·論著·
HIF-1α@Fe3O4納米顆粒標(biāo)記對(duì)胰腺癌干細(xì)胞增殖及凋亡的影響
謝曉東朱海濤吳熒熒黃文斯王穎吳愷迪張禮榮王冬青
【摘要】目的探討缺氧誘導(dǎo)因子-1α螯合四氧化三鐵(HIF-1α@Fe3O4)納米顆粒標(biāo)記胰腺癌干細(xì)胞的可行性,并觀察胰腺癌干細(xì)胞被標(biāo)記后的增殖及凋亡變化。方法采用無(wú)血清培養(yǎng)法培養(yǎng)胰腺癌細(xì)胞株P(guān)ANC1干細(xì)胞,將獲得的干細(xì)胞與5、15、45、135 μg/ml的HIF-1α@Fe3O4納米顆粒共孵育24 h,采用普魯士藍(lán)染色法觀察胰腺癌干細(xì)胞的標(biāo)記率,采用四甲基偶氮唑藍(lán)比色法檢測(cè)胰腺癌干細(xì)胞的增殖活性,采用流式細(xì)胞儀檢測(cè)胰腺癌干細(xì)胞的凋亡。結(jié)果HIF-1α@Fe3O4納米顆粒與胰腺癌干細(xì)胞共培養(yǎng)24 h后的胞質(zhì)內(nèi)可見(jiàn)不同程度的藍(lán)色鐵染顆粒,且隨著HIF-1α@Fe3O4濃度的增加而增加,濃度為45 μg/ml時(shí)標(biāo)記率達(dá)100%。未標(biāo)記的干細(xì)胞及5、15、45、135 μg/ml HIF-1α@Fe3O4標(biāo)記的胰腺癌干細(xì)胞的細(xì)胞凋亡率分別為(3.76±0.96)%、(4.38±0.84)%、(4.36±1.22)%、(3.80±0.11)%、(4.78±0.98)%,差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05);未標(biāo)記的干細(xì)胞與各濃度納米顆粒標(biāo)記的干細(xì)胞細(xì)胞存活率的差異亦無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。結(jié)論HIF-1α@Fe3O4納米顆??捎糜跇?biāo)記胰腺癌干細(xì)胞,且不影響胰腺癌干細(xì)胞的增殖及凋亡。
【關(guān)鍵詞】胰腺腫瘤;缺氧誘導(dǎo)因子-1α;四氧化三鐵;干細(xì)胞;細(xì)胞增殖;細(xì)胞凋亡
DOI:10.3760/cma.j.issn.1674-1935.2015.01.006
基金項(xiàng)目:江蘇省自然基金(BK2011487);鎮(zhèn)江市社會(huì)發(fā)展項(xiàng)目(SH2013026);江蘇大學(xué)大學(xué)生科研立項(xiàng)資助項(xiàng)目(12A209)
收稿日期:(2014-09-16)
Effect of HIF-1α@Fe3O4nanoparticles tag on proliferation and apoptosis of pancreatic cancer stem cellsXieXiaodong,ZhuHaitao,WuYingying,HuangWensi,WangYing,WuKaidi,ZhangLirong,WangDongqing.DepartmentofRadiology,AffiliatedHospitalofSuzhouUniversity,Zhenjiang212001,China
Correspondingauthor:ZhuHaitao,Email:zhht25@163.com
Abstract【】ObjectiveTo explore the feasibility of HIF-1α@Fe3O4 nanoparticles tag of pancreatic cancer stem cells, and to investigate the proliferation and apoptosis rate of the labeled pancreatic cancer stem cells. MethodsPANC1 pancreatic cancer stem cells were cultured in serum-free medium, and then the pancreatic cancer stem cells were co-cultured with different concentrations of HIF-1α@Fe3O4(5 、15、45 、135 μg/ml) for 24 h. Prussian blue staining method was used to determine the labeling efficiency. MTT method was used to analyze the proliferation of pancreatic cancer stem cells. Also, flow cytometry was used to evaluate apoptosis. ResultsPrussian blue staining showed various number of blue-stained iron particles in the cells after co-culture of HIF-1α@Fe3O4 nanoparticles and pancreatic cancer stem cells. And the number of blue-stained iron particles increased with the concentration of HIF-1α@Fe3O4, and the labeled rate reached 100% with the concentration of 45 μg/ml or above. The apoptosis rates of stem cells of 5, 15, 45, 135 μg/ml HIF-1α@Fe3O4 tag and stem cells of without tag were (4.38±0.84)%, (4.36±1.22)%, (3.8±0.11)%, (4.78±0.98)%, (3.76±0.96)%, and the difference between the two groups was not statistically significant (P>0.05); and the survival rates between the two groups was not statistically significant (P>0.05), either. ConclusionsHIF-1α@Fe3O4 nanoparticles can be used for tagging pancreatic cancer stem cells, and they do not affect the proliferation and apoptosis of pancreatic cancer stem cells.
【Key words】Pancreatic neoplasms;Hypoxia-inducible factor 1, alpha subunit;Ferrosoferric oxide;Stem cells;Cells proliferation;Apoptosis
作者單位:212001鎮(zhèn)江,江蘇大學(xué)附屬醫(yī)院影像科
通信作者:朱海濤,Email:zhht25@163.com
缺氧誘導(dǎo)因子-1α(hypoxiainduciblefactor-1α,HIF-1α)是在缺氧誘導(dǎo)的細(xì)胞核抽提物中發(fā)現(xiàn)的,最先由Semenza等[1]報(bào)道。它是缺氧誘導(dǎo)基因轉(zhuǎn)錄和信息傳遞中最重要的因子,在調(diào)控有氧能量代謝、腫瘤血管生成、侵襲轉(zhuǎn)移、細(xì)胞凋亡及放療和化療抵抗等生物學(xué)效應(yīng)中都發(fā)揮著重要作用[2-3]。磁共振成像(MRI)是一種無(wú)創(chuàng)、信號(hào)敏感和組織分辨率高的顯像方式,納米磁粒子Fe3O4因良好的生物相容性和MRI信號(hào)敏感性而常用于離體和活體的生物學(xué)研究[4]。本研究根據(jù)已知的HIF-1α可識(shí)別的核酸序列5′-RCGTG-3′設(shè)計(jì)、合成核酸序列,并使其螯合于納米磁粒子Fe3O4上合成HIF-1α@Fe3O4,用于探索該納米顆粒對(duì)胰腺癌干細(xì)胞標(biāo)記的可行性以及對(duì)細(xì)胞增殖、凋亡的影響,為后期應(yīng)用于磁共振靶向成像及靶向治療奠定基礎(chǔ)。
材料與方法
一、試劑及材料
胎牛血清(FBS)、細(xì)胞培養(yǎng)基DMEM/F12、培養(yǎng)基添加劑B27、表皮生長(zhǎng)因子(EGF)、堿性成纖維母細(xì)胞生長(zhǎng)因子(bFGF)、胰島素及轉(zhuǎn)鐵蛋白均購(gòu)于美國(guó)Gibco公司。胰蛋白酶購(gòu)于Amersco 公司。 二甲亞砜(DMSO)以及四甲基偶氮唑藍(lán)(MTT)購(gòu)于Sigma 公司,Annexin V-PI細(xì)胞凋亡檢測(cè)試劑盒購(gòu)自南京凱基生物公司。HIF-1α螯合于納米磁粒子Fe3O4的合成物HIF-1α@Fe3O4由江蘇大學(xué)化工學(xué)院合成。胰腺癌細(xì)胞系PANC1購(gòu)于中國(guó)科學(xué)院上海細(xì)胞研究所。
二、方法
1.胰腺癌干細(xì)胞培養(yǎng):PANC1細(xì)胞常規(guī)培養(yǎng)、傳代。培養(yǎng)三代到細(xì)胞狀態(tài)穩(wěn)定后采取本課題組前期實(shí)驗(yàn)采用的無(wú)血清培養(yǎng)方式,并在培養(yǎng)液中加入20 ng/ml EGF、20 ng/ml B27及bFGF、5 μg/ml胰島素、2.75 μg/ml轉(zhuǎn)鐵蛋白等培養(yǎng)胰腺癌干細(xì)胞[5]。
2.HIF-1α@Fe3O4標(biāo)記胰腺癌干細(xì)胞:收集胰腺癌干細(xì)胞,用培養(yǎng)液吹打成單細(xì)胞懸液,調(diào)整細(xì)胞密度為5×105/ml,接種于6孔板,每孔2 ml。4個(gè)孔中分別加入5、15、45、135 μg/ml的HIF-1α@Fe3O4納米顆粒,剩余2孔加入等體積的培養(yǎng)基作為對(duì)照,實(shí)驗(yàn)設(shè)置3組6孔板。6孔板置培養(yǎng)箱中孵育24 h后取出,棄培養(yǎng)上清,用預(yù)冷的PBS清洗3遍去除游離的HIF-1α@Fe3O4,再用4%戊二醛溶液固定細(xì)胞約20 min,用預(yù)冷的PBS洗滌細(xì)胞3遍,加入Perl試劑(2%亞鐵氰化鉀和6%鹽酸混合溶液)孵育30 min,用PBS再次清洗細(xì)胞3遍,最后用1%核固紅水溶液復(fù)染5 min,用蒸餾水洗去多余的核固紅,置顯微鏡下觀察,用Image J軟件分析圖片,結(jié)果以細(xì)胞內(nèi)顆粒數(shù)表示。
3.細(xì)胞增殖檢測(cè):取上述標(biāo)記的各組胰腺癌干細(xì)胞及未標(biāo)記的胰腺癌干細(xì)胞制備成單個(gè)細(xì)胞懸液,接種于96孔板中,每孔200 μl,1×105個(gè)細(xì)胞,以只加培養(yǎng)基孔作為空白對(duì)照。置37℃、5% CO2及飽和濕度的培養(yǎng)箱中培養(yǎng)1~6 d,每個(gè)時(shí)間點(diǎn)設(shè)3個(gè)復(fù)孔。到培養(yǎng)時(shí)間時(shí)在每孔中加入5 mg/ml MTT溶液20 μl,繼續(xù)孵育4 h,棄上清液,加入150 μl DMSO,置搖床振蕩10 min,上酶標(biāo)儀檢測(cè)波長(zhǎng)490 nm處的吸光度值(A490值),以空白對(duì)照孔調(diào)零,繪制細(xì)胞生長(zhǎng)曲線。
4.細(xì)胞凋亡測(cè)定:取標(biāo)記的各組胰腺癌干細(xì)胞和未標(biāo)記的胰腺癌干細(xì)胞,棄去上清,用3 ml預(yù)冷的PBS漂洗2次,將細(xì)胞重懸于預(yù)冷的70%乙醇中4℃固定1~2 h,離心棄去固定液,用3 ml PBS重懸細(xì)胞,400目篩網(wǎng)過(guò)濾、離心收集細(xì)胞,加含有1 μl PI和5 μl AnnexinV 的緩沖液,室溫下放置15 min,加400 μl的緩沖液混勻,上流式細(xì)胞儀檢測(cè)細(xì)胞凋亡。
三、統(tǒng)計(jì)學(xué)處理
結(jié)果
一、HIF-1α@Fe3O4標(biāo)記胰腺癌干細(xì)胞的效率
在無(wú)血清培養(yǎng)基條件下腫瘤細(xì)胞呈懸浮半貼壁的球形生長(zhǎng),細(xì)胞表面CD133高表達(dá)(圖1)。HIF-1α@Fe3O4納米顆粒與胰腺癌干細(xì)胞共培養(yǎng)24 h后,胞質(zhì)內(nèi)可見(jiàn)不同程度的藍(lán)色鐵染顆粒,且隨著HIF-1α@Fe3O4濃度的增加而增加。當(dāng)濃度為45 μg/ml HIF-1α@Fe3O4時(shí),標(biāo)記率達(dá)100%,而未標(biāo)記的干細(xì)胞的胞質(zhì)內(nèi)未見(jiàn)藍(lán)色鐵染顆粒(圖2)。
圖1 無(wú)血清培養(yǎng)基條件下的胰腺癌干細(xì)胞 1A:細(xì)胞呈半貼壁的球形生長(zhǎng);1B:細(xì)胞表面CD 133高表達(dá)(免疫熒光 ×200)
圖2 5(2A)、15(2B)、45(2C)、135 (2D)μg/ml HIF-1α@Fe 3O 4納米顆粒標(biāo)記的胰腺癌干細(xì)胞
二、胰腺癌干細(xì)胞的增殖
隨著時(shí)間的增加,各濃度標(biāo)記組干細(xì)胞的A490值均逐漸增加,同一時(shí)間點(diǎn)未標(biāo)記組及各濃度標(biāo)記組之間的A490值差異無(wú)統(tǒng)計(jì)學(xué)意義(t值分別為1.004、2.417、0.348、0.174、0.076、0.295,P值均>0.05),表明HIF-1α@Fe3O4納米顆粒毒性較小,不影響干細(xì)胞的活力(圖3)。
三、胰腺癌干細(xì)胞的凋亡
未標(biāo)記的胰腺癌干細(xì)胞及5、15、45、135 μg/ml HIF-1α@Fe3O4標(biāo)記的胰腺癌干細(xì)胞早期及晚期的總細(xì)胞凋亡率分別為(3.76±0.96)%、(4.38±0.84)%、(4.36±1.22)%、(3.80±0.11)%、(4.78±0.98)%(圖4),各組間的差異無(wú)統(tǒng)計(jì)學(xué)意義(t=1.807,P>0.05),表明 HIF-1α@Fe3O4納米顆粒不影響胰腺癌干細(xì)胞的正常凋亡。
討論
眾所周知,腫瘤干細(xì)胞在腫瘤的發(fā)生、發(fā)展以及復(fù)發(fā)方面起著決定性的作用[6-9],因此對(duì)干細(xì)胞的監(jiān)測(cè)及檢測(cè)對(duì)于患者的治療及預(yù)后判斷有著至關(guān)重要的作用。Fe3O4納米粒子對(duì)MRI的顯像具有信號(hào)強(qiáng)度變化顯著及對(duì)人體無(wú)傷害的優(yōu)勢(shì)[10-12],因此采用Fe3O4納米粒子標(biāo)記干細(xì)胞,為其成像提供新的方法與思路。
圖3 不同濃度HIF-1α@Fe 3O 4納米顆粒標(biāo)記的胰腺癌干細(xì)胞的生長(zhǎng)曲線
圖4 未標(biāo)記(4A)及5、15、45、135 μg/ml(4B~4E)HIF-1α@Fe 3O 4納米顆粒標(biāo)記的胰腺癌干細(xì)胞的細(xì)胞凋亡率
HIF-1α是缺氧條件下的一種核心轉(zhuǎn)錄因子,參與多種腫瘤細(xì)胞的轉(zhuǎn)錄以及新生血管形成和轉(zhuǎn)移過(guò)程[13-14],增強(qiáng)腫瘤細(xì)胞在缺氧條件下的生存能力。Zhong等[15]檢測(cè)了179例19種腫瘤標(biāo)本的HIF-1α表達(dá),結(jié)果顯示包括胰腺癌在內(nèi)的13種腫瘤的HIF-1α均有不同程度的表達(dá),而在良性腫瘤和癌旁正常組織中則未見(jiàn)表達(dá)。Akakurat等[16]報(bào)道,在所選取的20種胰腺癌細(xì)胞株中均有HIF-1α表達(dá)。Semenza等[17]發(fā)現(xiàn)5′-RCGTG-3′是HIF-1α可識(shí)別的基因序列。由于HIF在腫瘤細(xì)胞中高表達(dá),因此在納米磁粒子上修飾含有5′-RCGTG-3′核酸序列不僅可以與高表達(dá)HIF-1α的腫瘤細(xì)胞結(jié)合,更可以在磁共振上實(shí)時(shí)監(jiān)測(cè)腫瘤細(xì)胞的發(fā)生和發(fā)展,為早期檢測(cè)和治療腫瘤提供了新的檢測(cè)方法和治療思路。
本研究結(jié)果顯示,HIF-1α@Fe3O4成功地標(biāo)記到胰腺癌干細(xì)胞,細(xì)胞標(biāo)記率隨HIF-1α@Fe3O4濃度的增加而增加。HIF-1α@Fe3O4標(biāo)記后胰腺癌干細(xì)胞的增殖及凋亡均未受影響,為后期MRI檢測(cè)胰腺癌干細(xì)胞提供了實(shí)驗(yàn)基礎(chǔ)。同時(shí),根據(jù)轉(zhuǎn)錄因子HIF-1α識(shí)別的序列可設(shè)計(jì)出更加特異性結(jié)合的探針,以提高診斷的靈敏度和特異性。
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(本文編輯:屠振興)