Notch1對(duì)神經(jīng)膠質(zhì)瘤U251細(xì)胞干性及化療藥物敏感性的調(diào)節(jié)*

張麗柯，咸娜，林玲，龔雨晴，葉志強(qiáng)，鄭志竑△

(福建醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院生物化學(xué)與分子生物學(xué)系，神經(jīng)生物學(xué)研究中心，福建 福州 350108)

[摘要]目的： 探討Notch1對(duì)人膠質(zhì)瘤U251細(xì)胞干性和藥物敏感性的影響。方法: 用高表達(dá)Notch1胞內(nèi)段(Notch1 intracellular domain， NICD1)和Notch1-shRNA慢病毒表達(dá)載體感染體外培養(yǎng)的人膠質(zhì)瘤U251細(xì)胞，Western blot和免疫熒光染色法鑒定高表達(dá)NICD和Notch1沉默細(xì)胞。通過(guò)流式細(xì)胞術(shù)檢測(cè)分析CD133+細(xì)胞的比例、免疫熒光染色法檢測(cè)nestin和GFAP的表達(dá)情況、檢測(cè)腫瘤細(xì)胞球的形成率和SCID小鼠體內(nèi)種植致瘤情況，分析Notch1對(duì)細(xì)胞干性的調(diào)節(jié)。并采用MTT法檢測(cè)各組細(xì)胞對(duì)化療藥物替尼泊苷(VM-26)和卡莫司汀(BCNU)的敏感性。結(jié)果: NICD表達(dá)增加的瘤細(xì)胞干性表型增強(qiáng)，如CD133+細(xì)胞的比例增加、nestin表達(dá)增強(qiáng)而GFAP表達(dá)減弱、腫瘤細(xì)胞球的形成率和SCID小鼠種植致瘤率增加，并伴有對(duì)VM-26和BCNU的敏感性降低。而Notch1基因表達(dá)下調(diào)的瘤細(xì)胞干性表型受到明顯抑制，而對(duì)VM-26和BCNU的敏感性增高。結(jié)論: Notch1高表達(dá)可增加人膠質(zhì)瘤U251細(xì)胞的干性，減弱U251細(xì)胞對(duì)化療藥物的敏感性。

[關(guān)鍵詞]膠質(zhì)瘤U251細(xì)胞； Notch1； 化療藥物敏感性

[中圖分類(lèi)號(hào)]R392.12[文獻(xiàn)標(biāo)志碼]A

doi:10.3969/j.issn.1000-4718.2015.11.004

[文章編號(hào)]1000-4718(2015)11-1950-06

[收稿日期]2015-07-07[修回日期] 2015-09-10

[基金項(xiàng)目]*廣東省醫(yī)學(xué)科研基金資助項(xiàng)目(No.B2013133)

通訊作者△陸 英 Tel: 020-85252526； E-mail: xiaolu1196@163.com; 林東軍 Tel:020-85252227； E-mail: lindongjun0168 @ 163.com

Notch1 regulates stemness and chemotherapeutic sensitivity of human glioma U251 cellsZHANG Li-ke, XIAN Na, LIN Ling, GONG Yu-qing, YE Zhi-qiang, ZHENG Zhi-hong

(ResearchCenterofNeurobiology,DepartmentofBiochemistryandMolecularBiology,SchoolofBasicMedicalSciences,FujianMedicalUniversity,Fuzhou350108,China.E-mail:zhzheng@fjmu.edu.cn)

ABSTRACT[]AIM: To investigate whether Notch1 changes stemness and chemotherapeutic sensitivity in human glioma U251 cells. METHODS: The lentiviral vectors, which expressed Notch1-shRNA or Notch1 intracellular domain (NICD), were transfected into U251 cells . Western blot and immunofluorescence staining were applied to monitor the validity of the cells, down-regulation of Notch1 expression or over-expression of NICD. The proportion of CD133+ cells was analyzed by flow cytometry. The expression of nestin and GFAP was identified by immunofluorescence staining. The formation rate of tumor cell spheres and the implanted tumor growth in SCID mice were observed. MTT assay was performed to evaluate the chemotherapeutic sensitivity to VM-26 and BCNU of the cells with different treatments. RESULTS: Stemness was significantly enhanced in the cells over-expressing NICD. For example, the proportion of CD133+ cells was increased, the expression of nestin was up-regulated, the expression of GFAP was down-regulated, and the formation rate of tumor cell spheres and implanted tumor growth were increased. The chemotherapeutic sensitivity to VM-26 and BCNU of the cells was decreased. In the cells with Notch1 gene down-regulation by RNAi, the stemness was inhibited and chemotherapeutic sensitivity was increased. CONCLUSION: Notch1, which leads to the change of stemness and chemotherapeutic sensitivity in human glioma U251 cells, is likely to be a potential molecular target for treatment of glioma.

[KEY WORDS]Glioma U251 cells; Notch1; Chemotherapeutic sensitivity

神經(jīng)膠質(zhì)瘤是中樞神經(jīng)系統(tǒng)最常見(jiàn)的惡性腫瘤，手術(shù)結(jié)合放療或化療是膠質(zhì)瘤治療的重要手段[1]。目前，該腫瘤的常規(guī)治療預(yù)后仍不盡人意，其中化療效果欠佳的重要因素是化療后殘存的部分腫瘤細(xì)胞的繼發(fā)耐藥性逐漸形成，從而導(dǎo)致化療失效和腫瘤復(fù)發(fā)。而這一部分的細(xì)胞可能就是具有化療抵抗能力的腫瘤干細(xì)胞(cancer stem cells，CSCs)，研究發(fā)現(xiàn)，膠質(zhì)瘤內(nèi)CSCs的比例越高，膠質(zhì)瘤抵抗化療的能力就越強(qiáng)[2]。Notch信號(hào)通路參與了調(diào)節(jié)干細(xì)胞結(jié)構(gòu)和決定細(xì)胞命運(yùn)，在多種癌癥存在Notch信號(hào)通路的異常[3]，而且在結(jié)腸直腸癌、胰腺癌、乳腺癌均參與腫瘤干細(xì)胞群的調(diào)節(jié)。眾多研究已提示，Notch1及其配體在腦膠質(zhì)瘤中呈過(guò)度表達(dá)，將其敲除可抑制腦膠質(zhì)瘤細(xì)胞的增殖和生存。那么在與Notch1關(guān)系極為密切的神經(jīng)膠質(zhì)瘤，是否Notch1也是瘤細(xì)胞產(chǎn)生干細(xì)胞樣表型的重要因素？是否影響到細(xì)胞對(duì)化療藥物的敏感性？本研究利用Notch1基因修飾技術(shù)，旨在對(duì)上述問(wèn)題進(jìn)行探討，為膠質(zhì)瘤的治療提供實(shí)驗(yàn)依據(jù)。

材料和方法

1實(shí)驗(yàn)材料

Notch 1 胞內(nèi)段(Notch1 intracellular domain，NICD)表達(dá)載體pLVX-IRES2-ZsGreen-NICD由本實(shí)驗(yàn)室構(gòu)建和鑒定，簡(jiǎn)稱(chēng)pLVX-NICD，空對(duì)照載體稱(chēng)為pLVX。攜帶ZsGreen的3質(zhì)粒慢病毒表達(dá)系統(tǒng)以及Notch1基因RNA干擾載體由上海交通大學(xué)郭亞博士惠贈(zèng)，RNA干擾載體為pLKO.1-puro，干擾靶序列5’-CCGGGACATCACGGATCATAT-3’(簡(jiǎn)稱(chēng)pLKO-Notch1-ND),以無(wú)義干擾序列5’-CAACAAGATGAAGAGCACCAA-3’作為對(duì)照(簡(jiǎn)稱(chēng)pLKO-Notch1-NC)。

SCID小鼠購(gòu)自上海斯萊克實(shí)驗(yàn)動(dòng)物公司，合格證編號(hào)為2007000579362、2007000574540。

高糖DMEM培養(yǎng)基、DMEM/F12培養(yǎng)基、胎牛血清、無(wú)血清細(xì)胞培養(yǎng)添加劑B27(Gibco)；表皮生長(zhǎng)因子(epidermal growth factor,EGF)、堿性成纖維細(xì)胞生長(zhǎng)因子(basic fibroblast growth factor,bFGF)(PeproTech)；脂質(zhì)體Lipofectamine 2000、MTT(Invitrogen);兔抗人NICD多克隆抗體、兔抗膠質(zhì)細(xì)胞原纖維酸性蛋白(glial fibrillary acidic protein，GFAP)多克隆抗體、Rhodamine標(biāo)記的抗兔IgG抗體、Rhodamine標(biāo)記的抗小鼠IgG抗體(Millipore)；小鼠抗CD133/1(AC133)-PE 抗體(Miltenyi Biotec)；小鼠抗nestin單克隆抗體(R&D)；兔抗GAPDH多克隆抗體(Santa Cruz)；HRP-偶聯(lián)抗兔IgG抗體、HRP-偶聯(lián)抗小鼠IgG抗體(北京中杉金橋公司)；質(zhì)粒抽提純化試劑盒(QIAGEN)；ECL化學(xué)發(fā)光試劑盒(CST)；卡莫司汀[carmustine;即1,3-雙(2-氯乙基)-1-亞硝基脲，1，3-bis(2-chloroethyl)-1-nitrosourea,BCNU]和替尼泊苷(VM-26)購(gòu)自Enzo Life Science；嘌呤霉素(Amresco)；其它試劑購(gòu)于Sigma或碧云天公司。

2方法

2.1高表達(dá)NICD 的U251細(xì)胞和Notch1敲低U251細(xì)胞的獲得按本實(shí)驗(yàn)室常規(guī)方法將pLVX-NICD、pLVX、pLKO-Notch1-ND、pLKO-Notch1-NC質(zhì)粒，分別加用包裝質(zhì)粒pMD2.G和psPAX2，以及Lipo- fectamine 2000轉(zhuǎn)染293T細(xì)胞，進(jìn)行病毒包裝。轉(zhuǎn)染48～72 h收集和濃縮含病毒培養(yǎng)上清。

膠質(zhì)瘤細(xì)胞U251常規(guī)培養(yǎng)于含10%胎牛血清的DMEM培養(yǎng)液，每2～3 d用0.25%胰酶+0.03% EDTA消化傳代1次，感染前1 d接種細(xì)胞于6孔板，每孔約5×104細(xì)胞，第2天大約30% 融合時(shí)，除去培養(yǎng)液，每孔加入含血清培養(yǎng)液600 μL，慢病毒液10 μL，polybreen(1 g/L)4.8 μL，置于培養(yǎng)箱中培養(yǎng)，12 h后吸去含病毒的培養(yǎng)液，更換新鮮的培養(yǎng)液2 mL，置于37 ℃、5% CO2、飽和濕度下繼續(xù)培養(yǎng)48 h。高表達(dá)NICD 的U251細(xì)胞及其相應(yīng)的對(duì)照細(xì)胞，病毒感染48 h后熒光顯微鏡下判定感染成功率。Notch1基因RNA干擾細(xì)胞及其相應(yīng)對(duì)照細(xì)胞，用嘌呤霉素篩選出感染成功的細(xì)胞株。

2.2Notch1表達(dá)水平的鑒定Western blot檢測(cè)按下列步驟進(jìn)行： 用RIPA細(xì)胞裂解液提取細(xì)胞總蛋白，BCA法測(cè)定蛋白濃度。取40 μg蛋白樣品行SDS-PAGE電泳后轉(zhuǎn)膜印跡，經(jīng)封閉液4 ℃作用過(guò)夜后，用兔抗人NICD抗體或GAPDH抗體與膜上抗原結(jié)合，用羊抗兔HRP偶聯(lián)的II抗與其反應(yīng)后，加ECL化學(xué)發(fā)光試劑，在凝膠成像系統(tǒng)(Bio-Rad)下獲取圖像，用ImageJ 2x軟件計(jì)算蛋白條帶的灰度值，以目的條帶/內(nèi)參照條帶灰度比值作為Notch1的相對(duì)表達(dá)量。

免疫熒光染色檢測(cè)按下列步驟進(jìn)行： 將細(xì)胞培養(yǎng)于置入蓋玻片的12孔板，取對(duì)數(shù)生長(zhǎng)期的細(xì)胞，調(diào)整密度后接種，常規(guī)培養(yǎng)48 h。吸除培養(yǎng)液，用預(yù)冷的PBS洗滌3次后，4% 多聚甲醛室溫固定30 min，PBS洗滌后，0.1% Triton X-100細(xì)胞透化處理30 min，按常規(guī)方法用相應(yīng)的抗體進(jìn)行免疫熒光染色，然后再用DAPI(5 mg/L)復(fù)染10 min?？篃晒忖缫悍馄す夤簿劢癸@微鏡(Leica)下觀察、拍照。

2.3各組細(xì)胞干性的檢測(cè)和比較

2.3.1各組細(xì)胞中CD133+細(xì)胞比例的檢測(cè)胰酶消化并收集各組細(xì)胞，計(jì)數(shù)并調(diào)整細(xì)胞濃度為1×109/L。設(shè)空白對(duì)照組及同型對(duì)照組。各組細(xì)胞吸取100 μL至1.5 mL EP管，300×g離心10 min，用100 μL預(yù)冷PBA重懸。實(shí)驗(yàn)組加入1 μL PE標(biāo)記的CD133抗體，同型對(duì)照組加入1 μL PE標(biāo)記的同型對(duì)照抗體，空白對(duì)照組加入1 μL PBA?；靹?，置4 ℃冰箱避光孵育1 h。加入1 mL PBA洗滌細(xì)胞，300×g離心10 min，完全吸棄上清。500 μL PBA重懸各組細(xì)胞，流式細(xì)胞儀檢測(cè)。

2.3.2各組細(xì)胞nestin和GFAP表達(dá)的檢測(cè)分析免疫熒光染色方法同上述，將細(xì)胞培養(yǎng)于置入蓋玻片的12孔板，培養(yǎng)48 h后。用預(yù)冷的PBS洗滌3次后，用4% 多聚甲醛固定，PBS洗滌后，0.1% Triton X-100細(xì)胞透化處理，用抗nestin和GFAP抗體及相應(yīng)的 II抗進(jìn)行免疫熒光染色，再用DAPI復(fù)染。于激光共聚焦顯微鏡下觀察、拍照。

2.3.3各組腫瘤球的形成率實(shí)驗(yàn)取對(duì)數(shù)生長(zhǎng)期的各組細(xì)胞，胰酶消化，收集細(xì)胞，PBS洗滌細(xì)胞2次，1 000 r/min離心4 min。用干細(xì)胞培養(yǎng)液(DMEM/F12培養(yǎng)液含2% B27、20 μg/L EGF和20 μg/L bFGF)重懸各組細(xì)胞，計(jì)數(shù)并調(diào)整細(xì)胞密度為107/L。接種于24孔板，每孔1 mL，即每孔細(xì)胞數(shù)為104個(gè), 每組細(xì)胞接種3孔。37 ℃、5% CO2中培養(yǎng)，隔天半量更換干細(xì)胞培養(yǎng)液，觀察細(xì)胞變化及腫瘤球形成。培養(yǎng)10 d后，顯微鏡下計(jì)數(shù)直徑大于75 μm的腫瘤球個(gè)數(shù)，腫瘤球球形成率(%)=每孔中直徑大于75 μm的腫瘤球的個(gè)數(shù)/每孔中原始接種細(xì)胞的總數(shù)×100%。

2.3.4SCID小鼠皮下種植致瘤實(shí)驗(yàn)SCID小鼠恒溫(25～27 ℃)、恒溫和SPF條件下飼養(yǎng)。取對(duì)數(shù)生長(zhǎng)期的NICD高表達(dá)組細(xì)胞和其對(duì)照組細(xì)胞，制備成單細(xì)胞懸液，每0.2 mL的細(xì)胞數(shù)為1×103、1×105或5×105。小鼠分為6組，每組5只，以碘伏消毒SCID鼠腋窩皮膚，用無(wú)菌注射器(6號(hào)針頭)抽吸0.2 mL細(xì)胞接種于腋窩區(qū)皮下，繼續(xù)飼養(yǎng)。共觀察10周，然后采用頸髓離斷法處死。完整地剝出瘤體、去除表面脂肪組織、稱(chēng)重，取游標(biāo)卡尺分別測(cè)量腫瘤長(zhǎng)徑和短徑(mm)，按公式計(jì)算腫瘤體積，體積(mm3)=4/3×π×(長(zhǎng)徑/2)×(短徑/2)2。

2.4MTT法檢測(cè)各組細(xì)胞的多藥耐藥性選用2種化療藥物VM-26和BCNU，參考其它文獻(xiàn)及本實(shí)驗(yàn)室經(jīng)驗(yàn)，分別用1.5 μmol/L VM-26和150 μmol/L BCNU處理細(xì)胞。取對(duì)數(shù)生長(zhǎng)期的細(xì)胞，以每孔7 000個(gè)細(xì)胞接種于96孔板。37 ℃、5%CO2中培養(yǎng)12 h后，按確定濃度加入藥物，每組細(xì)胞做6個(gè)復(fù)孔。細(xì)胞加入藥物培養(yǎng)48 h后，每孔加入20 μL 5 g/L的MTT溶液，培養(yǎng)4 h后小心吸棄孔內(nèi)培養(yǎng)上清液，每孔加入150 μL DMSO，搖床上振蕩10 min，使結(jié)晶充分溶解。490 nm波長(zhǎng)測(cè)吸光度(A)值，計(jì)算4組細(xì)胞的生存率。細(xì)胞生存率(%)=(給藥組-空白對(duì)照組)/(陰性對(duì)照組-空白對(duì)照組)×100%。

3統(tǒng)計(jì)學(xué)處理

采用SPSS 17.0統(tǒng)計(jì)分析軟件，計(jì)量數(shù)據(jù)檢驗(yàn)正態(tài)性和方差齊性后，以均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示，各組間均數(shù)采用多樣本單因素方差分析，兩兩組間比較采用SNK-q檢驗(yàn),以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié)果

1獲得NICD高表達(dá)和Notch1基因敲低的U251細(xì)胞

按照前述轉(zhuǎn)染和篩選方法，共獲得4組細(xì)胞：NICD高表達(dá)U251細(xì)胞(pLVX-NICD)、高表達(dá)對(duì)照空載細(xì)胞(pLVX)、Notch1基因敲低的U251細(xì)胞(pLKO-Notch1-ND)和RNA干擾對(duì)照細(xì)胞(pLKO-Notch1-NC)。采用Western blot和免疫熒光染色法檢測(cè)結(jié)果均顯示，NICD高表達(dá)和Notch1基因敲低的U251細(xì)胞中Notch1的表達(dá)出現(xiàn)明顯變化，見(jiàn)圖1、2。

Figure 1.The protein expression of Notch1 in each group detected by Western blot. Mean±SD.n=3.**P<0.01vspLVX;##P<0.01vspLKO-Notch1-NC.

圖1Western blot檢測(cè)各組細(xì)胞Notch1蛋白的表達(dá)水平

2Notch1表達(dá)對(duì)U251細(xì)胞干性表型的調(diào)節(jié)

2.1Notch1表達(dá)影響U251細(xì)胞CD133+表型流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示NICD高表達(dá)的U251細(xì)胞中CD133+細(xì)胞百分率明顯高于對(duì)照組；而Notch1基因RNA干擾的U251細(xì)胞CD133+細(xì)胞百分率與對(duì)照組相比則降低，見(jiàn)圖3。

Figure 2.The expression of NICD (red) in each group detected by immunofluorescence staining.

圖2免疫熒光染色法鑒定各組細(xì)胞NICD的表達(dá)

Figure 3.The percentage of CD133+cells in each group. Mean±SD.n=3.**P<0.01vspLVX；##P<0.01vspLKO-Notch1-NC.

圖3各組細(xì)胞中CD133+細(xì)胞的百分率

2.2Notch1表達(dá)影響U251細(xì)胞中nestin和GFAP的表達(dá)免疫熒光強(qiáng)度檢測(cè)結(jié)果顯示，NICD高表達(dá)細(xì)胞的nestin蛋白表達(dá)明顯強(qiáng)于其對(duì)照組，而GFAP表達(dá)則明顯減弱；Notch1基因RNA干擾組細(xì)胞的nestin表達(dá)明顯減弱，而GFAP表達(dá)則明顯增強(qiáng)，見(jiàn)圖4、5及表1。

2.3Notch1表達(dá)影響U251細(xì)胞腫瘤球的形成率NICD高表達(dá)組的腫瘤球形成率，明顯高于其對(duì)照組，為對(duì)照組2.05倍。而Notch1基因RNA干擾組的腫瘤球形成率則明顯低于對(duì)照組,見(jiàn)圖6。

2.4NICD高表達(dá)的U251細(xì)胞在SCID小鼠中的成瘤能力增加觀察移植的SCID小鼠10周，發(fā)現(xiàn)接種1×103和1×105個(gè)細(xì)胞的NICD高表達(dá)組和對(duì)照組均未長(zhǎng)瘤；接種5×105個(gè)細(xì)胞的NICD高表達(dá)組5只SCID小鼠全部長(zhǎng)瘤，瘤大小為(120.64±42.51)mm3，瘤重(0.21±0.11) g，而對(duì)照組5只SCID小鼠中只有1只長(zhǎng)瘤，瘤大小為78 mm3，瘤重0.10 g，見(jiàn)圖7。

Figure 4.The expression of nestin (red) in each group detected by immunofluorescence staining.

圖4各組細(xì)胞nestin的表達(dá)情況

Figure 5.The expression of GFAP (red) in each group detected by immunofluorescence staining.

圖5各組細(xì)胞GFAP的表達(dá)情況

表1各組細(xì)胞中Nestin及GFAP的表達(dá)情況

Table 1.The expression of Nestin and GFAP in each group (Mean±SD.n=3)

GroupValueofimmunofluorescenceNestinGFAPpLVX36.23±7.8425.55±3.73pLVX-NICD60.75±11.98**12.06±1.79**pLKO-Notch1-NC34.40±6.5631.36±5.64pLKO-Notch1-ND22.40±6.48##89.00±15.28##

**P<0.01vspLVX；##P<0.01vspLKO-Notch1-NC

3Notch1表達(dá)影響U251細(xì)胞對(duì)化療藥物的敏感性

MTT法實(shí)驗(yàn)結(jié)果顯示，在相同濃度VM-26或BCNU作用下NICD高表達(dá)組細(xì)胞生存率明顯高于其對(duì)照組；而Notch1基因RNA干擾組細(xì)胞生存率則明顯低于干擾對(duì)照組，見(jiàn)圖8。

Figure 6.The formation rate of tumor spheres in each goup cultured with stem cell medium. Mean±SD.n=3.*P<0.05vspLVX;#P<0.05vspLKO-Notch1-NC.

圖6各組細(xì)胞在干細(xì)胞培養(yǎng)液培養(yǎng)后腫瘤球形成率的比較

討論

已有研究表明，Notch信號(hào)通路與膠質(zhì)瘤發(fā)生和發(fā)展有關(guān)，在膠質(zhì)瘤中發(fā)現(xiàn)Notch信號(hào)呈過(guò)表達(dá)，它在膠質(zhì)瘤細(xì)胞增殖分化及凋亡等中起著重要作用。本研究通過(guò)轉(zhuǎn)基因調(diào)節(jié)Notch1信號(hào)的方式，探討了Notch1表達(dá)對(duì)膠質(zhì)瘤U251細(xì)胞的干細(xì)胞樣特性和對(duì)化療藥物VM-26、BCNU的敏感性的調(diào)節(jié)，結(jié)果顯示：當(dāng)Notch1信號(hào)增強(qiáng)時(shí)，U251細(xì)胞表現(xiàn)較強(qiáng)的腫瘤干細(xì)胞的特征，如CD133+瘤細(xì)胞增多、細(xì)胞中nestin表達(dá)增加而GFAP表達(dá)減弱、腫瘤球形成率增加、SCID小鼠皮下移植成瘤性增強(qiáng)，而且對(duì)VM-26和BCNU的抗性也都明顯增強(qiáng)。這表明在膠質(zhì)瘤研究和治療中應(yīng)重視Notch1表達(dá)水平的檢測(cè)，它可望作為調(diào)節(jié)膠質(zhì)瘤化療敏感性的潛在靶點(diǎn)。

克服腫瘤細(xì)胞對(duì)化療藥物的耐藥性是提高腫瘤療效的重要內(nèi)容，CSCs概念的提出為腫瘤細(xì)胞耐藥研究拓展出一片新的空間，CSCs被認(rèn)為在藥物抵抗及腫瘤的轉(zhuǎn)移過(guò)程中發(fā)揮了重要的作用，因?yàn)镃SCs能表達(dá)藥物轉(zhuǎn)運(yùn)蛋白及增強(qiáng)DNA修復(fù)系統(tǒng)從而使CSCs產(chǎn)生耐藥性[4]。有證據(jù)表明Notch信號(hào)通路與CSCs相關(guān)，例如，該通路參與調(diào)節(jié)結(jié)直腸癌的干細(xì)胞群[5]，也在維持胰腺癌CSCs樣表型中發(fā)揮重要作用[6]。Farnie等[7]證明了Notch表達(dá)上調(diào)與乳腺癌干細(xì)胞相關(guān)的證據(jù)，說(shuō)明Notch和乳腺癌干細(xì)胞樣特征有關(guān)。Zhang等[8]的研究發(fā)現(xiàn)，Notch1的激活形式NICD在SHG-44和U87細(xì)胞系中可被檢測(cè)到，并且這2種腦膠質(zhì)瘤細(xì)胞株的增殖快于未檢測(cè)到NICD的膠質(zhì)瘤細(xì)胞株；SHG-44細(xì)胞中NICD的過(guò)度表達(dá)促進(jìn)了SHG-44細(xì)胞的生長(zhǎng)和集落形成；這些集落表達(dá)巢蛋白，為具有神經(jīng)干細(xì)胞表型的細(xì)胞。Hulleman等[9]發(fā)現(xiàn)轉(zhuǎn)錄因子HEY1作為Notch信號(hào)通路的一個(gè)下游靶分子，在膠質(zhì)瘤中顯著上調(diào)，且多形性膠質(zhì)母細(xì)胞瘤中HEY1的表達(dá)與腫瘤分級(jí)和生存相關(guān)，而通過(guò)RNA干擾技術(shù)沉默HEY1，將使組織培養(yǎng)中的膠質(zhì)母細(xì)胞瘤增殖減弱。在膠質(zhì)瘤干細(xì)胞中，干擾素調(diào)節(jié)因子7可抑制白細(xì)胞介素6-Janus激酶信號(hào)轉(zhuǎn)導(dǎo)與Jagged-Notch信號(hào)通路活化，導(dǎo)致膠質(zhì)瘤干細(xì)胞標(biāo)志物表達(dá)下降，腫瘤細(xì)胞球形成能力及致瘤性降低[10]。本實(shí)驗(yàn)通過(guò)轉(zhuǎn)基因過(guò)表達(dá)NICD進(jìn)一步證實(shí)了Notch1信號(hào)高表達(dá)能夠使膠質(zhì)瘤細(xì)胞呈干細(xì)胞樣的表型。

Figure 7.Over-expression of NICD enhanced the formation of xenograft tumor in SCID mice.

圖7高表達(dá)NICD增加U251細(xì)胞在SCID小鼠移植成瘤

Figure 8.The effects of chemotherapeutics on the survival rate of the cells with different treatments. Mean±SD.n=3.**P<0.01vsplvx;##P<0.01vspLKO-Notch1-NC.

圖8各組瘤細(xì)胞在化療藥物作用下的生存率

化療是癌癥治療中重要的治療手段。然而，因?yàn)樗幬锟剐缘淖饔?，化療不能消滅所有的腫瘤細(xì)胞，這也是腫瘤復(fù)發(fā)最主要的原因。最近，有研究報(bào)道Notch信號(hào)通路與藥物抗性有關(guān)。更重要的是，Notch調(diào)節(jié)腫瘤干細(xì)胞的形成，促進(jìn)細(xì)胞獲得上皮-間質(zhì)轉(zhuǎn)變表型，它和藥物抗性顯著相關(guān)[11-12]。許多研究發(fā)現(xiàn)，在乳腺癌、胰腺癌、結(jié)腸癌等許多腫瘤中，抑制Notch1的表達(dá)，會(huì)提高腫瘤細(xì)胞對(duì)化療藥物的敏感性[13-15]。沉默Notch1基因可通過(guò)激活JNK1信號(hào)通路活化p53,促進(jìn)PUMA和NOXA蛋白表達(dá),進(jìn)而通過(guò)線粒體途徑導(dǎo)致人乳腺癌MCF-7細(xì)胞凋亡[16]。在膠質(zhì)瘤中，抑制Notch信號(hào)通路可增強(qiáng)CD133+膠質(zhì)瘤細(xì)胞對(duì)化療藥物替莫唑胺的敏感性[17]。本實(shí)驗(yàn)結(jié)果也提示，當(dāng)Notch1信號(hào)增強(qiáng)時(shí)，U251細(xì)胞表現(xiàn)較強(qiáng)的腫瘤干細(xì)胞的特征，而且對(duì)VM-26和BCNU的抗性也明顯增強(qiáng)。據(jù)研究報(bào)道腫瘤耐藥最常見(jiàn)的原因是表達(dá)一種或更多種能量依賴(lài)的轉(zhuǎn)運(yùn)蛋白(它可以發(fā)現(xiàn)細(xì)胞中的化療藥物并將它排出)、藥物誘導(dǎo)的凋亡及藥物誘導(dǎo)的解毒功能失常[18]。例如，ABC藥物轉(zhuǎn)運(yùn)蛋白可以保護(hù)腫瘤細(xì)胞免受化療藥物的傷害。ABC轉(zhuǎn)運(yùn)蛋白將毒性藥物排出癌細(xì)胞，使藥物殺死腫瘤細(xì)胞的作用下降。ABCC1(多藥耐藥相關(guān)蛋白1,multidrug resistance-associated protein 1.MRP1)、ABCB1(P-糖蛋白)和ABCG2(乳腺癌耐藥蛋白)3種ABC轉(zhuǎn)運(yùn)蛋白已被鑒定[19]。已有研究發(fā)現(xiàn)，MRP1與神經(jīng)膠質(zhì)瘤的耐藥性有關(guān)。在神經(jīng)膠質(zhì)瘤組織和膠質(zhì)瘤細(xì)胞株中均檢測(cè)到MRP1的表達(dá)[20]。Calatozzolo等[21]發(fā)現(xiàn)在人腦膠質(zhì)瘤組織切片中，MRP1的陽(yáng)性率達(dá)70%，且膠質(zhì)瘤Ⅱ、Ⅲ、Ⅳ級(jí)沒(méi)有明顯的等級(jí)差異性，在原發(fā)性和復(fù)發(fā)性膠質(zhì)瘤中也沒(méi)有顯著區(qū)別。Spiegl-Kreinecker等[22]的研究表明隨著膠質(zhì)瘤惡性級(jí)別的增加，MRP1表現(xiàn)出陽(yáng)性率逐漸增加的趨勢(shì)。

本實(shí)驗(yàn)研究表明Notch1信號(hào)的增強(qiáng)可以促進(jìn)腫瘤干細(xì)胞樣細(xì)胞的形成與增殖，而且也影響神經(jīng)膠質(zhì)瘤細(xì)胞對(duì)化療藥物的敏感性。因此，Notch1表達(dá)水平可作為研判膠質(zhì)瘤的干細(xì)胞性和預(yù)測(cè)化療敏感性的指標(biāo)，干預(yù)Notch1信號(hào)可望成為膠質(zhì)瘤治療的干預(yù)靶點(diǎn)，以克服其藥物抗性作用和殺死膠質(zhì)瘤干細(xì)胞樣細(xì)胞，提高對(duì)膠質(zhì)瘤的化療效果。

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(責(zé)任編輯： 盧萍， 羅森)