肖恒，趙成嶺，陳詩(shī)軍，張庚艷，陳余清

（蚌埠醫(yī)學(xué)院第一附屬醫(yī)院呼吸與危重癥醫(yī)學(xué)科，呼吸系病基礎(chǔ)與臨床安徽省重點(diǎn)實(shí)驗(yàn)室，蚌埠233000）

臍帶間充質(zhì)干細(xì)胞惡性分化并促進(jìn)肺癌A549細(xì)胞移植瘤生長(zhǎng)及轉(zhuǎn)移

肖恒，趙成嶺，陳詩(shī)軍，張庚艷，陳余清*

（蚌埠醫(yī)學(xué)院第一附屬醫(yī)院呼吸與危重癥醫(yī)學(xué)科，呼吸系病基礎(chǔ)與臨床安徽省重點(diǎn)實(shí)驗(yàn)室，蚌埠233000）

目的 探討人臍帶來(lái)源的間充質(zhì)干細(xì)胞（human umbilical cord mesenchymal stem cells，hUCMSCs）對(duì)裸鼠肺癌移植瘤生長(zhǎng)及轉(zhuǎn)移的影響。方法 將24只裸鼠隨機(jī)分為4組，分別為A549細(xì)胞單獨(dú)注射組（A549組）、hUCMSCs單獨(dú)注射組（MSCs組）、A549細(xì)胞與正常人皮膚成纖維細(xì)胞（skin fibroblasts, SKs）共同注射組（A549+SKs組）、hUCMSCs與A549細(xì)胞共注射組（A549+MSCs組）。HE染色和免疫組織化學(xué)染色確定hUCMSCs注射裸鼠皮下結(jié)節(jié)性質(zhì)；流式細(xì)胞分選技術(shù)分選出各組移植瘤瘤組織中的A549細(xì)胞；實(shí)時(shí)熒光定量PCR和Westernblot檢測(cè)A549細(xì)胞中上皮-間質(zhì)轉(zhuǎn)化（epithelial-mesenchymal transition，EMT）相關(guān)分子標(biāo)志物的表達(dá)差異。結(jié)果 hUCMSCs注射后，hUCMSCs發(fā)生惡性分化，hUCMSCs與A549細(xì)胞共注射后肺癌移植瘤體積明顯大于單獨(dú)A549注射組和A549與SKs共注射組；2只A549與hUCMSCs共注射組裸鼠出現(xiàn)了肺癌肝臟轉(zhuǎn)移，而A549組與A549+SKs組的全部 6只裸鼠均沒(méi)有發(fā)現(xiàn)肝臟轉(zhuǎn)移。A549與MSCs共注射組A549細(xì)胞中上皮表型特征分子E-cadherin、β-catenin mRNA相對(duì)表達(dá)量和β-catenin蛋白表達(dá)水平高于A549組與A549+SKs組，E-cadherin蛋白表達(dá)水平明顯高于A549+SKs組，間質(zhì)表型特征分子N-cadherin和vimentin蛋白表達(dá)水平明顯低于A549組與A549+SKs組，EMT轉(zhuǎn)錄調(diào)控因子twist蛋白表達(dá)水平低于A549組與A549+SKs組。結(jié)論 hUCMSCs可以自我惡性分化；hUCMSCs能促進(jìn)肺癌A549細(xì)胞移植瘤的生長(zhǎng)與轉(zhuǎn)移；hUCMSCs增強(qiáng)A549細(xì)胞侵襲、轉(zhuǎn)移的機(jī)制不是通過(guò)誘導(dǎo)A549細(xì)胞發(fā)生EMT（上皮-間質(zhì)轉(zhuǎn)化）。

人臍帶間充質(zhì)干細(xì)胞；惡性轉(zhuǎn)化；A549細(xì)胞；肺癌；上皮-間質(zhì)轉(zhuǎn)化

間充質(zhì)干細(xì)胞（mesenchymal stem cells，MSCs）是一種無(wú)造血功能的干細(xì)胞，具有多向分化的潛能。近期研究顯示，MSCs具有向腫瘤組織趨化的能力，被認(rèn)為是理想的藥物載體應(yīng)用于癌癥治療[1]。但是，也有研究結(jié)果表明，MSCs可以發(fā)生染色體異變后表現(xiàn)出惡性分化，參與腫瘤的發(fā)生與發(fā)展[2]。因此，深入研究間充質(zhì)干細(xì)胞與腫瘤之間的關(guān)系對(duì)于闡明其在腫瘤治療中的作用，指導(dǎo)臨床合理應(yīng)用意義重大。當(dāng)前研究較多的MSCs多為骨髓來(lái)源，而人臍帶來(lái)源的間充質(zhì)干細(xì)胞（human umbilical cord mesenchymal stem cells，hUCMSCs）對(duì)供體損傷較小，基本不受倫理學(xué)的限制[3]，同時(shí)具有胚胎干細(xì)胞及成體干細(xì)胞的優(yōu)點(diǎn)。本實(shí)驗(yàn)將檢測(cè)hUCMSCs是否發(fā)生惡性轉(zhuǎn)化及其對(duì)A549細(xì)胞移植瘤生長(zhǎng)及轉(zhuǎn)移能力的影響。

材料與方法

1 主要材料和試劑

在產(chǎn)婦本人及其家屬知情同意的基礎(chǔ)上，無(wú)菌采集、提取培養(yǎng)臍帶來(lái)源的MSCs細(xì)胞；人肺腺癌細(xì)胞株A549細(xì)胞，由軍事醫(yī)學(xué)科學(xué)院饋贈(zèng)，蚌埠醫(yī)學(xué)院呼吸內(nèi)科實(shí)驗(yàn)室保存。BALB/C裸鼠購(gòu)自于揚(yáng)州大學(xué)比較醫(yī)學(xué)中心（質(zhì)量合格證編號(hào)：NO.201603478）。胰蛋白酶、青霉素/鏈霉素購(gòu)自碧云天生物公司，Trizol A+試劑購(gòu)自北京天根生化科技有限公司，DMEM/F12培養(yǎng)基、優(yōu)質(zhì)胎牛血清購(gòu)自購(gòu)自美國(guó)Hyclone公司；鼠抗人TTF-1-FⅠTC和CD73-PE購(gòu)自美國(guó)eBioscience公司，兔抗人CK、E-鈣粘蛋白、β-catenin、N-鈣粘蛋白、波形蛋白、twist及內(nèi)參β-肌動(dòng)蛋白一抗購(gòu)自美國(guó)Abcam公司，辣根過(guò)氧化物酶標(biāo)記的山羊抗兔ⅠgG、5×SDS-PAGE蛋白上樣緩沖液、SDS-PAGE 凝膠配制試劑盒、10 ×Tris-甘氨酸蛋白電泳緩沖液、BCA 蛋白分析試劑盒、ECL檢測(cè)試劑盒、DAB顯色劑均購(gòu)自南京東極生物科技有限公司。

2 肺癌裸鼠移植瘤模型建立

裸鼠隨機(jī)分為4組，每組6只。A549細(xì)胞單獨(dú)注射組（A549組），腋下皮下組織內(nèi)接種5×106個(gè)A549細(xì)胞；hUCMSCs單獨(dú)注射組，腋下接種5×106個(gè)hUCMSC（MSCs組）；A549細(xì)胞與正常人皮膚成纖維細(xì)胞共同注射組（A549+SKs組），腋下接種5×106個(gè)A549細(xì)胞與5×106個(gè)SKs；hUCMSCs與A549細(xì)胞共注射組（A549+MSCs組），腋下接種5×106個(gè)hUCMSCs與5×106個(gè)A549細(xì)胞。裸鼠置于SPF級(jí)條件下飼養(yǎng)，約2周后可見(jiàn)裸鼠皮下出現(xiàn)腫瘤結(jié)節(jié)，以后每周2次測(cè)裸鼠皮下移植瘤直徑變化，游標(biāo)卡尺測(cè)量移植瘤最長(zhǎng)徑a，最短徑b，計(jì)算腫瘤體積變化（V=0.5ab2）繪制生長(zhǎng)曲線圖。于第5周處死裸鼠，留取移植瘤組織進(jìn)行后續(xù)實(shí)驗(yàn)。

3 免疫組織化學(xué)

取裸鼠肺癌移植瘤組織，PBS洗凈后以10%中性福爾馬林液固定，梯度乙醇脫水、二甲苯透明、浸蠟、石蠟包埋；4μm連續(xù)切片，常規(guī)脫蠟、水化，抗原修復(fù)；滴加3%過(guò)氧化氫于37℃濕盒孵育15min抑制內(nèi)源性過(guò)氧化物酶活性；PBS漂洗3次，每次5min；分別滴加CK（1:200）、vimentin（1:200）一抗，置4℃冰箱孵育過(guò)夜，PBS漂洗3次，每次5min；滴加生物素標(biāo)記的山羊抗兔ⅠgG（1:500）37℃濕盒孵育30min，PBS漂洗3次，每次5min；滴加鏈霉親和素-過(guò)氧化物酶復(fù)合物于37℃濕盒孵育30min，PBS漂洗3次，每次5min。滴加DAB顯色劑顯色3～5min，沖洗終止顯色后，復(fù)染，脫水，透明，中性樹脂封片。用高倍視野（40倍物鏡）觀察裸鼠移植瘤組織，評(píng)價(jià)CK、vimentin的表達(dá)情況，以胞質(zhì)中或胞膜出現(xiàn)棕色至深棕色顆粒且其染色強(qiáng)度高于背景非特異染色為陽(yáng)性細(xì)胞。

4 流式細(xì)胞術(shù)

取A549組、A549+SKs組和A549+MSCs組移植瘤組織，去除組織表面的結(jié)締組織，用含500U/ml青霉素、500mg/ml鏈霉素的PBS清洗兩次后剪成約1mm3的小組織，盡量剪碎；含雙抗的PBS清洗兩次后，加入0.25%胰酶在37℃搖晃消化30min后加入0.5%Ⅰ型膠原酶37℃繼續(xù)消化1h；隨后離心，PBS洗兩次，過(guò)70um細(xì)胞篩；分別加入FⅠTC-TTF-1和PE-CD73抗體（1:100稀釋），室溫避光20min，流式細(xì)胞儀分選A549細(xì)胞。

5 實(shí)時(shí)熒光定量PCR（qPCR）

Trizol法分別提取各組肺癌移植瘤組織中A549細(xì)胞的總RNA，采用SYBR Green熒光染料，參照試劑盒說(shuō)明構(gòu)建反應(yīng)體系。反應(yīng)條件：95℃預(yù)變性10min，95℃ 5s，60℃1min，共40個(gè)循環(huán)。PCR引物序列如下：

E-cadherin：5’-GACCGAGAGAGTTTCCCTACG-3’（F），5’-TCAGGCACCTGACCCTTGTA-3’（R）；

β-catenin，5’-TCTGAGGACAAGCCACAAGATTACA-3’（F）；5’-TGGGCACCAATATCAAGTCCAA-3’（R）；

N-cadherin：5’-GAGATCCTACTGGACGGTTCG-3’（F），5’-TCTTGGCGAATGATCTTAGGA-3’（R）；

vimentin：5’-CCTTGAACGCAAAGTGGAATC-3’（F），5’-TGAGGTCAGGCTTGGAAACAT-3’（R）；

twist：5’-GGAGTCCGCAGTCTTACGAG-3’，5’-TCTGGAGGACCTGGTAGAGG-3’（R）；

GAPDH：5’-CAGGGCTGCTTTTAACTCTGGTAA-3’（F），R:5’-GGGTGGAATCATATTGGAACATGT-3’（R）。

2-ΔΔct用于分析相對(duì)表達(dá)水平。

6 Western blot

分別提取A549組、A549+SKs組和A549+MSCs組中A549細(xì)胞的總蛋白， 二喹啉甲酸（bicinchonininc acid，BCA）法測(cè)定蛋白濃度，每組樣本取等量蛋白進(jìn)行SDS-聚丙烯酰胺凝膠電泳，電泳后PVDF 轉(zhuǎn)膜，經(jīng)5%脫脂奶粉封閉1 h（室溫）后，分別加入一抗4 ℃過(guò)夜，之后孵育二抗，以β-肌動(dòng)蛋白（工作濃度1∶500）為內(nèi)參，Thermo ECL顯色后，應(yīng)用Tanon凝膠成像儀測(cè)定各蛋白條帶光密度，以目的蛋白和β-肌動(dòng)蛋白（β-actin）條帶光密度比值代表各蛋白的相對(duì)水平。

7 統(tǒng)計(jì)學(xué)分析

所有實(shí)驗(yàn)至少重復(fù)3次。采用SPSS17．0軟件進(jìn)行統(tǒng)計(jì)學(xué)處理，計(jì)量資料用平均數(shù)±標(biāo)準(zhǔn)差（±s）表示，組間比較采用t檢驗(yàn)，以P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1 hUCMSCs的形態(tài)學(xué)特征與免疫表型

臍帶組織塊培養(yǎng)15d左右可以見(jiàn)到臍帶周圍梭形細(xì)胞爬出，20d左右細(xì)胞融合逐漸鋪滿25cm2的培養(yǎng)瓶。細(xì)胞傳代后可以觀察到細(xì)胞形態(tài)均一，呈旋渦狀生長(zhǎng)。流式細(xì)胞儀分析顯示，所提取的hUCMSCs表達(dá)基質(zhì)細(xì)胞相關(guān)表面標(biāo)記CD73、CD90、CD105；不表達(dá)單核細(xì)胞表面標(biāo)記CD14、淋巴細(xì)胞表面標(biāo)記CD19、造血干細(xì)胞表面標(biāo)記CD34、人類白細(xì)胞共同抗原CD45及MHC-Ⅱ類分子HLA-DR（圖1）。

圖1 hUCMSCs免疫表型Fig.1 Ⅰmmunophenotypes of hUCMSCs

2 hUCMSCs惡性自發(fā)分化及促進(jìn)裸鼠皮下移植瘤生長(zhǎng)與轉(zhuǎn)移

至實(shí)驗(yàn)結(jié)束（5周），腫瘤體積分別為：A549組（86.6±38.40）mm3，hUCMSCs單獨(dú)注射組（13.9±2.0）mm3，A549+SKs組（26.2±17.2）mm3，A549+MSCs組（354.65±177.01）mm3（ 圖2），hUCMSCs與A549細(xì)胞共注射組裸鼠皮下移植瘤生長(zhǎng)速度明顯快于A549組與SKs與A549細(xì)胞共注射組。另外，在hUCMSCs單獨(dú)注射組，hUCMSCs發(fā)生了自發(fā)分化并表現(xiàn)出惡性轉(zhuǎn)化，HE染色鏡下可見(jiàn)呈腫瘤組織形態(tài)，表現(xiàn)為異形細(xì)胞廣泛分布，胞質(zhì)豐富，核深染，核分裂相多（圖3A）；免疫組織化學(xué)染色示hUCMSCs自發(fā)分化后表達(dá)上皮和間質(zhì)標(biāo)記物CK和vimentin（圖3B，圖3C）；2只hUCMSCs與A549細(xì)胞共注射組裸鼠出現(xiàn)了肺癌肝臟轉(zhuǎn)移；A549組與A549+SKs組的全部 6只裸鼠均沒(méi)有發(fā)現(xiàn)肝臟轉(zhuǎn)移（圖4）。

圖2 皮下移植瘤的體積變化對(duì)比。與A549細(xì)胞單獨(dú)注射組比較：*，0.01＜P＜0.05；**，P＜0.01Fig. 2 Comparison on the changes in volume of subcutaneous xenografts. A549+MSCs group showed significantly higher volume compared to A549 group: *, 0.01＜P＜0.05; **, P ＜0.01

圖3 hUCMSCs自發(fā)惡性轉(zhuǎn)化。A，單獨(dú)注射hUCMSCs組織的HE染色；B，單獨(dú)注射hUCMSCs組織CK表達(dá)的免疫組織化學(xué)檢測(cè)；C，單獨(dú)注射hUCMSCs組織的vimentin表達(dá)；比例尺，100μmFig. 3 Spontaneous malignant transformation of hUCMSCs. A, HE staining of the tissue injected with hUCMSCs only; B, Ⅰmmunohistochemical stain of CK in the tissue injected with hUCMSCs only; C, Ⅰmmunohistochemical stain of vimentin in the tissue injected with hUCMSCs only; scale bar, 100μm

圖4 A549+MSCs組裸鼠肺癌肝臟轉(zhuǎn)移及HE染色檢測(cè)。A，肺癌肝轉(zhuǎn)移標(biāo)本肉眼觀；B和C，肺癌肝轉(zhuǎn)移標(biāo)本HE染色；比例尺：A，1cm；B和C，400μmFig. 4 Detection of liver metastasis and HE staining in group A549 + MSCs. A, naked eye view of lung cancer liver metastasis in group A549 cells co-injection with MSCs; B and C, HE staining of lung cancer liver metastasis; scale bar: 1cm in A; 400μm in B and C

3 hUCMSCs上調(diào)A549細(xì)胞E-cadherin、β-catenin mRNA表達(dá)

應(yīng)用流式細(xì)胞儀分選A549組、A549+SKs組和A549+MSCs組移植瘤組織中的A549細(xì)胞（圖5）后進(jìn)行qPCR檢測(cè)，結(jié)果顯示A549+MSCs組中的A549細(xì)胞上皮表型標(biāo)志E-cadherin、β-catenin mRNA相對(duì)表達(dá)量顯著高于A549組，而間質(zhì)表型特征分子N-cadherin、vimentin及EMT轉(zhuǎn)錄調(diào)控因子twist mRNA表達(dá)水平無(wú)明顯影響（圖6）。

圖5 移植瘤組織中TTF-1標(biāo)記的A549細(xì)胞的流式細(xì)胞分選。Fig. 5 Flow cytometry sorting of TTF-1 labeled A549 cells from the transplanted tumors

4 hUCMSCs使A549細(xì)胞β-catenin和E-cadherin上調(diào)，N-cadherin、vimentin和twist下調(diào)

Western blot檢測(cè)顯示，A549+MSCs組A549細(xì)胞內(nèi)β-catenin水平明顯高于A549組與A549+SKs組，E-cadherin水平明顯高于A549+SKs組與A549組無(wú)顯著差異；A549+MSCs組A549細(xì)胞內(nèi)胞間質(zhì)表型特征分子N-cadherin與vimentin水平和EMT轉(zhuǎn)錄調(diào)控因子twist水平明顯低于A549組和與A549+SKs組（圖7）。

圖6 hUCMSCs細(xì)胞對(duì)A549細(xì)胞EMT相關(guān)分子mRNA表達(dá)的影響；*，P＜0.01Fig. 6 Effect of hUCMSCs on the expression of EMT- related molecules in A549 cells at mRNA level. *, P ＜0.01

圖7 hUCMSCs對(duì)A549細(xì)胞EMT相關(guān)蛋白水平的影響. A，Western blot檢測(cè)EMT相關(guān)蛋白表達(dá)水平；B，Western blotting檢測(cè)的統(tǒng)計(jì)學(xué)分析Fig. 7 Effect of hUCMSCs on the expression of EMT-related proteins in A549 cells at protein level. A, Representative western blot to determine EMT-related protein level; B, Statistical analysis of EMT-related proteins level determined by Western blot. *, 0.01＜P ＜0.05; **, P ＜0.01

討 論

本實(shí)驗(yàn)采用組織塊法從人臍帶組織中成功分離、培養(yǎng)出hUCMSCs，細(xì)胞形態(tài)均一，漩渦狀生長(zhǎng)。流式細(xì)胞表型鑒定結(jié)果顯示，表達(dá)基質(zhì)細(xì)胞相關(guān)表面標(biāo)記CD73、CD90、CD105；不表達(dá)單核細(xì)胞表面標(biāo)記CD14、淋巴細(xì)胞表面標(biāo)記CD19、造血干細(xì)胞表面標(biāo)記CD34、人類白細(xì)胞共同抗原CD45及MHC-Ⅱ類分子HLA-DR，符合國(guó)際細(xì)胞治療協(xié)會(huì)對(duì)間充質(zhì)干細(xì)胞的鑒定標(biāo)準(zhǔn)[4]。

關(guān)于MSCs在肺癌生長(zhǎng)、轉(zhuǎn)移和耐藥等惡性表型的功能學(xué)方面的研究，報(bào)道并不多，而且相關(guān)報(bào)道的結(jié)果也不太一致。本研究結(jié)果表明，MSCs促進(jìn)腫瘤的生長(zhǎng)、侵襲能力，但也有研究得出了相反的結(jié)論[5]。Suzuki[6]等觀察到，在MSCs與Lewis肺癌細(xì)胞共注射至C57BL/6小鼠皮下后，MSCs通過(guò)促進(jìn)腫瘤的血管生成而促進(jìn)腫瘤的生長(zhǎng)。而Shin等[7]的研究則表明，在肺癌轉(zhuǎn)移患者的血清中溶血磷脂酸（LPA）的含量較高，LPA可以誘導(dǎo)脂肪組織來(lái)源的MSCs通過(guò)增強(qiáng)癌細(xì)胞的粘附和增殖能力從而在肺癌發(fā)生和發(fā)展中發(fā)揮關(guān)鍵作用。但是，也有研究指出MSCs可以抑制肺癌細(xì)胞的生長(zhǎng)[8]。脂肪來(lái)源的MSCs與A549細(xì)胞共注射至裸鼠皮下后，抑制了移植瘤的生長(zhǎng)。Li等[9]發(fā)現(xiàn)MSCs通過(guò)分泌某些可溶性因子影響腫瘤的血管生成從而在體內(nèi)外抑制SK-MES-1細(xì)胞與A549細(xì)胞的增殖，并促進(jìn)肺癌細(xì)胞的凋亡。另外有研究顯示[10]，MSCs在體內(nèi)外實(shí)驗(yàn)中對(duì)肺癌細(xì)胞的影響完全不同，MSCs在體外實(shí)驗(yàn)中可以抑制A549細(xì)胞及H446細(xì)胞的增殖能力，但卻促進(jìn)皮下移植瘤的生長(zhǎng)。出現(xiàn)上述不同研究結(jié)果的原因可能是由于選用的MSCs的來(lái)源不同，所選用的肺癌細(xì)胞亞型也存在著差異。在本次實(shí)驗(yàn)中，我們觀察到hUCMSCs在裸鼠皮下自發(fā)分化并表現(xiàn)出惡性轉(zhuǎn)化，并且hUCMSCs與A549細(xì)胞共注射后促進(jìn)了肺癌移植瘤的生長(zhǎng)及轉(zhuǎn)移。

我們的本次實(shí)驗(yàn)結(jié)果顯示臍帶來(lái)源的MSCs可以通過(guò)自我惡性分化參與到腫瘤發(fā)生過(guò)程。Rubio[11]等人發(fā)現(xiàn)脂肪來(lái)源的MSCs在體外長(zhǎng)期傳代后，也可以發(fā)生自我分化，并且進(jìn)一步確定了自我惡性分化過(guò)程中涉及的機(jī)制。他們發(fā)現(xiàn)通過(guò)上調(diào)c-myc及抑制p16的水平，MSCs獲得了端粒酶活性，繞過(guò)了衰老的過(guò)程發(fā)生了自我分化。為了進(jìn)一步探討hUCMSCs促進(jìn)肺癌轉(zhuǎn)移機(jī)制，我們利用流式細(xì)胞分選技術(shù)將各組移植瘤組織中的A549細(xì)胞分選出來(lái)進(jìn)行EMT相關(guān)分子標(biāo)志物檢測(cè)。EMT（上皮-間質(zhì)轉(zhuǎn)化）被認(rèn)為在腫瘤的發(fā)展和轉(zhuǎn)移中發(fā)揮關(guān)鍵性的作用，EMT的主要特點(diǎn)是上皮細(xì)胞黏附分子（如E-cadherin等）表達(dá)的減少及間質(zhì)表型分子（如N-cadherin等）的表達(dá)增高。通過(guò)EMT過(guò)程，腫瘤細(xì)胞可以遷移、侵入周圍組織并進(jìn)入血流，使得原發(fā)腫瘤可以轉(zhuǎn)移到其他器官[12]。在關(guān)于胰腺腺癌和黑色素瘤的研究中[13,14]，MSCs可以通過(guò)誘導(dǎo)腫瘤細(xì)胞發(fā)生EMT進(jìn)而增強(qiáng)胰腺腺癌及黑色素瘤的侵襲、轉(zhuǎn)移能力。然而，也有實(shí)驗(yàn)結(jié)果表明EMT過(guò)程并非腫瘤轉(zhuǎn)移的必需條件，在上皮原發(fā)性腫瘤中，只有一小部分腫瘤細(xì)胞發(fā)生EMT過(guò)程。值得注意的是，肺轉(zhuǎn)移灶主要由非EMT腫瘤細(xì)胞組成，這些腫瘤細(xì)胞仍然保持了它們的上皮細(xì)胞特征。有研究通過(guò)過(guò)表達(dá)miR-200來(lái)抑制EMT過(guò)程，并不影響肺轉(zhuǎn)移灶的發(fā)育[15]。本實(shí)驗(yàn)的研究結(jié)果發(fā)現(xiàn)在hUCMSCs與A549細(xì)胞共注射至裸鼠皮下形成移植瘤后，A549細(xì)胞的E-cadherin和β-catenin的表達(dá)水平上調(diào)，同時(shí)N-cadherin、vimentin及EMT轉(zhuǎn)錄調(diào)控因子twist的表達(dá)水平下調(diào)，hUCMSCs并沒(méi)有誘導(dǎo)A549細(xì)胞發(fā)生EMT從而增強(qiáng)肺癌的轉(zhuǎn)移能力。由此說(shuō)明hUCMSCs細(xì)胞促進(jìn)肺癌發(fā)生轉(zhuǎn)移可能存在其他的途徑及機(jī)制。

盡管目前利用MSCs做為治療腫瘤的藥物載體已應(yīng)用至包括肺癌[16]、乳腺癌[17]在內(nèi)的多種腫瘤治療的臨床實(shí)踐中。但是本研究結(jié)果提示hUCMSCs可發(fā)生自我分化并表現(xiàn)出惡性表型，同時(shí)促進(jìn)肺癌的生長(zhǎng)及轉(zhuǎn)移，這為我們?cè)诶肕SCs做為載藥工具時(shí)敲響了警鐘。在開發(fā)MSCs作為新的治療肺癌的方法時(shí)，明確在肺癌微環(huán)境下MSCs對(duì)肺癌細(xì)胞生長(zhǎng)、轉(zhuǎn)移的作用就顯得至關(guān)重要，更加需要在其安全性的問(wèn)題上進(jìn)一步研究。

[1] Park JS, Suryaprakash S, Lao YH. Engineering mesenchymal stem cells for regenerative medicine and drug delivery. Methods, 2015, 84: 3-16.

[2] Rosland GV, Svendsen A, Torsvik A, et al. Long-term cultures of bone marrow derived human mesenchymal stem cells frequently undergo spontaneous malignant transforma-tion. Cancer Res, 2009, 69(13): 5331-5339.

[3] Hendijani F, Javanmard SH, Rafiee L. Effect of human Wharton’s jelly mesenchymal stem cell secretome on proliferation, apoptosis and drug resistance of lung cancer cells. Res Pharm Sci, 2015, 10(2): 134-142.

[4] Dominici M, Le Blanc K, Mueller Ⅰ, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006, 8(4): 315-317.

[5] Klopp AH, Gupta A, Spaeth E, et al. Concise review: Dissecting a discrepancy in the literature: do mesenchymal stem cells support or suppress tumor growth? Stem Cells, 2011, 29(1): 11-19.

[6] Suzuki K, Sun R, Origuchi M, et al. Mesenchymal stromal cells promote tumor growth through the enhancement of neovascularization. Mol Med, 2011, 17(7-8): 579-587.

[7] Shin SH, Kim J, Heo SC, et al. Proteomic identification of betaig-h3 as a lysophosphatidic acidinduced secreted protein of human mesenchymal stem cells: paracrine activation of A549 lung adenocarcinoma cells by betaig-h3. Mol Cell Proteomics, 2012, 11(2): M111.012385.

[8] Rhyu JJ, Yun JW, Kwon E, et al. Dual effects of human adipose tissue derived mesenchymal stem cells in human lung adenocarcinoma A549 xenografts and colorectal adenocarcinoma HT-29 xenografts in mice. Oncol Rep, 2015, 34(4): 1733-1744.

[9] Li L, Tian H, Chen Z, et al. Ⅰnhibition of lung cancer cell proliferation mediated by human mesenchymal stem cells. Acta Biochim Biophys Sin, 2011, 43(2): 143-148.

[10] Luo D, Yan X, Liu D, et al. Differential effects of mesenchymal stem cells on a heterogeneous cell population within lung cancer cell lines. Mol Cell Biochem, 2013, 378(1-2): 107-116.

[11] Rubio D, Garcia S, Paz MF, et al. Molecular characterization of spontaneous mesenchymal stem cell transformation. PLoS One, 2008, 3(1): e1398.

[12] Kothari AN, Mi Z, Zapf M. Novel clinical therapeutics targeting the epithelial to mesenchymal transition. Clin Transl Med, 2014, 3: 35.

[13] Zhou HS, Su XF, Fu XL, et al. Mesenchymal stem cells promote pancreatic adenocarcinoma cells invasion by transforming growth factor-β1 induced epithelial-mesenchymal transition. Oncotarget, 2016, 7(27): 41294-41305.

[14] Lv C, Dai H, Sun M, et al. Mesenchymal stem cells induce epithelial mesenchymal transition in melanoma by paracrine secretion of transforming growth factor-β. Melanoma Res, 2017, 27(2): 74-84.

[15] Fischer KR, Durrans A, Lee S, et al. Epithelial-to-mesenchymal transition is notrequired for lung metastasis but contributes to chemoresistance. Nature, 2015, 527(7579): 472-476.

[16] Yan C, Song X, Yu W, et al. Human umbilical cord mesenchymal stem cells delivering sTRAⅠL home to lung cancer mediated by MCP-1/CCR2 axis and exhibit antitumor effects. Tumour Biol, 2016, 37(6): 8425-8435.

[17] Shen CJ, Chan TF, Chen CC, et al. Human umbilical cord matrix-derived stem cells expressing interferon-β gene inhibit breast cancer cells via apoptosis. Oncotarget, 2016, 7(23): 34172-34179.

HUCMSCs undergo malignant transformation and promote the growth as well as the metastasis of A549 subcutaneous xenografts in nude mice

Xiao Heng, Zhao Chengling, Chen Shijun, Zhang Genyan, Chen Yuqing*
(Department of Respiratory Disease, First Affiliated Hospital of Bengbu Medical College, Provincial Key Laboratory of Respiratory disase in Anhui, Bengbu 233000, China)

Objective The present study aimed to investigate the effect of human umbilical cord-derived mesenchymal stem cells (hUCMSCs) on lung cancer xenograft growth and metastasis in nude mice. Methods Twenty-four nude mice were randomly divided into four groups: A549 cells injection alone (group A549), hUCMSCs injection alone (group MSCs), A549 cells co-injection with normal human skin fibroblasts (group A549 and SKs), A549 cells co-injection with hUCMSCs (group A549 and MSCs). HE staining and immunohistochemistry were used to characterize subcutaneous nodules in group MSCs. A549 cells from tumor tissues were sorted out by flow cytometry. The expression of epithelial-mesenchymal transition (EMT) molecular markers in A549 cells was evaluated by quantitative real-time PCR and Western-blotting. Results HUCMSCs undergo spontaneous malignant transformation after being injected into nude mice. The volume of transplanted tumor in group A549 and MSCs was significantly higher than that in group A549 as well as group A549 and SKs. While two nude mice in group A549 and MSCs had liver metastases, none was observed in group A549 or group A549 and SKs. The expression of E-cadherin, β-catenin at mRNA level and β-catenin at protein level were significantlyhigher in group A549 and MSCs than in group A549 and group A549 and SKs. The expression levels of N-cadherin, vimentin and EMT transcription factor Twist were significantly lower in group A549 and MSCs than in group A549 and group A549 and SKs. Conclusion HUCMSCs undergo spontaneous malignant transformation and promote the growth as well as the metastasis of subcutaneous xenografts in nude mice, the mechanism of which is unlikely through inducing EMT in A549 cells.

Human umbilical cord derived mesenchymal stem cells, malignant transformation, A549cells, Lung cancer, EMT

R734

A DOⅠ：10.16705/ j. cnki. 1004-1850.04.005

2017-03-29

2017-07-20

國(guó)家自然科學(xué)基金項(xiàng)目（81172213）；安徽省自然科學(xué)基金青年項(xiàng)目（1608085QH189）；蚌埠醫(yī)學(xué)2015年研究生科研創(chuàng)新計(jì)劃立項(xiàng)項(xiàng)目（Byycxz1502）

肖恒，男( 1991年)，漢族，碩士研究生

*通訊作者(To whom correspondence should be addressed)：bbmccyq@126.com