pEGFP-C1-HSP27真核表達(dá)載體的構(gòu)建及其穩(wěn)定轉(zhuǎn)染人胰腺癌SW1990細(xì)胞株的篩選

張松 陳敏 黃淑玲 許春紅 李建琦 王軍 鄒曉平

·論著·

pEGFP-C1-HSP27真核表達(dá)載體的構(gòu)建及其穩(wěn)定轉(zhuǎn)染人胰腺癌SW1990細(xì)胞株的篩選

張松 陳敏 黃淑玲 許春紅 李建琦 王軍 鄒曉平

目的構(gòu)建表達(dá)熱休克蛋白27(HSP27)的攜帶增強(qiáng)型綠色熒光蛋白(EGFP)的真核載體，建立穩(wěn)定表達(dá)HSP72的人胰腺癌SW1990細(xì)胞株。方法采用RT-PCR法從SW1990細(xì)胞中擴(kuò)增出帶有BamH I 、HindⅢ酶切位點(diǎn)的HSP27基因片段，插入真核表達(dá)載體pEGFP-C1，鑒定正確后用重組質(zhì)粒轉(zhuǎn)染SW1990細(xì)胞，并篩選穩(wěn)轉(zhuǎn)細(xì)胞株。熒光顯微鏡觀察HSP72的定位，RT-PCR、蛋白質(zhì)印跡法檢測(cè)轉(zhuǎn)染細(xì)胞HSP27的表達(dá)。結(jié)果雙酶切法鑒定和測(cè)序證實(shí)重組載體pEGFP-C1-HSP27的DNA序列完全正確，并成功轉(zhuǎn)染SW1990細(xì)胞，篩選獲得穩(wěn)轉(zhuǎn)細(xì)胞株。熒光顯微鏡見(jiàn)EGFP主要分布在胞質(zhì)中，轉(zhuǎn)染細(xì)胞的HSP27mRNA表達(dá)明顯增加(1.458±0.160比0.897±0.051，P<0.05),且表達(dá)HSP27與EGFP的融合蛋白。結(jié)論成功構(gòu)建真核表達(dá)載體pEGFP-C1-HSP27，并篩選獲得穩(wěn)轉(zhuǎn)的細(xì)胞株。

胰腺腫瘤； 熱休克蛋白類(lèi)； 轉(zhuǎn)染； SW1990細(xì)胞株； 基因攜帶體

以吉西他濱作為核心藥物的化療已成為胰腺癌治療的重要策略并可使患者受益[1-2]，但胰腺癌化療耐藥已經(jīng)成為當(dāng)前亟待解決的難題。熱休克蛋白27(heat shock protein 27，HSP27)，又名熱休克蛋白B1(heat shock protein B1，HSPB1)，是熱休克蛋白家族中的小分子熱休克蛋白亞家族的重要一員。目前，已有研究證實(shí)HSP27在食管、胃、肝、胰腺、結(jié)腸、乳腺、前列腺、卵巢、膀胱等多種癌細(xì)胞中異常高表達(dá)[3-5]。同時(shí)，多項(xiàng)研究證實(shí)在胃癌、直腸癌、肝癌及前列腺癌等腫瘤中HSP27的高表達(dá)與腫瘤轉(zhuǎn)移、不良預(yù)后等相關(guān)[6-9]。進(jìn)一步研究表明，HSP27在胃癌、胰腺癌、肝癌、結(jié)腸癌等耐藥細(xì)胞株中表達(dá)較敏感細(xì)胞株明顯升高[10-13]，而HSP27與胰腺癌吉西他濱化療耐藥等相關(guān)性及其機(jī)制研究鮮有報(bào)道。本實(shí)驗(yàn)構(gòu)建表達(dá)增強(qiáng)型綠色熒光蛋白(enhanced green fluorescence protein，EGFP)與HSP27融合蛋白的載體pEGFP-C1-HSP27，并轉(zhuǎn)染人胰腺癌細(xì)胞株SW1990，為今后研究HSP27對(duì)胰腺癌生物學(xué)行為的影響奠定實(shí)驗(yàn)基礎(chǔ)。

材料和方法

一、pEGFP-C1-HSP27重組質(zhì)粒構(gòu)建

SW1990細(xì)胞為本實(shí)驗(yàn)室保存，常規(guī)培養(yǎng)。收集對(duì)數(shù)生長(zhǎng)期細(xì)胞，應(yīng)用Trizol提取細(xì)胞總RNA。根據(jù)GenBank中HSP27 mRNA序列(NM_001540.3)設(shè)計(jì)引物，上、下游引物的5′端分別引入HindⅢ、BamHⅠ酶切位點(diǎn)和保護(hù)堿基，上游為5′-GTTGTCAAGCTTCTGAGCAGACGTCCAGAGCA-3′，下游為5′-GTGGATCCGCATCCGGGCTAAGGCTTT-3′，擴(kuò)增產(chǎn)物687 bp。引物由上海生工公司合成。先反轉(zhuǎn)錄合成cDNA，再行PCR擴(kuò)增。PCR條件：94℃ 5 min，94℃ 30 s、58℃ 30 s、72℃ 1 min，32個(gè)循環(huán)，最后72℃ 5 min。產(chǎn)物經(jīng)電泳分離、鑒定，切膠回收、純化。

HSP27片段和攜帶熒光蛋白EGFP的pEGFP-C1載體(南京鼓樓醫(yī)院生殖中心惠贈(zèng))行HindⅢ和BamHⅠ雙酶切，酶切產(chǎn)物經(jīng)凝膠電泳回收、純化，在T4 DNA連接酶作用下，16℃連接4 h。連接產(chǎn)物轉(zhuǎn)化感受態(tài)DH-5α，在含卡那霉素的LB平板上篩選單克隆并擴(kuò)增，提取質(zhì)粒DNA，行雙酶切鑒定及送華大基因生物有限公司測(cè)序。重組質(zhì)粒命名為pEGFP-C1-HSP27。轉(zhuǎn)化的細(xì)菌大量擴(kuò)增，收集細(xì)菌，抽提質(zhì)粒，置-20℃保存。

二、重組質(zhì)粒轉(zhuǎn)染SW1990細(xì)胞及穩(wěn)轉(zhuǎn)細(xì)胞篩選

SW1990接種至6孔板培養(yǎng)。待細(xì)胞生長(zhǎng)至70%～80%融合時(shí)，用無(wú)血清培養(yǎng)基饑餓細(xì)胞6 h，將pEGFP-C1及pEGFP-C1-HSP27按4 μg質(zhì)粒∶10 μl LipofectamineTM2000比例分別轉(zhuǎn)染SW1990細(xì)胞，6 h后換無(wú)抗生素完全培養(yǎng)基培養(yǎng)48 h，置倒置熒光顯微鏡下觀察熒光表達(dá)情況。轉(zhuǎn)染的細(xì)胞分別命名為SW1990-C1及SW1990-HSP27細(xì)胞。

應(yīng)用G418(500 μg/ml)篩選后，利用有限稀釋法將存活細(xì)胞接種于96孔板獲得穩(wěn)轉(zhuǎn)單克隆，同時(shí)用含維持濃度G418(250 μg/ml)的培養(yǎng)基繼續(xù)培養(yǎng)28 d，獲得穩(wěn)轉(zhuǎn)細(xì)胞株。將穩(wěn)轉(zhuǎn)細(xì)胞株經(jīng)PBS洗滌及換液后，置于倒置熒光顯微鏡488 nm激發(fā)波長(zhǎng)下觀察蛋白的表達(dá)及細(xì)胞定位。

三、細(xì)胞HSP27 mRNA和蛋白表達(dá)檢測(cè)

收集兩轉(zhuǎn)染組細(xì)胞及親本細(xì)胞，應(yīng)用Trizol提取細(xì)胞總RNA。先反轉(zhuǎn)錄獲得cDNA。HSP27特異性PCR引物上游5′-CAAGGATGGCGTGGTGGA-3′，下游5′-TCTCGTTGGACTGCGTGGC-3′，產(chǎn)物197 bp；內(nèi)參β-actin上游5′-GCCTCGCTGTCCACCTTCCA-3′，下游5′-CACCTTCACCGTTCCAGTTT-3′，產(chǎn)物253 bp。PCR擴(kuò)增程序同前。

收集上述3組細(xì)胞，經(jīng)裂解液裂解，低溫離心，獲取細(xì)胞總蛋白，常規(guī)行蛋白質(zhì)印跡法檢測(cè)HSP27蛋白。

四、統(tǒng)計(jì)學(xué)分析

應(yīng)用Quantity one、SPASS12.0統(tǒng)計(jì)軟件，采用單因素方差分析進(jìn)行檢驗(yàn)。P<0.05為差異具有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

一、重組質(zhì)粒的鑒定

目的基因HSP27經(jīng)擴(kuò)增后，獲得的片段約687 bp，與設(shè)計(jì)的目的片段長(zhǎng)度相當(dāng)(圖1)。重組質(zhì)粒經(jīng)HindⅢ、BamH Ⅰ雙酶切，酶切產(chǎn)物約679 bp(圖2)，且測(cè)序證實(shí)無(wú)移碼改變，提示重組質(zhì)粒構(gòu)建成功。

圖1HSP27基因擴(kuò)增產(chǎn)物電泳圖(左)圖2pEGFP-C1(2)及pEGFP-C1-HSP27(3)雙酶切后的電泳圖(右)

二、穩(wěn)轉(zhuǎn)細(xì)胞株的顯微鏡下表現(xiàn)

熒光顯微鏡下，SW1990-C1穩(wěn)轉(zhuǎn)細(xì)胞表達(dá)的EGFP廣泛分布于整個(gè)細(xì)胞(圖3a)；SW1990-HSP27穩(wěn)轉(zhuǎn)細(xì)胞表達(dá)的EGFP與HSP27的融合蛋白主要分布在胞質(zhì)中(圖3b)。

圖3pEGFP-C1(a)和pEGFP-C1-HSP27(b)轉(zhuǎn)染的SW1990細(xì)胞(熒光顯微鏡 ×200)

三、細(xì)胞HSP27 mRNA及蛋白的表達(dá)

SW1990、SW1990-C1及SW1990-HSP27細(xì)胞均表達(dá)HSP27mRNA(圖4)，表達(dá)量分別為0.956±0.066、0.897±0.051、1.458±0.160，SW1990-HSP27細(xì)胞的表達(dá)水平較SW1990及SW1990-C1細(xì)胞明顯增高(P值均<0.05)。

圖4SW1990(1)、SW1990-C1(2)、SW1990-HSP27(3)細(xì)胞的HSP27 mRNA表達(dá)

SW1990、SW1990-C1、SW1990-HSP27細(xì)胞均表達(dá)內(nèi)源性HSP27蛋白，分子質(zhì)量為27000，SW1990-HSP27細(xì)胞還表達(dá)HSP27與EGFP融合蛋白，分子質(zhì)量為54000(圖5)。

圖5SW1990(1)、SW1990-C1(2)、SW1990-HSP27(3)細(xì)胞表達(dá)的HSP27蛋白

討 論

HSP27作為一種分子伴侶廣泛存在于原核和真核生物中，并可在病理、生理及應(yīng)激狀態(tài)下被誘導(dǎo)表達(dá)。HSP27在生物體內(nèi)存在結(jié)構(gòu)型和誘導(dǎo)型兩種形式。生理狀態(tài)下機(jī)體表達(dá)為結(jié)構(gòu)型HSP27，參與調(diào)控細(xì)胞增殖、分化以及神經(jīng)系統(tǒng)的發(fā)育等生理過(guò)程。在高溫、紫外線、藥物等刺激下，機(jī)體表達(dá)的是誘導(dǎo)型HSP27，其通過(guò)調(diào)節(jié)細(xì)胞死亡的關(guān)鍵信號(hào)通路、減少活性氧物質(zhì)形成等多種方式發(fā)揮細(xì)胞保護(hù)作用[14]。Yang等[10, 15]報(bào)道，HSP27在耐長(zhǎng)春新堿胃癌細(xì)胞株SGC790l/VCR中的表達(dá)較SGC790l明顯增強(qiáng)，通過(guò)反義寡核苷酸抑制HSP27表達(dá)可增強(qiáng)SGC7901/VCR中長(zhǎng)春新堿和阿霉素化療敏感性。Peng等[12]研究表明，HSP27與結(jié)腸癌5-FU耐藥密切相關(guān)，沉默結(jié)腸癌細(xì)胞株LoVo及HCT15的HSP27表達(dá)可緩解其對(duì)5-FU的耐藥。Baylot等[16]報(bào)道，吉西他濱耐藥的胰腺癌細(xì)胞株(KLM1-R)高表達(dá)HSP27，通過(guò)siRNA沉默HSP27表達(dá)可恢復(fù)其對(duì)吉西他濱的敏感性。作者隨訪了11例胰腺癌患者，發(fā)現(xiàn)胰腺癌組織高表達(dá)HSP27的患者對(duì)吉西他濱的敏感性差，且預(yù)后不良。

熒光蛋白標(biāo)記技術(shù)是近年來(lái)發(fā)展迅速的新型細(xì)胞示蹤技術(shù)。來(lái)源于水母的GFP由238個(gè)氨基酸殘基組成，分子質(zhì)量為27000[17]。它在450～490 nm紫外線激發(fā)下發(fā)出綠色熒光。因其分子質(zhì)量小、無(wú)毒、可形成融合蛋白而不影響配體蛋白功能，目前已成為監(jiān)測(cè)完整細(xì)胞和組織內(nèi)基因表達(dá)以及蛋白定位的理想標(biāo)記物[18]。EGFP是GFP的一個(gè)突變體，其熒光強(qiáng)度提高了35倍，應(yīng)用前景更廣泛[19]。本實(shí)驗(yàn)利用基因重組技術(shù)將HSP27基因克隆到EGFP真核表達(dá)載體pEGFP-C1，構(gòu)建的質(zhì)粒綜合了EGFP的示蹤功能和HSP27的生物學(xué)特性，通過(guò)脂質(zhì)體成功轉(zhuǎn)染人胰腺癌SW1990細(xì)胞株，并篩選獲得穩(wěn)轉(zhuǎn)細(xì)胞株，為進(jìn)一步研究HSP27與胰腺癌的生物學(xué)行為、化療耐藥及耐藥機(jī)制奠定基礎(chǔ)。

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ConstructionofpEGFP-C1-HSP27recombinanteukaryoticexpressionvectorandscreeningofhumanpancreaticcancerSW1990celllinestablyexpressingHSP27

ZHANGSong,CHENMin,HUANGShu-ling,XUChun-hong,LIJian-qi,WANGJun,ZOUXiao-ping.

DepartmentofGastroenterology,DrumTowerHospital,NanjingUniversityMedicalSchool,Nanjing210008,China

ZOUXiao-ping,Email:zouxiaoping795@hotmail.com

ObjectiveTo construct pEGFP-C1-HSP27 recombinant eukaryotic expression vector and establish human pancreatic cancer SW1990 cell line stably expressing HSP27.MethodsRT-PCR was applied to amplify human HSP27 cDNA from human pancreatic cancer SW1990 cells with a pair of specific primers carrying a restriction enzyme site BamH I or Hind Ⅲ on each 5′ end. HSP27 cDNA was inserted into pEGFP-C1 vector and then identified by restriction enzyme digestion and sequencing. Successful constructed pEGFP-C1-HSP27 or empty vector was transfected into SW1990 cells by lipofectamine 2000, respectively. The location of HSP72 was determined by fluoroscopy, RT-PCR and Western blot was used to detect the expression of HSP27 in transfected cell.ResultsThe DNA sequence of pEGFP-C1-HSP27 recombinant plasmid was completely correct, and it was successfully transfected into SW1990 cell lines and stably transfected SW1990 cell lines were obtained, which were confirmed by restriction enzyme and sequencing. The expression of EGFP was distributed in cytoplasm, the HSP27mRNA expression was significantly increased (1.458±0.160vs0.897±0.051,P<0.05). In addition, it was showed that EGFP-HSP27 fusion protein was expressed.ConclusionsThe eukaryotic expression vector pEGFP-C1-HSP27 was constructed successfully and stably transfected SW1990 cell line expressing HSP27 was obtained.

Pancreatic neoplasms； Heat-shock proteins; Transfection; SW1990 cell line； Genetic carriers

10.3760/cma.j.issn.1674-1935.2012.03.011

國(guó)家自然科學(xué)基金(81071816)

210008 南京，南京大學(xué)醫(yī)學(xué)院附屬鼓樓醫(yī)院消化科

鄒曉平，Email: zouxiaoping795@hotmail.com

2011-12-10)

(本文編輯：屠振興)