘤作用,是未分類結(jié)腸癌細(xì)胞致瘤細(xì)胞富集率的200倍以上。Ricci-Vitian等[29]以CD133+為標(biāo)記進(jìn)行結(jié)腸癌干細(xì)胞分離鑒定,得出了基本相同的結(jié)果。Haraguchi等[30]則認(rèn)為,CD133+CD44+細(xì)胞才存在致瘤性,這也從另一方面說明單純CD133+細(xì)胞并不一定就是結(jié)腸癌干細(xì)胞。而在Dalerba等[31]的研究中,CD133在不同腫瘤中表達(dá)并不相同,而CD44表達(dá)陽性的細(xì)胞通常伴有CD133的陽性表達(dá)。以上皮細(xì)胞黏附分子(EpCAM)和CD44作為表面標(biāo)記鑒定,EpCAMhighCD44+細(xì)胞群接種NOD/SCID鼠,可形成移植瘤,且移植瘤的表型分化比例、異質(zhì)性以及形態(tài)特征均與原代腫瘤類似,而EpCAMlowCD44-細(xì)胞則無移植瘤形成。因此他們認(rèn)為CD44可作為結(jié)腸癌干細(xì)胞篩選的更優(yōu)越的特異性標(biāo)記物。

7 其他腫瘤干細(xì)胞

除了上述腫瘤干細(xì)胞外,更多的腫瘤干細(xì)胞被眾多學(xué)者廣泛研究。Collins等[32]從原代培養(yǎng)的前列腺癌細(xì)胞中分離出高純度的CD44+/整合素α 2β1/CD133+細(xì)胞,該細(xì)胞具有自我更新、多向分化和高增殖等干細(xì)胞特點(diǎn),并具有較強(qiáng)的體外致瘤性。Fang等[33]則在黑色素瘤中分離出具有干細(xì)胞特性的細(xì)胞亞群,體外培養(yǎng)后可使NOD/SCID小鼠產(chǎn)生腫瘤。

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