王少川(綜述),宋　武(審校)

(中山大學附屬第一醫(yī)院胃腸胰外科,廣州 510080)

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骨髓間質干細胞在結直腸腫瘤微環(huán)境中的作用

王少川△(綜述),宋武※(審校)

(中山大學附屬第一醫(yī)院胃腸胰外科,廣州 510080)

摘要：骨髓間質干細胞(MSCs)是由中胚層發(fā)育而來的一類成體干細胞，存在于全身結締組織和器官間質中，屬于骨髓非造血干細胞，具有干細胞的一般特性，即自我更新以及多向分化能力。MSCs能夠促進結直腸腫瘤的生長。在腫瘤條件下，骨髓中的MSCs隨血運遷至腫瘤微環(huán)境，參與腫瘤的構建過程。MSCs促進腫瘤侵襲的機制是復雜的，包括MSCs轉化為腫瘤相關纖維母細胞參與腫瘤間質的形成以及MSCs對微環(huán)境的調(diào)節(jié)作用。MSCs在結直腸癌靶向治療策略中的應用前景是廣闊的，通過不斷的研究，將為MSCs在臨床中的安全應用提供新的理論依據(jù)。

關鍵詞：結直腸腫瘤；骨髓間質干細胞；腫瘤相關纖維母細胞

早在1889年，Paget[1]即提出腫瘤生長的“種子-土壤學說”，器官微環(huán)境(“土壤”)可影響特定腫瘤細胞(“種子”)的種植、侵襲、存活、生長。回顧近年來腫瘤學研究進展不難發(fā)現(xiàn)，腫瘤的發(fā)生及轉移并不僅和腫瘤細胞自身有關，腫瘤細胞的周邊環(huán)境對腫瘤的生長也發(fā)揮重要作用[2]。腫瘤微環(huán)境包含多種成分，骨髓間質干細胞(mesenchymal stem cells,MSCs)是其中重要的一群，MSCs在腫瘤微環(huán)境中的作用一直是學者們關注的焦點。MSCs是由中胚層發(fā)育而來的一類成體干細胞，具有干細胞的一般特性，即自我更新及多向分化的能力，這些特點尤其體現(xiàn)在間質干細胞對損傷組織的歸巢與修復作用上[3]。隨著干細胞工程技術的進步，間質干細胞與腫瘤間的關系備受矚目。已經(jīng)證實，間質干細胞對于腫瘤組織亦能表現(xiàn)出類似于炎性白細胞的歸巢作用[4]。近年來，隨著診斷技術的進步，癌前病變檢出率顯著提高，結直腸癌的病死率大幅降低[5]。美國癌癥聯(lián)合會公布的結果顯示，Ⅰ期結直腸癌患者5年生存率為93.2%，而Ⅳ期只有8.1%[6]?？梢姡砥诮Y直腸癌的預后不容樂觀，其死因多為腫瘤向肝、肺、腹腔淋巴結等多器官轉移[7]。該文回顧并總結了MSCs與結直腸腫瘤微環(huán)境之間關系的研究方法及結果，旨在為結直腸癌早期診斷、預測轉移風險、評估預后、研發(fā)新型靶向治療策略等方面提供新的思路和認識。

1MSCs的細胞學特性

MSCs存在于全身結締組織和器官間質中，屬于骨髓非造血干細胞，具有干細胞的一般特性；在適當?shù)臈l件下，MSCs可分化為軟骨細胞、脂肪細胞、肌細胞等多種細胞[8]。在細胞培養(yǎng)中，MSCs具有貼壁生長的特點，表達細胞表面標志(CD105、CD90、CD73等)，不表達典型的造血細胞標志(如CD14、CD34、CD19、CD45等)[9]?；谶@些特性，在組織發(fā)生損傷時，MSCs能夠被募集到損傷組織參與組織修復，遂被廣泛應用于再生醫(yī)學等領域。研究人員通過比較腫瘤與組織損傷過程，發(fā)現(xiàn)其相似之處，即腫瘤同樣可以釋放細胞因子、趨化因子以及生長因子等炎性介質[10]。試驗表明，在神經(jīng)膠質瘤[11]、乳腺[12-14]、結腸[15]、卵巢[16]、肺及其他轉移性腫瘤[17-18]的微環(huán)境中，這種定向遷移的機制同樣存在；骨髓中的MSCs隨血運遷移至腫瘤部位，參與腫瘤的構建過程[14]。Chamberlain等[8]推測，整合蛋白和黏附分子參與調(diào)控MSCs的遷移。研究者們通過構建其他類型的腫瘤模型，發(fā)現(xiàn)了一系列參與這一過程的調(diào)節(jié)因子(如內(nèi)皮生長因子、趨化因子)[4]。但目前針對結直腸腫瘤構建模型的研究尚不多見，有待進行更深入的研究去探索MSCs在結直腸腫瘤微環(huán)境下的作用和機制。

2MSCs與腫瘤相關纖維母細胞

MSCs類似白細胞可定向遷移至腫瘤部位。MSCs在腫瘤微環(huán)境中的間接作用基于一種假設，即MSCs是腫瘤相關纖維母細胞(tumor associated fibroblast,TAF)的前體[19]。TAF是腫瘤微環(huán)境中的重要細胞群，在腫瘤微環(huán)境中參與血管構建，促進腫瘤細胞浸潤、轉移[20]。研究者通過免疫組織化學方法證實,TAF能夠表達α平滑肌蛋白、大量膠原以及其他基質成分，是膠原積聚和組織結構改變的重要原因[21]。此外，TAF分泌可溶性因子(如轉化生長因子、血小板衍生生長因子等)促進腫瘤細胞的生長和侵襲，且兩者共同反饋，構成腫瘤生長、浸潤和轉移的微環(huán)境[22]。在腫瘤微環(huán)境中，兩者均可利用腫瘤細胞代謝出的乳酸鹽作為能量來源，參與腫瘤的構建[23]?？梢?，MSCs和TAF的關系是十分密切的。據(jù)Shinagawa等[24]的研究報道，MSCs在結直腸癌微環(huán)境中表達α平滑肌蛋白；而Nakagawa等[25]通過免疫熒光檢測出轉移性結直腸癌TAF中亦表達此蛋白；不僅如此，他還通過聚合酶鏈式反應檢測到結直腸TAF中波形蛋白的表達。波形蛋白是存在于MSCs中的一種中間絲蛋白,能夠調(diào)節(jié)細胞骨架蛋白、細胞黏附分子等蛋白間的相互作用,參與腫瘤細胞和腫瘤相關內(nèi)皮細胞、巨噬細胞的黏附、遷移、侵襲和細胞信號轉導等[26]。可見，波形蛋白與腫瘤發(fā)生、轉移密切相關，是MSCs的重要標志。這些MSCs與TAF的共性表達，均支持“MSCs是TAF的前體”這一假說。

3MSCs與結直腸腫瘤的形成和轉移

體外實驗證實，MSCs能夠同時抑制腫瘤細胞增殖和凋亡；然而體內(nèi)試驗發(fā)現(xiàn)，MSCs通過免疫抑制作用，能夠降低淋巴細胞活性，重構腫瘤微環(huán)境，使其更利于腫瘤的生長[27]。Tsai等[28]將MSCs與結直腸癌細胞混合后進行體外培養(yǎng)，發(fā)現(xiàn)MSCs能夠通過旁分泌作用直接促進腫瘤生長。Shinagawa等[24]將人類骨源性MSCs按不同比例與癌細胞混合后接種于小鼠體內(nèi)，并與單獨接種癌細胞的小鼠進行對照，結果發(fā)現(xiàn)較高比例的MSCs明顯促進了結直腸癌的生長和轉移，而較低比例的MSCs不能催生結直腸癌的轉移。另據(jù)Liu等[29]的研究，較之未經(jīng)處理過的MSCs,干擾素γ和腫瘤壞死因子α預刺激過的MSCs通過活化低氧誘導因子1α信號通路，表達更高水平的血管內(nèi)皮細胞生長因子,從而更好地促進腫瘤血管構建；對照組數(shù)據(jù)提示，即便混合培養(yǎng)的MSCs比例較低，但在某些炎性因子刺激下，MSCs亦能夠發(fā)揮更大的促腫瘤作用。上述研究均構建于小鼠模型之中，其人為設定的MSCs與癌細胞比例，不能充分反映MCSs在人體腫瘤微環(huán)境中的作用，未來應通過模擬更趨近于人體結直腸腫瘤微環(huán)境中的MSCs比例，來證實這一觀點。轉化生長因子β是另一類重要的慢性炎性因子，傳統(tǒng)觀點認為轉化生長因子β的表達可以抑制腫瘤細胞的繁殖。據(jù)Shangguan等[30]報道，抑制轉化生長因子β/Smad信號通路可以有效阻止MSCs向TAF轉化，繼而達到抗腫瘤的目的。Calon等[31]的研究卻提示，轉化生長因子β在微環(huán)境中的表達能夠促進結直腸腫瘤的轉移。MSCs與轉化生長因子β信號通路間的復雜關系值得進一步探索。MSCs具有免疫調(diào)節(jié)功能，在某些因子的作用下，可以對淋巴細胞產(chǎn)生抑制作用，繼而促進腫瘤逃避免疫監(jiān)視[32]。另有學者研究發(fā)現(xiàn)，MSCs可以增強B細胞的活力，但抑制B細胞的增殖[33-34]。而Corcione等[35]認為，B細胞分泌細胞因子的活動并不受MSCs的影響。Schioppa等[36]發(fā)現(xiàn)一種調(diào)節(jié)性B細胞(tumor-evoked Bregs，tBreg)，在腫瘤微環(huán)境中抑制了抗腫瘤免疫，使腫瘤壞死因子α促進腫瘤生長。腫瘤微環(huán)境中產(chǎn)生的腫瘤壞死因子α通過誘導核因子κB抗凋亡分子的表達而促進腫瘤細胞存活、促進腫瘤血管新生，抑制T細胞反應逃避免疫監(jiān)視[37]。Olkhanud等[38]通過小鼠模型發(fā)現(xiàn)，tBreg借助生長因子β，使靜止的CD4+T細胞向叉頭狀轉錄因子3+調(diào)節(jié)性T細胞轉化，進而促進腫瘤的轉移。這進一步說明tBreg可以促進腫瘤生長。然而，腫瘤微環(huán)境中的MSCs是否能夠促進或抑制tBreg產(chǎn)生，兩者之間有何聯(lián)系，具體發(fā)揮何種作用，這些問題仍不明確。盡管上述研究是基于人乳腺癌細胞株小鼠構建的模型，但已有文獻報道叉頭狀轉錄因子3+調(diào)節(jié)性T細胞與結腸癌細胞之間的緊密聯(lián)系[39]。因此，tBreg與MSCs的關系及作用機制更加值得關注。

4MSCs與上皮細胞間質轉化

上皮細胞間質轉化(epithelial to mesenchymal transition,EMT)的概念于20世紀80年代被提出，后被學者闡釋出在結直腸癌原發(fā)性浸潤以及繼發(fā)性轉移中占舉足輕重的地位[40]。EMT通過減弱依賴于E2鈣黏蛋白的胞間黏附并且提高癌細胞機動性,從而增強癌細胞浸潤能力，使上皮細胞失去原有特性，并獲得間質轉化[41]。Loboda等[42]通過調(diào)查326例結腸癌患者得出結論，結腸癌發(fā)生過程中主要的基因表達過程與EMT高度相關。Varnat等[43]的研究認為，人結直腸癌上皮細胞激活Hedgehog/Gli1信號通路，引發(fā)致瘤性EMT的作用。致瘤性EMT通過活化Wnt/β聯(lián)蛋白信號通路，賦予癌細胞一系列干細胞學特性和轉移侵襲的能力[44]。據(jù)此認為，在結直腸腫瘤微環(huán)境中，癌細胞通過EMT作用獲得了遷移性的表型。癌細胞由EMT作用而獲得干細胞特性，使得研究人員對MSCs和EMT之間存在的聯(lián)系產(chǎn)生了興趣，Martin等[45]的體外研究表明，乳腺癌微環(huán)境中的MSCs可以刺激上皮細胞發(fā)生EMT；Bhattacharya等[46]將MSCs與肝癌細胞混合培養(yǎng)，發(fā)現(xiàn)MSCs增強了EMT標志性波形蛋白的表達、參與鈣黏素的調(diào)控，同時癌細胞轉移侵襲機制的轉錄因子Snail和Slug表達亦見增多。這進一步說明MSCs在腫瘤微環(huán)境中促生EMT，而MSCs在結直腸癌微環(huán)境中是否具有類似的作用有待于進一步探索。

5小結

MSCs促進腫瘤侵襲的機制是復雜的，包括MSCs轉化為TAF參與間質形成以及MSCs對微環(huán)境的調(diào)節(jié)。MSCs與結直腸腫瘤的浸潤、轉移間的關系，目前認識仍不夠清晰。MSCs旁分泌因子的作用及相關調(diào)控機制，需要進一步探明。同時，在結直腸癌微環(huán)境中，MSCs與調(diào)節(jié)性免疫細胞間的潛在聯(lián)系也是值得探索的。未來應更加注重MSCs的體內(nèi)研究，構建出更好的生物模型以更加真實地反映出人體內(nèi)環(huán)境中的MSCs分布，來闡釋MSCs結直腸癌環(huán)境下的作用機制。

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Study on the Role of Mesenchymal Stem Cells in Colorectal Tumor Microenvironment

WANGShao-chuan,SONGWu.

(DepartmentofGastrointestinalPancreaticSurgery,theFirstAffiliatedHospitalofSunyat-senUniversity,Guangzhou510080,China)

Abstract：Mesenchymal stem cells(MSCs) are a type of adult stem cells,which are developed from the mesoderm.They exist in the interstitium of connective tissues and organs in the whole body,and belong to the bone marrow non-hematopoietic stem cells.It has the general characteristics of stem cells,namely the ability of self-renewal and multi-directional differentiation.MSCs could promote the growth of colorectal cancer.Under the condition of tumor,MSCs move along with the blood stream to the tumor microenvironment,and participate in the process of the construction of the tumor.The mechanism of MSCs in promoting tumor invasion is complex,which includes MSCs′ transformation into tumor associated fibroblast (TAF) to participate in the formation of stroma and their regulating effect on tumor microenviroment.The application prospect of MSCs in colorectal cancer targeted therapy strategy is extensive,further study in this area will provide new theoretical basis for its safety applications.

Key words：Colorectal tumor； Mesenchymal stem cells； Tumor associated fibroblast

doi：10.3969/j.issn.1006-2084.2015.03.019

中圖分類號:R735

文獻標識碼：A

文章編號：1006-2084(2015)03-0434-04

收稿日期：2014-03-20修回日期：2014-08-08編輯:辛欣