pshuttle-Egr-1-hSmac質(zhì)粒聯(lián)合X線照射對(duì)乳腺癌MDA-MB-435細(xì)胞增殖的抑制作用

梁 碩,王志成,李艷博,,郭彩霞,龔守良,林承赫

(1.吉林大學(xué)公共衛(wèi)生學(xué)院衛(wèi)生部放射生物學(xué)重點(diǎn)實(shí)驗(yàn)室,吉林長春 130021;2.首都醫(yī)科大學(xué)公共衛(wèi)生與家庭醫(yī)學(xué)學(xué)院,北京 100069;3.吉林大學(xué)第一醫(yī)院核醫(yī)學(xué)科,吉林長春 130021)

pshuttle-Egr-1-hSmac質(zhì)粒聯(lián)合X線照射對(duì)乳腺癌MDA-MB-435細(xì)胞增殖的抑制作用

梁 碩1,王志成1,李艷博1,2,郭彩霞2,龔守良1,林承赫3

(1.吉林大學(xué)公共衛(wèi)生學(xué)院衛(wèi)生部放射生物學(xué)重點(diǎn)實(shí)驗(yàn)室,吉林長春 130021;2.首都醫(yī)科大學(xué)公共衛(wèi)生與家庭醫(yī)學(xué)學(xué)院,北京 100069;3.吉林大學(xué)第一醫(yī)院核醫(yī)學(xué)科,吉林長春 130021)

目的:構(gòu)建pshuttle-Egr-1-hSmac質(zhì)粒并轉(zhuǎn)染人乳腺癌MDA-MB-435細(xì)胞,觀察其抑制腫瘤細(xì)胞的輻射增敏作用。方法:轉(zhuǎn)染pshuttle-Egr-1-hSmac質(zhì)粒的MDA-MB-435細(xì)胞經(jīng)過2 Gy X線照射不同時(shí)間(4、8、12、24和48 h)和0.5～5.0 Gy X線照射后24 h收集細(xì)胞,采用RT-PCR和Western blotting法檢測Smac mRNA及其蛋白表達(dá)。將細(xì)胞分為對(duì)照組、pshuttle質(zhì)粒組、pshuttle-Egr-1-hSmac質(zhì)粒組、2 Gy組、pshuttle＋2.0 Gy組和pshuttle-Egr-1-hSmac＋2.0 Gy組,MTT法檢測各組細(xì)胞增殖;克隆形成實(shí)驗(yàn)檢測細(xì)胞存活能力;AnnexinⅤ-FITC雙染法檢測細(xì)胞凋亡;PI單染法檢測細(xì)胞周期。結(jié)果:對(duì)照組和pshuttle質(zhì)粒組MDA-MB-435細(xì)胞中Smac mRNA無表達(dá),而pshuttle-Egr-1-hSmac質(zhì)粒組MDA-MB-435細(xì)胞Smac m RNA表達(dá)水平隨時(shí)間延長逐漸升高,于24和48 h時(shí)表達(dá)水平最高;經(jīng)0.5～5.0 Gy X線照射后24 h MDA-MB-435細(xì)胞Smac m RNA表達(dá)水平隨照射劑量增加而逐漸增加,在2.0和5.0 Gy X線照射后Smac m RNA表達(dá)水平最高。pshuttle-Egr-1-hSmac質(zhì)粒組4、8、12和24 h后Smac蛋白表達(dá)水平逐漸升高,24 h后表達(dá)水平最高。經(jīng)過0、0.5、1.0、2.0和5.0 Gy X線照射后24 h Smac蛋白表達(dá)水平逐漸升高,尤其以5.0 Gy X線照射時(shí)表達(dá)水平最高。MTT法檢測時(shí)程效應(yīng),2.0 Gy、pshuttle＋2.0 Gy和pshuttle-Egr-1-hSmac＋2.0 Gy質(zhì)粒組24、48和72 h細(xì)胞A490值明顯低于對(duì)照組(P＜0.01);劑量效應(yīng),pshuttle-Egr-1-hSmac質(zhì)粒組1.0～5.0 Gy X線照射后, MDA-MB-435細(xì)胞A490值明顯低于0 Gy X線照射(P＜0.05或P＜0.01)。pshuttle-Egr-1-hSmac質(zhì)粒組細(xì)胞存活分?jǐn)?shù)明顯低于對(duì)照組(P＜0.01)。pshuttle-Egr-1-hSmac＋2.0 Gy組細(xì)胞凋亡率明顯高于2.0 Gy組(P＜0.01), G0/G1期和S期細(xì)胞百分率明顯低于2.0 Gy照射組(P＜0.01),G2/M期細(xì)胞百分率明顯高于2.0 Gy組(P＜0.01)。結(jié)論:X線照射能增加pshuttle-Egr-1-hSmac質(zhì)粒轉(zhuǎn)染的MDA-MB-435細(xì)胞有效表達(dá)Smac m RNA及蛋白,能抑制細(xì)胞存活,且誘導(dǎo)G2/M期阻滯和凋亡增加;Smac基因聯(lián)合放射治療可明顯增加乳腺癌細(xì)胞的放射敏感性。

Smac基因;Egr-1啟動(dòng)子;X射線;基因-放射治療;細(xì)胞凋亡

臨床放射治療(放療)在乳腺癌治療中占有重要的地位,但是輻射的副作用不可避免,如何在提高乳腺癌放療療效的同時(shí)減少副損傷是腫瘤治療的研究熱點(diǎn)[1-3]。輻射敏感啟動(dòng)子Egr-1具有輻射誘導(dǎo)特性,即該啟動(dòng)子在正常條件下不增強(qiáng)下游基因的表達(dá),只有在輻射條件下,下游基因才能在Egr-1介導(dǎo)下表達(dá)增強(qiáng),增強(qiáng)該基因的效應(yīng)。因此Egr-1可以從時(shí)間和空間上調(diào)控下游基因的體內(nèi)表達(dá),提高放療的敏感性,減輕毒副作用,實(shí)現(xiàn)腫瘤綜合治療的目的[4-5]。與Egr-1相連接的基因有很多種類,但是有關(guān)Smac基因的研究卻未見報(bào)道。本研究利用放療能夠殺傷腫瘤細(xì)胞和誘導(dǎo)Egr-1啟動(dòng)子的轉(zhuǎn)錄作用以及Smac基因能夠促進(jìn)腫瘤細(xì)胞凋亡的作用[6-7],將pshuttle-Egr-1-hSmac質(zhì)粒聯(lián)合X線照射作用于乳腺癌MDA-MB-435細(xì)胞,以降低輻射劑量并能夠減輕或避免正常組織損傷,達(dá)到抑制腫瘤生長和殺傷腫瘤細(xì)胞的目的,為腫瘤治療開辟新途徑。

1 材料與方法

1.1 細(xì)胞培養(yǎng)及轉(zhuǎn)染人乳腺癌細(xì)胞株MDA-MB-435為吉林大學(xué)衛(wèi)生部放射生物學(xué)重點(diǎn)實(shí)驗(yàn)室保存,以含10%胎牛血清的DMEM培養(yǎng)液(美國Gibco公司)于37℃、5%CO2培養(yǎng)箱中常規(guī)培養(yǎng),待細(xì)胞80%～90%融合可傳代。取對(duì)數(shù)生長期細(xì)胞接種于6孔板,6×105個(gè)/孔。第2天待細(xì)胞達(dá)90%～95%,按照Lipofectamine 2000轉(zhuǎn)染試劑盒(美國Invitrogen公司)說明書轉(zhuǎn)染構(gòu)建正確的質(zhì)粒。

1.2 細(xì)胞照射采用國產(chǎn)X射線深部治療機(jī)進(jìn)行照射,電壓200 k V,電流10 m A,濾板為0.5 mm Cu和1.0 mm Al。照射時(shí)靶皮距50 cm,劑量率0.287 Gy·min－1。

1.3 Smac m RNA和蛋白表達(dá)規(guī)律將經(jīng)過脂質(zhì)體轉(zhuǎn)染空載體pshuttle和pshuttle-Egr-1-hSmac質(zhì)粒24 h的MDA-MB-435細(xì)胞經(jīng)過2.0 Gy X線照射后不同時(shí)間(4、8、12、24和48 h)和不同劑量(0.5、1.0、2.0和5.0 Gy)照射后24 h收集細(xì)胞,采用RT-PCR方法和Western blotting法檢測Smac m RNA和蛋白表達(dá)規(guī)律,Smac引物序列上游引物:5′-gctctagaatggcggctctgaagagttggctgt-3′, 含XbaⅠ酶切位點(diǎn);下游引物:5′-gcggatcctcaa tcctcacgcaggt-3′,含Bam HⅠ酶切位點(diǎn)。

1.4 細(xì)胞增殖時(shí)程及劑量效應(yīng)實(shí)驗(yàn)MDA-MB-435細(xì)胞分為對(duì)照組(control)、pshuttle質(zhì)粒組、pshuttle-Egr-1-hSmac質(zhì)粒組、2.0 Gy組、pshuttle＋2.0 Gy組和pshuttle-Egr-1-hSmac＋2.0 Gy組。MDA-MB-435細(xì)胞接種于96孔板, 1×104個(gè)/孔,每組6復(fù)孔。轉(zhuǎn)染質(zhì)粒24 h后,給予2.0 Gy照射,于照射后0、12、24、48和72 h加入MTT,每孔MTT(5 g·L－1)20μL,繼續(xù)培養(yǎng)4 h后棄上清,每孔加入150μL DMSO,待充分溶解后酶標(biāo)儀測定490 nm波長處吸光度(A490)值。以A490值表示細(xì)胞增殖情況,A490值降低,細(xì)胞生長受抑制。

MDA-MB-435細(xì)胞以6×104個(gè)/孔接種于96孔細(xì)胞培養(yǎng)板,分別設(shè)對(duì)照組、pshuttle組和pshuttle-Egr-1-hSmac組,經(jīng)脂質(zhì)體轉(zhuǎn)染后24 h給予照射,照射劑量分別為0、0.5、1.0、2.0和5.0 Gy,X線照射后24 h加入MTT測量A490值。以A490值表示細(xì)胞增殖情況,A490值降低,細(xì)胞生長受抑制。

1.5 細(xì)胞克隆形成實(shí)驗(yàn)對(duì)照細(xì)胞及轉(zhuǎn)染pshuttle和pshuttle-Egr-1-hSmac質(zhì)粒24 h后,按照預(yù)先設(shè)計(jì)的細(xì)胞密度接種到60 mm無菌塑料培養(yǎng)皿中,各組設(shè)3個(gè)平行樣,接種密度為100個(gè)/ 孔(0 Gy)、200個(gè)/孔(2 Gy)、600個(gè)/孔(4 Gy)、2 000個(gè)/孔(6 Gy)、5 000個(gè)/孔(8 Gy)和50 000個(gè)/孔(10 Gy)。加入5 m L培養(yǎng)液,繼續(xù)培養(yǎng)14 d,Giemsa染色,空氣中干燥,封片,計(jì)數(shù)克隆數(shù)。用未照射組的集落形成率(PE)進(jìn)行校正,計(jì)算細(xì)胞存活分?jǐn)?shù)(survival fraction,SF),計(jì)算導(dǎo)致細(xì)胞63%死亡所需劑量(D0)值。

1.6 細(xì)胞周期及凋亡檢測分組同1.4。MDAMB-435細(xì)胞接種于6孔板,6×105個(gè)/孔,細(xì)胞轉(zhuǎn)染24 h后給予2.0 Gy照射,24 h后胰酶消化收獲細(xì)胞,PBS洗2次,0.5 m L PBS重懸細(xì)胞,加入AnnexinⅤ/PI或PI避光染色1 h,立即上流式細(xì)胞儀進(jìn)行檢測。采用CellQuest軟件收取細(xì)胞(每份樣品收取1×104個(gè)細(xì)胞),采用ModFit軟件分析,結(jié)果以細(xì)胞凋亡率及各周期細(xì)胞百分率表示。

1.7 統(tǒng)計(jì)學(xué)分析采用SPSS 17.0軟件進(jìn)行統(tǒng)計(jì)分析。各組細(xì)胞A490值、細(xì)胞SF、凋亡率和各細(xì)胞周期細(xì)胞百分率以±s表示,均數(shù)兩兩比較采用t檢驗(yàn),多組均數(shù)比較采用完全隨機(jī)設(shè)計(jì)的單因素方差分析。

2 結(jié)果

2.1 X線照射后轉(zhuǎn)染pshuttle-Egr-1-hSmac質(zhì)粒的MDA-MB-435細(xì)胞中Smac mRNA表達(dá)的規(guī)律

2.0 Gy X線照射后,對(duì)照組和pshuttle質(zhì)粒組Smac mRNA在24 h時(shí)無表達(dá),而pshuttle-Egr-1-hSmac質(zhì)粒組MDA-MB-435細(xì)胞中Smac m RNA表達(dá)水平隨時(shí)間延長逐漸增高,4、8、12、24和48 h時(shí)Smac m RNA表達(dá)相對(duì)值分別為0.55、0.52、0.81、1.63和1.59,于24和48 h時(shí)表達(dá)較高,具有一定的時(shí)程效應(yīng)。見圖1。

重組質(zhì)粒轉(zhuǎn)染細(xì)胞后24 h進(jìn)行照射,照射劑量為0.5、1.0、2.0和5.0 Gy,照射后24 h檢測Smac m RNA表達(dá)。對(duì)照組和pshuttle質(zhì)粒組MDA-MB-435細(xì)胞中Smac m RNA無表達(dá)。pshuttle-Egr-1-hSmac質(zhì)粒組MDA-MB-435細(xì)胞經(jīng)0.5、1.0、2.0和5.0 Gy照射后Smac m RNA表達(dá)相對(duì)值分別為0.31、0.84、1.33和1.29,其中2.0和5.0 Gy照射后Smac m RNA表達(dá)較為明顯,具有一定的劑量效應(yīng)。見圖2。

圖1 2.0 Gy X線照射后不同時(shí)間MDA-MB-435細(xì)胞中Smac m RNA表達(dá)電泳圖Fig.1 Electrophoregram of Smac mRNA expressions in MDA-MB-435 cells at different time after 2.0 Gy X-ray irradiationLane 1:Pshuttle group;Lane 2:DL 2000 marker;Lane 3:Control group;Lane 4―8:4,8,12,24,and 48 h in pshuttle-Egr-1-hSmac＋2.0 Gy group.

圖2 不同劑量X線照射后24 h Smac m RNA表達(dá)電泳圖Fig.2 Electrophoregram of Smac m RNA expressions 24 h after different doses of X-ray irradiationLane 1:Control group;Lane 2:DL 2000 marker;Lane 3:Pshuttle group;Lane 4－7:5.0,2.0,10,and 0.5 Gy X-ray irradiation in pshuttle-Egr-1-hSmac plasmid group.

2.2 X線照射后轉(zhuǎn)染pshuttle-Egr-1-hSmac質(zhì)粒的MDA-MB-435細(xì)胞中Smac蛋白表達(dá)的規(guī)律轉(zhuǎn)染pshuttle-Egr-1-hSmac質(zhì)粒24 h后進(jìn)行照射,采用Western blotting法檢測細(xì)胞中Smac蛋白表達(dá)。Smac的相對(duì)分子質(zhì)量為25 000,從圖3中可見有明顯的蛋白條帶,與Smac蛋白大小一致,而β-actin的相對(duì)分子質(zhì)量為42 000。MDA-MB-435細(xì)胞經(jīng)2.0 Gy X線照射后,對(duì)照組Smac蛋白無表達(dá),pshuttle-Egr-1-hSmac質(zhì)粒組4、8、12和24 h后蛋白表達(dá)逐漸增加,灰度分析后蛋白表達(dá)相對(duì)值為0.28、0.31、0.44和0.62(圖3);不同劑量X線照射后,對(duì)照組無Smac蛋白表達(dá),0.5、1.0、2.0和5.0 Gy照射組Smac蛋白表達(dá)相對(duì)值分別為0.31、0.34、0.68和0.86(圖4)。說明質(zhì)粒pshuttle-Egr-1-hSmac在X射線誘導(dǎo)下可有效表達(dá)Smac蛋白,并且以5 Gy照射后24 h表達(dá)最為明顯。

2.3 重組質(zhì)粒聯(lián)合X線照射對(duì)MDA-MB-435細(xì)胞生長抑制的時(shí)程及劑量效應(yīng)時(shí)程效應(yīng)結(jié)果:轉(zhuǎn)染pshuttle和pshuttle-Egr-1-hSmac質(zhì)粒后MDA-MB-435細(xì)胞生長有抑制傾向,但不明顯, 2.0 Gy、pshuttle＋2.0 Gy和pshuttle-Egr-1-hSmac＋2.0 Gy組細(xì)胞A490值明顯降低,24、48 和72 h時(shí)與對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義(P＜0.05或P＜0.01),其中pshuttle-Egr-1-hSmac＋2.0 Gy組細(xì)胞A490值降低最明顯,與pshuttle-Egr-1-hSmac組比較差異也有統(tǒng)計(jì)學(xué)意義(P＜0.05或P＜0.01)。劑量效應(yīng)結(jié)果:MDA-MB-435細(xì)胞A490值隨照射劑量增加逐漸降低,對(duì)照組和pshuttle組降低不明顯;pshuttle-Egr-1-hSmac質(zhì)粒組MDA-MB-435細(xì)胞A490值明顯降低,1.0、2.0和5.0 Gy X線照射后,與0 Gy X線照射比較,細(xì)胞A490值明顯降低(P＜0.05或P＜0.01)。見表1和2。

圖3 2.0 Gy X線照射后不同時(shí)間Smac蛋白表達(dá)電泳圖Fig.3 Electrophoregram of Smac protein expressions at different time after 2.0 Gy X-ray irradiationLane 1―4:4,8,12,and 24 h after 2.0 Gy X-ray irradiation in pshuttle-Egr-1-hSmac＋2 Gy group;Lane 5:Control group.

圖4 不同劑量X線照射后24 h Smac蛋白表達(dá)電泳圖Fig.4 Electrophoregram of Smac protein expressions after different doses of X-ray irradiation for 24 hLane 1－4:0.5,1.0,2.0,and 5.0 Gy X-ray irradiation in pshuttle-Egr-1-hSmac plasmid group;Lane 5:Control group.

表1 2.0 Gy X線照射后不同時(shí)間各組細(xì)胞A490值Tab.1 A490values of cells after 2 Gy irradiation in various groups(n＝6±s)

表1 2.0 Gy X線照射后不同時(shí)間各組細(xì)胞A490值Tab.1 A490values of cells after 2 Gy irradiation in various groups(n＝6±s)

?P＜0.05,??P＜0.01 vs control group;△P＜0.05,△△P＜0.01 vs pshuttle-Egr1-hSmac group.

Group A490value (t/h) 0 12 24 48 72 Control 1 1 1 1 1 Pshuttle 0.97±0.04 0.97±0.05 0.95±0.05 0.98±0.04 0.91±0.08 Pshuttle-Egr-1-hSmac 0.95±0.08 0.99±0.04 1.02±0.04 0.97±0.03 0.95±0.02 2.0 Gy 0.91±0.01 0.89±0.05 0.89±0.05?0.86±0.08?0.89±0.02?Pshuttle＋2.0 Gy 0.92±0.09 0.95±0.05 0.86±0.03?0.93±0.08?0.87±0.06?Pshuttle-Egr-1-hSmac＋2.0 Gy 0.83±0.05?△0.73±0.02??△0.69±0.02??△△0.67±0.07??△△0.65±0.06??△△

表2 不同劑量X線照射后各組細(xì)胞A490值Tab.2 A490values of cells after different doses X-ray irradiation in various groups(n＝6,±s)

表2 不同劑量X線照射后各組細(xì)胞A490值Tab.2 A490values of cells after different doses X-ray irradiation in various groups(n＝6,±s)

?P＜0.05,??P＜0.01 vs control group.

Group A490value (D/Gy)0 0.5 1.0 2.0 5.0 Control 1 1 1 1 1 Pshuttle 1.01±0.04 1.02±0.05 1.02±0.03 1.01±0.02 0.99±0.07 Pshuttle-Egr-1-hSmac 1.01±0.02 0.94±0.05 0.91±0.02?0.87±0.02?0.71±0.02??

2.4 各組細(xì)胞劑量存活曲線與對(duì)照組比較, pshuttle組細(xì)胞SF未見明顯變化,而pshuttle-Egr-1-hSmac質(zhì)粒組細(xì)胞SF明顯降低(P＜0.01,見表3)。將各組細(xì)胞的SF進(jìn)行直線相關(guān)與回歸分析得出如下方程:Y＝－9.9316X＋102.45, R2＝0.9661(對(duì)照組);Y＝－9.6487X＋100.62,R2＝0.9559(pshuttle組);Y＝－9.2199X＋80.614,R2＝0.9126(pshuttle-Egr-1-hSmac組)。進(jìn)而計(jì)算得出各組細(xì)胞的D0值為3.31、3.29和2.70,對(duì)照組與pshuttle質(zhì)粒組細(xì)胞D0值相近,而pshuttle-Egr-1-hSmac質(zhì)粒組細(xì)胞D0值明顯降低,說明該組細(xì)胞放射敏感性高。

表3 各組MDA-MB-435細(xì)胞SFTab.3 SF of MDA-MB-435 cells in various groups(n＝3,±s,η/%)

表3 各組MDA-MB-435細(xì)胞SFTab.3 SF of MDA-MB-435 cells in various groups(n＝3,±s,η/%)

?P＜0.01 compared with control group.

Group SF (D/Gy)0 2 4 6 8 10 Control 99.00±1.11 85.20±3.32 70.30±4.07 40.20±1.88 12.30±1.44 9.76±0.89 Pshuttle 95.10±2.32 84.90±5.98 72.30±2.55 39.00±5.14 13.10±1.61 9.78±1.11 Pshuttle-Egr-1-hSmac 90.20±1.29?66.10±3.15?30.20±1.50?11.10±0.36?4.02±0.47?0.61±0.08?

2.5 重組質(zhì)粒聯(lián)合X線照射作用下MDA-MB-435細(xì)胞凋亡率MDA-MB-435細(xì)胞轉(zhuǎn)染pshuttle-Egr-1-hSmac質(zhì)粒后,細(xì)胞凋亡率未見明顯增加;與對(duì)照組比較,2.0 Gy、pshuttle＋2.0 Gy和pshuttle-Egr-1-hSmac＋2.0 Gy組細(xì)胞凋亡率明顯升高(P＜0.05或P＜0.01),其中以pshuttle-Egr-1-hSmac＋2.0 Gy組升高最為明顯;與2.0 Gy組比較,pshuttle-Egr-1-hSmac＋2.0 Gy組細(xì)胞凋亡率明顯升高(P＜0.01)。見表4。

表4 各組MDA-MB-435細(xì)胞凋亡率Tab.4 Apoptotic rates of MDA-MB-435 cells in various groups(n＝4,±s,η/%)

表4 各組MDA-MB-435細(xì)胞凋亡率Tab.4 Apoptotic rates of MDA-MB-435 cells in various groups(n＝4,±s,η/%)

?P＜0.05,??P＜0.01 vs control group;△P＜0.01 vs 2.0 Gy group.

Group Apoptotic rate Control 3.71±0.69 Pshuttle 4.13±0.88 Pshuttle-Egr-1-hSmac 4.03±0.45 2.0 Gy 5.12±1.31?Pshuttle＋2.0 Gy 5.23±1.09?Pshuttle-Egr-1-hSmac＋2.0 Gy 29.12±4.15??△

2.6 重組質(zhì)粒聯(lián)合X線照射作用下MDA-MB-435細(xì)胞周期的細(xì)胞百分率對(duì)照組、pshuttle組和pshuttle-Egr-1-hSmac組各期MDA-MB-435細(xì)胞百分率基本一致;2.0 Gy X線照射后,2.0 Gy組、pshuttle＋2.0 Gy組和pshuttle-Egr-1-hSmac＋2.0 Gy組G0/G1和S期細(xì)胞百分率明顯降低(P＜0.01),而G2/M期細(xì)胞百分率明顯升高(P＜0.01),其中pshuttle-Egr-1-hSmac＋2.0 Gy組細(xì)胞百分率與對(duì)照組、2.0 Gy組和pshuttle-Egr-1-hSmac組比較差異均有統(tǒng)計(jì)學(xué)意義(P＜0.01)。見表5。

3 討論

放療是乳腺癌治療的一個(gè)重要手段,但是放療所致鄰近部位的放射損傷或者患者的輻射耐受嚴(yán)重影響和限制其療效和廣泛應(yīng)用,如何增強(qiáng)乳腺癌細(xì)胞對(duì)射線的敏感性對(duì)提高乳腺癌預(yù)后非常重要[8-9]。輻射敏感啟動(dòng)子Egr-1具有輻射誘導(dǎo)特性,該啟動(dòng)子在正常條件下不增強(qiáng)下游基因的表達(dá),只有在照射條件下,下游基因在Egr-1介導(dǎo)下表達(dá)增強(qiáng),從而增強(qiáng)該基因的效應(yīng)[10-11]。Egr-1可以從時(shí)間和空間上調(diào)控下游基因的體內(nèi)表達(dá),提高放療的敏感性。Smac是2000年發(fā)現(xiàn)的一種存在于線粒體并且調(diào)節(jié)細(xì)胞凋亡的蛋白質(zhì),主要通過抑制凋亡抑制蛋白(inhibitor of apoptosis proteins,IAPs)的活性而發(fā)揮促凋亡作用,是一種線粒體依賴性凋亡途徑[12-15]。

表5 各組MDA-MB-435細(xì)胞周期細(xì)胞百分率Tab.5 Percentages of MDA-MB-435 cells at each cell cycle in various groups(n＝4,±s,η/%)

表5 各組MDA-MB-435細(xì)胞周期細(xì)胞百分率Tab.5 Percentages of MDA-MB-435 cells at each cell cycle in various groups(n＝4,±s,η/%)

?P＜0.05,??P＜0.01 vs control group;△P＜0.05,△△P＜0.01 vs pshuttle-Egr-1-hSmac group;＃P＜0.05,＃＃P＜0.01 vs 2.0 Gy group.

Group Percentage of MDA-MB-435 cells G0/G1S G2/M Control 61.28±1.64 31.56±1.11 8.16±0.64 Pshuttle 62.95±1.8 26.68±1.59 10.37±0.85 Pshuttle-Egr-1-hSmac 60.82±1.22 31.93±1.01 8.25±2.19 2.0 Gy 48.42±1.56?28.30±1.74?23.28±1.43??Pshuttle＋2.0 Gy 49.78±1.78?24.83±2.67?30.39±2.26??Pshuttle-Egr-1-hSmac＋2.0 Gy 36.92±1.83??△△＃22.18±0.85?△＃40.9±1.43??△＃

本研究利用已構(gòu)建成功的pshuttle-Egr-1-hSmac質(zhì)粒轉(zhuǎn)染人乳腺癌MDA-MB-435細(xì)胞,結(jié)果顯示:2.0 Gy X線照射后24 h,對(duì)照組和pshuttle質(zhì)粒組均無Smac m RNA表達(dá),而轉(zhuǎn)染pshuttle-Egr-1-hSmac質(zhì)粒的細(xì)胞經(jīng)過2.0 Gy照射后,Smac mRNA表達(dá)水平隨著時(shí)間的延長逐漸升高,于照射后24和48 h表達(dá)較高,具有一定的時(shí)程效應(yīng)關(guān)系;pshuttle-Egr-1-hSmac質(zhì)粒組轉(zhuǎn)染后進(jìn)行0.5～5.0 Gy X線照射,細(xì)胞中Smac mRNA表達(dá)具有一定的量效關(guān)系,在2.0和5.0 Gy照射后表達(dá)較為明顯;蛋白檢測結(jié)果顯示: pshuttle組MDA-MB-435細(xì)胞中無Smac蛋白表達(dá),經(jīng)0.5、1.0、2.0和5.0 Gy照射后表達(dá)逐漸增多,5.0 Gy照射時(shí)其蛋白表達(dá)最為明顯, 2.0 Gy照射后4 h開始即有表達(dá)且逐漸增多,照射后24 h時(shí)達(dá)到最高。本研究結(jié)果提示:在X線照射后,從Smac m RNA和蛋白水平上看,Egr-1啟動(dòng)子發(fā)揮了輻射誘導(dǎo)增強(qiáng)的作用,具有一定的時(shí)程效應(yīng)和劑量效應(yīng)關(guān)系,而空載體和正常對(duì)照則無表達(dá)。

本研究中MTT檢測結(jié)果顯示:MDA-MB-435細(xì)胞在轉(zhuǎn)染pshuttle和pshuttle-Egr-1-hSmac質(zhì)粒后0～72 h,細(xì)胞增殖能力稍有降低,但不明顯;而各組細(xì)胞經(jīng)過2.0 Gy X射線照射后,細(xì)胞增殖能力明顯降低,尤其以轉(zhuǎn)染了pshuttle-Egr-1-hSmac質(zhì)粒的細(xì)胞增殖能力降低的更明顯;0～5.0 Gy X射線照射后,對(duì)照組MDA-MB-435細(xì)胞增殖能力降低,但差異不明顯;轉(zhuǎn)染pshuttle質(zhì)粒后進(jìn)行相應(yīng)劑量照射,轉(zhuǎn)染了pshuttle-Egr-1-hSmac質(zhì)粒則明顯抑制細(xì)胞增殖。上述結(jié)果表明:轉(zhuǎn)染重組質(zhì)粒對(duì)MDA-MB-435細(xì)胞生長影響不明顯,而X射線照射能抑制MDA-MB-435細(xì)胞生長,在轉(zhuǎn)染后進(jìn)行照射則可加強(qiáng)其抑制效果,可能與輻射誘導(dǎo)Smac表達(dá)有關(guān)。本研究結(jié)果顯示:轉(zhuǎn)染空載體pshuttle后,MDA-MB-435細(xì)胞SF未見明顯改變,即轉(zhuǎn)染空載體未對(duì)細(xì)胞產(chǎn)生明顯影響,而轉(zhuǎn)染pshuttle-Egr-1-hSmac質(zhì)粒的細(xì)胞SF明顯下降,說明MDA-MB-435細(xì)胞存活能力明顯降低,輻射對(duì)其具有明顯影響。本研究結(jié)果顯示:對(duì)照組和pshuttle質(zhì)粒組D0值基本相同,說明放射敏感性無明顯差別;而pshuttle-Egr-1-hSmac質(zhì)粒組的D0值降低,說明細(xì)胞放射敏感性增高,提示轉(zhuǎn)染pshuttle-Egr-1-hSmac質(zhì)粒能增強(qiáng)MDA-MB-435細(xì)胞的放射敏感性;電離輻射可以抑制乳腺癌細(xì)胞的生長,轉(zhuǎn)染pshttle-Egr-1-hSmac質(zhì)粒后進(jìn)行相應(yīng)電離輻射細(xì)胞生長抑制更明顯,可能與電離輻射誘導(dǎo)Smac表達(dá)有關(guān)。

本研究中凋亡檢測結(jié)果顯示:MDA-MB-435細(xì)胞轉(zhuǎn)染pshuttle-Egr-1-hSmac質(zhì)粒后,凋亡率未增加;2.0 Gy照射后,與對(duì)照組比較,pshuttle-Egr-1-hSmac組細(xì)胞凋亡率明顯升高,與其他組比較差異均有統(tǒng)計(jì)學(xué)意義,說明基因聯(lián)合放療對(duì)乳腺癌MDA-MB-435細(xì)胞的促凋亡作用具有良好的效果,能有效地誘導(dǎo)細(xì)胞凋亡、殺傷細(xì)胞。Smac基因是線粒體內(nèi)釋放的凋亡促進(jìn)蛋白,而電離輻射也可以誘導(dǎo)細(xì)胞凋亡,二者的聯(lián)合應(yīng)用勢必增加細(xì)胞凋亡率。本研究結(jié)果顯示:轉(zhuǎn)染pshuttle和pshuttle-Egr-1-hSmac后MDA-MB-435細(xì)胞周期無明顯變化,2.0 Gy、pshuttle＋2.0 Gy和pshuttle-Egr-1-hSmac＋2.0 Gy組MDA-MB-435細(xì)胞G0/G1和S期細(xì)胞百分率明顯下降,G2/M期細(xì)胞百分率明顯升高,其中pshuttle-Egr-1-hSmac ＋2.0 Gy組變化最為明顯,導(dǎo)致MDA-MB-435細(xì)胞G2/M期阻滯。上述結(jié)果可能與電離輻射的作用有密切關(guān)聯(lián),也是輻射誘導(dǎo)細(xì)胞凋亡的主要機(jī)制之一。

綜上所述,pshuttle-Egr-1-hSmac聯(lián)合X線照射能有效地抑制MDA-MB-435細(xì)胞的生長,提高細(xì)胞放射敏感性,影響細(xì)胞周期進(jìn)程,導(dǎo)致MDA-MB-435細(xì)胞發(fā)生G2/M期阻滯,促進(jìn)細(xì)胞凋亡,從而達(dá)到更好的殺傷腫瘤的作用。本研究為提高基因-放療效果開辟了新途徑,為基因-放療的臨床應(yīng)用提供了實(shí)驗(yàn)依據(jù)。

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Inhibitory effect of pshuttle-Egr-1-hSmac plasmid combined with X-ray irradiation on proliferation of breast cancer MDA-MB-435 cells

LIANG Shuo1,WANG Zhi-cheng1,LI Yan-bo1,2,GUO Cai-xia2,GONG Shou-liang1,LIN Cheng-he3
(1.Key Laboratory of Radiobiology,Ministry of Health,School of Public Health,Changchun 130021, China;2.School of Public Health and Family Medicine,Capital Medical University,Beijing 100069, China;3.Department of Nuclear Medicine,First Hospital,Jilin University,Changchun 130021,China)

ObjectiveTo construct the pshuttle-Egr-1-hSmac plasmid and transfect human breast cancer MDA-MB-435 cells,and to observe its radiotherapy enhancing effect on tumor cells.MethodsThe empty vector pshuttle and pshuttle-Egr-1-hSmac plasmid were transfected into MDA-MB-435 cells by liposomal.At different time(4,8,12,24 and 48 h)after irradiation with 2.0 Gy X-ray and 24 h after irradiation with 0.5―5.0 Gy,the total RNA and protein were collected and extracted from these cells to analyze the Smac m RNA and protein expression levels with RT-PCR and Western blotting methods.The cells were divided into control,pshuttle, pshuttle-Egr-1-hSmac,2.0 Gy irradiation group,pshuttle＋2.0 Gy irradiation and pshuttle-Egr-1-hSmac＋2.0 Gy irradiation groups.MTT method was used to evaluate cell proliferation,and the cell survival ability was measured with clone formation assay;AnnexinⅤ/PI double staining and PI single staining were used to examine the apoptosis and cell cycle of MDA-MB-435 cells.ResultsThere was no Smac m RNA expression in MDA-MB-435 cells in control and pshuttle groups,but the Smac mRNA expression levels in MDA-MB-435 cells in pshuttle-Egr-1-hSmac plasmid group were gradually increased with the time prolongation,and reached the maximum at 24 and 48 h;the Smac m RNA expression levels in MDA-MB-435 cells were increased gradually 24 h after irradiation of 0.5―5.0 Gy X-ray with the increasing of irradiation doses,and reached the maximum after 2.0 and 5.0 Gy irradiation.The Smac protein expression levels in pshuttle-Egr-1-hSmac plasmid group were increased gradually with the time prolongation,and reached the maximum at 24 h.The Smac protein expression lervels were increased 24 h afer irradiation of 0,0.5,1.0,2.0 and 5.0 Gy X-ray,especially in 5.0 Gy group.The MTT results showed that the A490values in 2.0 Gy,pshuttle＋2.0 Gy and pshuttle-Egr-1-hSmac groups 24, 48,and 72 h after irradiation were lower than those in control group(P＜0.01);the A490values of MDA-MB-435 cells in pshuttle-Egr-1-hSmac group after 1.0－5.0 Gy X-ray irradiation were lower than those in 0 Gy group(P＜0.05 or P＜0.01);the survival fraction(SF)in pshuttle-Egr-1-hSmac group was lower than those in control group (P＜0.01).The percentages of the cells at G0/G1and S phase in pshuttle-Egr-1-hSmac group were lower than those in 2.0 Gy group(P＜0.01),the percentage of the cells at G2/M phase was higher than that in 2.0 Gy group(P＜0.01);the apoptotic rate of the cells in pshuttle-Egr-1-hSmac group was higher than that in 2.0 Gy group(P＜0.01).ConclusionX-ray irradiation can significantly increase the Smac mRNA and protein expression levels in MDA-MB-435 cells transfected with pshuttle-Egr-1-hSmac plasmid,inhibit the cell survival rate,and induce G2/M arrest and apoptotic increasing;Smac gene combined with radiotherapy could significantly increase the radiosensitivity of breast cancer cells.

Smac gene;Egr-1 promoter;X-ray;gene-radiotherapy;apoptosis

R737.9

A

2014-01-10

國家自然科學(xué)基金資助課題(30870747);吉林大學(xué)基本科研項(xiàng)目資助課題(2012)

梁 碩(1973－),男,吉林省長春市人,講師,醫(yī)學(xué)博士,主要從事腫瘤基因-放射治療方面的研究。

林承赫(Tel:0431-88782717,E-mail:linchh1967＠163.com)