張雪梅，孫延亮，楊揚，蘭輝，王喜梅

(1.湖南醫(yī)藥學(xué)院，湖南 懷化 418000；2.懷化市第一人民醫(yī)院，湖南 懷化 418000；3.湖南醫(yī)藥學(xué)院第一附屬醫(yī)院，湖南 懷化 418000)

·論 著·

轉(zhuǎn)染反義MIF對人胃癌細(xì)胞的影響

張雪梅1，孫延亮2，楊揚1，蘭輝3，王喜梅1

(1.湖南醫(yī)藥學(xué)院，湖南 懷化 418000；2.懷化市第一人民醫(yī)院，湖南 懷化 418000；3.湖南醫(yī)藥學(xué)院第一附屬醫(yī)院，湖南 懷化 418000)

目的 探討反義巨噬細(xì)胞移動抑制因子(MIF)對人胃癌MGC-803細(xì)胞的影響。方法將胃癌MGC-803細(xì)胞分為pcDNA3.1-Anti MIF組與空白對照組。pcDNA3.1-Anti MIF組采用轉(zhuǎn)染技術(shù)將MIF反義RNA真核表達質(zhì)粒(pcDNA3.1-AntiMIF)轉(zhuǎn)入MGC-803細(xì)胞；空白對照組轉(zhuǎn)染pcDNA3.1-sh-MIF質(zhì)粒。qRT-PCR與蛋白質(zhì)印跡法檢測轉(zhuǎn)染效率；采用MTT法、侵襲實驗、AnnexinV-FITC和PI染色法分別檢測反義MIF對MGC-803細(xì)胞增殖、侵襲、凋亡的影響。結(jié)果qRT-PCR結(jié)果顯示，pcDNA3.1-Anti MIF組MIF mRNA表達量(2.086±0.248)較空白對照組(6.992±0.342)明顯下調(diào)；Western blot顯示，pcDNA3.1vAntiMIF組MIF蛋白表達水平量(0.361±0.043)較空白對照組(1.171±0.091)明顯下調(diào)；MTT實驗結(jié)果顯示，pcDNA3.1-AntiMIF組MGC-803細(xì)胞的OD值(0.436± 0.017)較空白對照組(0.563±0.019)明顯下降；侵襲實驗結(jié)果顯示，pcDNA3.1-AntiMIF組穿過基質(zhì)膠的MGC-803細(xì)胞數(shù)(73.67±8.54)較空白對照組(137.30±11.91)明顯減少；AnnexinV-FITC和PI染色法結(jié)果顯示，pcDNA3.1-AntiMIF組MGC-803細(xì)胞的凋亡率(21.61±4.62)%較空白對照組(7.67±0.63)%明顯增加。以上各項指標(biāo)比較差異均具有顯著統(tǒng)計學(xué)意義(P<0.01)。結(jié)論反義MIF能抑制人胃癌MGC-803細(xì)胞的增殖與侵襲，并能誘導(dǎo)凋亡。

巨噬細(xì)胞移動抑制因子；胃癌；增殖；侵襲；凋亡

胃癌的發(fā)病率與死亡率居消化道惡性腫瘤之首[1]，嚴(yán)重危害人類的生命與健康，其對人體的危害不僅在于腫瘤細(xì)胞生長失控而引起的克隆性異常增生，同時，腫瘤的侵襲和轉(zhuǎn)移更是導(dǎo)致胃癌患者死亡的重要原因。因此，尋求一種有效抑制胃癌細(xì)胞生長，防止胃癌轉(zhuǎn)移和復(fù)發(fā)的新方法顯得極為緊迫。目前，基因治療作為一種新的治療手段已逐漸成為腫瘤治療的一種重要策略[2]。

巨噬細(xì)胞移動抑制因子(macrophage migration inhibitory factors，MIF)是一種多功能細(xì)胞因子，廣泛表達于人體多種細(xì)胞和組織，與多種炎癥性疾病、自體免疫性疾病及腫瘤等密切相關(guān)。研究顯示，MIF可從多個層次促進腫瘤的發(fā)生發(fā)展，主要通過促進細(xì)胞增殖、抑制細(xì)胞凋亡、促進血管生成等共同促進腫瘤的發(fā)生發(fā)展和侵襲轉(zhuǎn)移[3-5]。抑制MIF分泌及活性可抑制惡性腫瘤的生長和轉(zhuǎn)移。本研究利用基因重組技術(shù)構(gòu)建MIF反義RNA真核表達質(zhì)粒pcDNA3.1-AntiMIF，將其轉(zhuǎn)染胃癌細(xì)胞株MGC-803細(xì)胞，觀察MIF對胃癌細(xì)胞的作用，從而為以MIF為靶點抗胃癌的基因治療提供新思路。

1 材料與方法

1.1 細(xì)胞系 人胃癌MGC-803由南華大學(xué)腫瘤研究惠贈，細(xì)胞用含10%胎牛血清的RPMI-1640培養(yǎng)，置于37℃、5%CO2的恒溫箱中培養(yǎng)。

1.2 主要材料 Lipofectamine 2000購自美國Invitrogen公司，pcDNA3.1-Anti-MIF與pcDNA3.1-sh-MIF (空載體)由Invitrogen公司設(shè)計構(gòu)建，RNA抽提試劑盒購于Applied Biosystems公司，MIF和β-actin抗體購于美國Santa Cruz公司，MTT粉購自美國Sigma公司，Transwell小室購于美國BD公司。

1.3 pcDNA3.1-Anti MIF轉(zhuǎn)染及細(xì)胞分組 培養(yǎng)人胃癌MGC-803細(xì)胞，將MGC-803細(xì)胞分為pcDNA3.1-Anti MIF組與空白對照組。pcDNA3.1-Anti MIF組轉(zhuǎn)染pcDNA3.1-Anti MIF質(zhì)粒，空白對照組轉(zhuǎn)染pcDNA3.1-sh-MIF質(zhì)粒。于6孔板內(nèi)按2 mL/孔鋪加MGC-803細(xì)胞懸液，放置于37℃、5%CO2的細(xì)胞培養(yǎng)箱中培養(yǎng)至細(xì)胞匯合度達30%～50%。在無菌EP管中配好pcDNA3.1-Anti MIF質(zhì)粒、pcDNA3.1-sh-MIF質(zhì)粒以及Lipofectamin2000；室溫中放置20 min，使脂質(zhì)體與質(zhì)粒DNA形成復(fù)合體。用無血清培養(yǎng)液輕洗待轉(zhuǎn)細(xì)胞后加入無血清RPMI-1640(1 mL)，再將孵育好的pcDNA3.1-Anti MIF質(zhì)粒與pcDNA3.1-sh-MIF質(zhì)粒脂質(zhì)體混合液加至6孔板中，將細(xì)胞置于37℃、5%CO2培養(yǎng)箱中繼續(xù)培養(yǎng)6 h后換成有RPMI-1640完全細(xì)胞培養(yǎng)基，再繼續(xù)培養(yǎng)48 h。

1.4 qRT-PCR 用RNA抽提試劑盒抽提上述兩組細(xì)胞中總RNA，逆轉(zhuǎn)錄合成cDNA于-80℃冰箱保存。PCR擴增反應(yīng)體系為20μL，其中包括PCR primers(5 mmol/L)0.4μL，正向引物：5'-AGTGGTGTCCGAGAAGTCAG-3'；反向引物：5'-TTAGGCGAAGGTGGAGTTGT-3'，RT product2.0μL，Taq DNA polymerase(5 U/μL)0.2μL，2×SYBRMix 10μL，滅菌蒸餾水7.4μL?；靹?，反應(yīng)條件如下：50℃2 min，95℃10 min后95℃15 s，60℃60 s，40個循環(huán)，溶解曲線條件：95℃15 s，60℃60 s，95℃15 s。以U6 snRNA為內(nèi)參，所測定的MIF的相對表達量采用2-ΔΔCT法分析。

1.5 Western blot檢測 收集上述兩組細(xì)胞，提取細(xì)胞總蛋白，采用BCA法測定蛋白濃度。每組取等量蛋白樣本進行SDS-PAGE凝膠電泳，再將蛋白轉(zhuǎn)至PVDF膜上，采用5%脫脂牛奶進行封閉2 h，再加MIF抗體或β-actin抗體，于4℃下繼續(xù)過夜。TBST洗膜30 min，加入二抗室溫孵育1 h，TBST洗膜30 min，然后加ECL發(fā)光劑，X片曝光、顯影、定影。

1.6 MTT法檢測細(xì)胞增殖 消化上述兩組細(xì)胞，取200μL即5 000個細(xì)胞接種于96孔板中，設(shè)置6個復(fù)孔，培養(yǎng)48 h后取出，每孔加20μL MTT液，繼續(xù)培養(yǎng)4 h后取出，每孔中加150μL DMSO，低速振蕩10 min，選擇波長為570 nm，在酶標(biāo)儀上測定各孔吸光值，實驗重復(fù)3次。

1.7 Transwell侵襲實驗 在Transwell小室中鋪加適量基質(zhì)膠稀釋液，過夜并成膜。上述兩組細(xì)胞中分別取100μL即含1×105個細(xì)胞/mL的細(xì)胞稀釋液接種至Transwell小室的上室，取含10%胎牛血清的RPMI-1640培養(yǎng)液500μL加入下室，置于37℃、5%CO2的細(xì)胞培養(yǎng)箱中培養(yǎng)36 h后取出，用棉簽擦棄小室中上層未穿過基質(zhì)膠的細(xì)胞，磷酸鹽緩沖液(PBS)輕洗，再將Transwell小室放置于4%多聚甲醛中，以固定小室背面穿出的細(xì)胞，結(jié)晶祡染色，PBS液洗，倒置，晾干。光學(xué)顯微鏡下觀察并攝相，隨機選取4個高倍視野進行細(xì)胞計數(shù)，取平均值，實驗重復(fù)3次。

1.8 細(xì)胞凋亡實驗 采用AnnexinV-FITC/PI染色法。將上述兩組組培養(yǎng)至80%匯合度，用PBS液洗滌細(xì)胞2次并離心收集，取1×106個細(xì)胞，加入100μL的結(jié)合緩沖液懸浮細(xì)胞，加Annexin V-FITC 5μL混勻，再加PI1μL混勻。室溫下避光反應(yīng)15～30 min，上機，流式細(xì)胞儀檢測。

1.9 統(tǒng)計學(xué)方法 應(yīng)用SPSS-13.0軟件進行統(tǒng)計學(xué)分析，計量資料以均數(shù)±標(biāo)準(zhǔn)差(x±s)表示，兩組比較采用t檢驗，以P<0.05為差異有統(tǒng)計學(xué)意義。

2 結(jié) 果

2.1 反義MIF抑制人胃癌MGC-803細(xì)胞的增殖與侵襲 qRT-PCR與Western blot顯示結(jié)果顯示，pcDNA3.1-AntiMIF組MIF mRNA與蛋白表達水平較空白對照組均明顯下調(diào)，差異有顯著統(tǒng)計學(xué)意義(P<0.01)，提示轉(zhuǎn)染成功；MTT實驗結(jié)果顯示，pcDNA3.1-AntiMIF組MGC-803細(xì)胞的OD值較空白對照組明顯下降，差異有顯著統(tǒng)計學(xué)意義(P<0.01)；侵襲實驗結(jié)果顯示，pcDNA3.1-AntiMIF組穿過基質(zhì)膠的MGC-803細(xì)胞數(shù)較空白對照組明顯減少，差異有顯著統(tǒng)計學(xué)意義(P<0.01)。見表1。

2.2 反義MIF誘導(dǎo)人胃癌細(xì)胞凋亡 凋亡實驗結(jié)果顯示，pcDNA3.1-AntiMIF組MGC-803細(xì)胞的凋亡率為(21.61±4.62)%，與空白對照組的(7.67±0.63)%比較，差異均具有顯著統(tǒng)計學(xué)意義(t=8.020，P<0.01)。

表1 兩組細(xì)胞mRNA、蛋白灰度值、OD值和穿過基質(zhì)膠的細(xì)胞數(shù)比較

表1 兩組細(xì)胞mRNA、蛋白灰度值、OD值和穿過基質(zhì)膠的細(xì)胞數(shù)比較

組別pcDNA3.1-Anti MIF組空白對照組t值P值mRNA 2.086±0.248 6.992±0.342 28.300 <0.01蛋白灰度值0.361±0.043 1.171±0.091 9.637 <0.01 OD值0.436±0.017 0.563±0.019 4.961 <0.01穿過基質(zhì)膠的細(xì)胞數(shù)73.67±8.54 137.30±11.91 8.593 <0.01

3 討論

自分泌MIF信號刺激巨噬細(xì)胞產(chǎn)生腫瘤壞死因子、白介素-1、一氧化氮、過氧化氫以及其他炎癥因子[6]。在大鼠模型胃炎的發(fā)病機制中研究發(fā)現(xiàn)，MIF是重要的調(diào)節(jié)因子，尤其在幽門螺桿菌(Helicobacterpylori，HP)感染的胃炎中MIF的表達量明顯上調(diào)[7-8]。研究顯示，沉默MIF后能預(yù)防HP誘導(dǎo)的胃炎發(fā)生[9]。此外，大量的研究亦顯示，MIF在多種腫瘤中高表達，如肺癌[10]、肝癌[11]、乳腺癌[12]、胃癌[13]、結(jié)腸癌[14]以及前列腺癌[15]等。且MIF高表達通過旁分泌途徑能促進腫瘤細(xì)胞增殖與抑制凋亡[16-17]。這些結(jié)果表明，MIF在慢性炎癥與癌癥性疾病中可能扮演著重要角色。

研究顯示，MIF在正常胃黏膜、慢性胃竇炎、腸上皮化生以及胃癌組織黏膜中的表分別為12.19%、52.12%、66.11%、95.51%，且其在胃癌中的表達水平與胃癌患者的臨床分期、淋巴結(jié)轉(zhuǎn)移以及預(yù)后明顯相關(guān)[13]。還有研究顯示，MIF在胃癌患者血清中的表達濃度亦明顯增高[18]。研究表明MIF可能是腫瘤治療的重要分子靶點。目前，已有以MIF為靶點，使用MIF特異性抑制劑、阻斷MIF受體、制備MIF單抗、靶向MIF的基因治療等方法來降低MIF分泌和活性，進而抑制惡性腫瘤生長和轉(zhuǎn)移的相關(guān)研究報道[19-20]。研究顯示，在結(jié)腸癌細(xì)胞中轉(zhuǎn)染MIF反義質(zhì)粒下調(diào)MIF后，癌細(xì)胞的增殖能力明顯減弱[14]。在前列腺癌DU-145細(xì)胞中，抑制MIF與其受體CD74能明顯減弱癌細(xì)胞的增殖與侵襲能力[15]。然而，沉默MIF是否影響胃癌的發(fā)展以及MIF在胃癌的治療中是否是一個潛在的治療靶分子，有待驗證。

本研究首先采用轉(zhuǎn)染技術(shù)將MIF反義RNA真核表達質(zhì)粒轉(zhuǎn)于人胃癌MGC-803細(xì)胞，以沉默該細(xì)胞中MIF的表達。qRT-PCR與蛋白質(zhì)印跡法驗證轉(zhuǎn)染效率，結(jié)果顯示，轉(zhuǎn)染組MIF的蛋白與mRNA水平較對照均明顯下調(diào)，提示轉(zhuǎn)染成功。接下來進一步采用MTT法、侵襲以及凋亡實驗明確其對胃癌細(xì)胞生物活行為的影響。MTT與侵襲實驗結(jié)果顯示，在胃癌細(xì)胞中沉默MIF后，細(xì)胞的增殖與侵襲明顯下降。而凋亡實驗結(jié)果顯示，在胃癌細(xì)胞中沉默MIF后，細(xì)胞的凋亡誘導(dǎo)作用明顯增強。

綜上所述，在胃癌細(xì)胞中沉默MIF能抑制癌細(xì)胞的增殖與侵襲，并能誘導(dǎo)癌細(xì)胞凋亡，接下來的研究重點將放在構(gòu)建胃癌體內(nèi)模型上，以進一步研究在體內(nèi)沉默MIF的表達對胃癌細(xì)胞生物學(xué)行為的影響，且進一步探討其具體機制，為胃癌的臨床靶向治療提供重要的理論基礎(chǔ)和實驗依據(jù)。

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Effects of antisense macrophage migration inhibitory factor in human gastric cancer cells.

ZHANG Xue-mei1,SUN Yan-liang2,YANG Yang1,LAN Hui3,WANG Xi-mei1.1.Hunan University of Medicine,Huaihua 418000,Hunan,CHINA; 2.First Affiliated Hospital of Hunan University of Medicine,Huaihua 418000,Hunan,CHINA;3.First People's Hospital of Huaihua City,Huaihua 418000,Hunan,CHINA

ObjectiveTo explore the effect of antisense macrophage migration inhibitory factor(MIF)on human gastric cancer MGC-803 cells.MethodsHuman gastric cancer MGC-803 cells were assigned into two groups to receive transfection of antisense MIF plasmid pcDNA3.1-Anti MIF(pcDNA3.1-Anti MIF group)and pcDNA3.1-sh-MIF(control group).The qRT-PCR and Western blot analysis were employed for detecting transfection efficiency.The ability of proliferation and invasion,as well as apoptosis rate of MGC-803 cells regulated by antisense MIF were evaluated by MTT,Transwell invasion assays,and Annexin V/propidium iodide staining.ResultsqRT-PCR results showed thatthe expression of MIF mRNAin pcDNA3.1-Anti MIF group was significantly lower than thatin control group,(2.086±0.248)vs(6.992±0.342).Western blot analysis results showed that the expression of MIF protein in pcDNA3.1-Anti MIF group was significantly lower than thatin controlgroup,(0.361±0.043)vs(1.171±0.091).MTT assay showed thatthe OD value of MGC-803 cells in the pcDNA3.1-Anti MIF group was significantly lower than that in control group,(0.436±0.017)vs(0.563±0.019).Transwell invasion assays results showed that the number of cells through the matris glue in pcDNA3.1-Anti MIF group was significantly lower than that in the control group,(73.67± 8.54)vs(137.30±11.91).Annexin V/propidium iodide staining showed that the apoptosis rate of MGC-803 cells in the pcDNA3.1-Anti MIF group was significantly higher than thatin controlgroup,(21.61±4.62)%vs(7.67±0.63)%.The differences were all statistically significant(P<0.01).ConclusionAntisense MIF can inhibit the proliferation and invasion of human gastric cancer MGC-803 cells,as wellas induce apoptosis.

Macrophage migration inhibitory factor;Gastric cancer;Proliferation;Invasion;Apoptosis

R735.2

A

1003—6350（2017）09—1377—04

10.3969/j.issn.1003-6350.2017.09.001

2017-01-10)

湖南省教育廳科學(xué)研究項目(編號：13C741)

王喜梅。E-mail：447409108@qq.com