呂一峰，程明榮，吳衍

(上海市浦東新區(qū)周浦醫(yī)院 普通外科，上海 201318)

?

GM-SCF，IL-21和Rae-1基因聯(lián)合治療對肝癌小鼠模型的影響

呂一峰，程明榮Δ，吳衍

(上海市浦東新區(qū)周浦醫(yī)院 普通外科，上海 201318)

目的 將基因巨噬細胞集落刺激因子(GM-SCF)，白介素-21(IL-21)和視黃酸早期轉(zhuǎn)錄因子-1(Rae-1)構(gòu)建成重組質(zhì)粒，觀察重組質(zhì)粒對小鼠肝癌皮下模型的療效。方法 利用RT-PCR的方法將GM-SCF，IL-21和Rae-1構(gòu)建成重組質(zhì)粒，將小鼠皮下植模型分6組，分別為control組，IRES/GFP，IRES/IL21，IRES/GM-SCF，IRES/GM-SCF-IL21和IRES/combination組，每組15只，分別予以相應(yīng)的基因治療，觀察各組60 d的生存率，并觀察小鼠機體干擾素-γ(IFN-γ)和白介素-2(IL-2)水平的變化。結(jié)果 pGM-CSF-GFP-IRES-Rae-1-IL-21已經(jīng)成功構(gòu)建，control組到第14天小鼠全部死亡；IRES/GFP組到第16天小鼠全部死亡；治療60 d后，IRES/GM-SCF組剩2只模型小鼠存活，其存活率為13.33%；IRES/IL21組的模型存活僅剩1只，其存活率為6.67%；IRES/GM-SCF-IL21模型存活11只模型小鼠，其存活率最高為73.33%。IRES/GM-SCF-IL21組的存活率明顯高于其他各組(P<0.05或<0.01)。治療后1～6 d IRES/combination，IRES/GM-SCF-IL21，IRES/GM-SCF和IRES/IL21組的IL-2和INF-γ水平出現(xiàn)逐漸升高，以IRES/combination水平最高，IRES/GM-SCF-IL21次之，IRES/GM-SCF和IRES/IL21組水平相對較低(P<0.01)，6～10 d，IRES/combination組的IL-2和INF-γ水平仍為穩(wěn)步增長，而IRES/GM-SCF-IL21，IRES/GM-SCF和IRES/IL21組出現(xiàn)逐漸下降。治療結(jié)束時，IRES/GM-SCF-IL21組的IL-2和INF-γ水平較IRES/GM-SCF和IRES/IL21組明顯增高(P<0.01)，而IRES/GM-SCF和IRES/IL21組的IL-2和INF-γ水平較IRES/GFP和control組明顯增高(P<0.01)，以IRES/combination在的IL-2和INF-γ水平最高(P<0.01)。結(jié)論 聯(lián)合GM-SCF，IL-21和Rae-1基因具有明顯抑制小鼠肝癌的作用，其機理與機體的免疫激活有關(guān)。

巨噬細胞集落刺激因子；白介素-21；視黃酸早期轉(zhuǎn)錄因子-1；肝癌

腫瘤細胞能夠通過多種機制逃避機體免疫系統(tǒng)識別和攻擊，從而得以在體內(nèi)生存和增殖。提高機體免疫細胞活性，或者促使腫瘤細胞表達能被免疫細胞識別的抗體或者配體，就能夠促進機體自身識別和殺滅腫瘤細胞，從而達到腫瘤治療的目的。前期實驗及文獻均發(fā)現(xiàn)巨噬細胞集落刺激因子(GM-SCF)和白介素-21(IL-21)基因融合質(zhì)粒能明顯抑制腫瘤的生長，并能促進NK細胞及CTL細胞的免疫活性[1-4]。體外實驗發(fā)現(xiàn)高表達視黃酸早期轉(zhuǎn)錄因子-1(Rae-1)的腫瘤細胞更易被免疫細胞所識別[5]。本研究在前期實驗的基礎(chǔ)上，將Rae-1基因和綠色熒光蛋白(GFP)基因與融合質(zhì)粒GM-CSF-IL-21通過基因拼接技術(shù)拼接成多功能融合質(zhì)粒，其中GM-CSF和IL-21基因的功能是促進機體免疫系統(tǒng)的激活，尤其是NK細胞和CTL細胞的激活。Rae-1基因轉(zhuǎn)染到腫瘤細胞后，主要促進腫瘤細胞表達Rae-1，后者促進腫瘤細胞被NK細胞和CTL細胞的識別。GFP基因主要用于監(jiān)測基因體內(nèi)外的轉(zhuǎn)染情況。采用GM-SCF、IL-21和Rae-1三基因聯(lián)合治療小鼠原位肝癌模型，現(xiàn)報道如下。

1 材料與方法

1.1 材料及儀器 Balb/c小鼠購自上海復(fù)旦大學(xué)動物中心[SCXK(滬)2007-0002]，雌性，7周齡，體質(zhì)量約20 g左右。所有小鼠飼養(yǎng)條件為SPFB級，小鼠肝癌細胞(H22)購自 the China Center for Type Culture Collection (CCTCC,武漢, 中國)。RPMI 1640培養(yǎng)基(四季青生物工程材料有限公司產(chǎn)品，杭州)。實驗得到上海市周浦醫(yī)院和復(fù)旦大學(xué)醫(yī)學(xué)院倫理委員會審查批準。四甲基偶氮唑藍(MTT，Sigma有限公司, 上海)；大劑量質(zhì)粒抽提試劑盒購自Promega公司；干擾素(IFN-γ)和白細胞介素2(IL-2)、酶聯(lián)免疫吸附(ELISA)測定試劑盒(圣克魯斯生物工學(xué), 美國)，酶標儀(美國Bio-rad公司)。

1.2 方法

1.2.1 重組質(zhì)粒pGM-CSF-GFP -IRES-IL-21-Rae-1的構(gòu)建：從小鼠脾臟中調(diào)取GM-CSF、IL-21基因，化學(xué)合成Rae-1，GFP基因。針對GM-CSF和IL-21基因的CDS區(qū)序列設(shè)計并合成PCR 引物(見表1)，在GM-CSF基因的5’端添加XhoI酶切位點，3’端添加EcoRI酶切位點。在GFP基因的5’端添加EcoRI酶切位點，3’端添加MluI酶切位點。在Rae-1基因的5’端添加Xba1酶切位點，3’端添加SalI酶切位點。在IL-21基因的5’端添加SalI酶切位點，3’端添加Not1酶切位點。GFP片段與目的載體pIRES的連接，獲得pGFP-IRES；GM-CSF基因與目的載體pGFP-IRES的連接，獲得pGM-CSF-GFP-IRES載體；將Rae-1基因PCR產(chǎn)物插入pGM-CSF-GFP-IRES載體，獲得pGM-CSF-GFP-IRES-Rae-1載體；將IL21片段插入pGM-CSF-GFP-IRES-Rae-1載體，獲得pGM-CSF-GFP-IRES-Rae-1-IL-21真核表達載體。MCS A處是GM-CSF-GFP，MCS B處是Rae-1-IL-21。

表1 擴增GM-CSF、GFP、Rae-1和 IL-21基因全長的引物序列Tab.1 Amplification of GM-CSF, GFP, Rae-1, and IL-21 gene sequences

加粗字體分別為酶切位點XhoI和EcoRI；EcoRI和MluI；XbaI和SalI；SalI和NotI

1.3 動物模型建立 用小鼠肝癌細胞株(H22)建立小鼠肝癌皮下模型[3]。將小鼠處死后解剖出腫瘤組織，挑選生長旺盛新鮮的腫瘤組織，制成6×107個/mL 的腫瘤細胞懸液。用1 mL注射器注射腫瘤細胞懸液50 μL注入右前肢腋窩皮下。

1.3.1 重組質(zhì)粒治療小鼠皮下肝癌模型：待小鼠肝癌皮下模型成模后第5天，肝癌組織約4～6 mm，按隨機數(shù)字法，將小鼠分為6組，分別為control組，IRES/GFP，IRES/GM-SCF，IRES/IL21，IRES/GM-SCF-IL21和IRES/combination組，每組15只。control組：腫瘤內(nèi)注射PBS 200 μL；IRES/GFP，IRES/GM-SCF，IRES/IL21，IRES/GM-SCF-IL21和IRES/combination組分為在腫瘤內(nèi)注射200 μL(分別含100 μg相應(yīng)的質(zhì)粒)，每天1次，連續(xù)5天，到治療第10天，60天后，觀察并記錄各組小鼠的生存率。

1.4 IL-2和INF-γ的ELISA檢測 各組分別于治療0、2、4、6、8、10 d檢測模型IL-2和INF-γ的水平，其具體方法為：小鼠中分離各組小鼠的血清標本，-20 ℃保存，取血清標本于37 ℃恒溫箱解凍，雙蒸餾水稀釋至500 mL,倍比稀釋標準品至8000 μg/L,每孔加入測定稀釋液150 μL，繼加入標準品或測定樣品50 μL，15 min內(nèi)完成，充分振蕩搖勻后室溫孵育2 h；取盡液體后，加入400 μL洗滌液重復(fù)。洗滌4次，加入辣根過氧化物酶標記IL-2和INF-γ多抗200 μL用上述方法振蕩搖勻，室溫孵育2 h;再取盡液體后，加入400 μL洗滌液重復(fù)洗滌4次;將顯色劑A,B等量混勻后加入200 μL酶標抗體，室溫避光孵育30 min；加50 μL終止液終止酶反應(yīng)；立即置酶標儀中測定，以450 nm波長讀光密度值，根據(jù)標準曲線計算樣品的含量。

2 結(jié)果

2.1 pGM-CSF-GFP-IRES-Rae-1-IL21重組載體酶切鑒定 從圖1可知，采用EcoRI和MluI雙酶切pGM-CSF-GFP-IRES-Rae-1-IL21得目的條帶(紅色箭頭)與目的基因GFP大小一致的片段；采用XhoI和EcoRI雙酶切pGM-CSF-GFP-IRES-Rae-1-IL21得目的條帶(紅色箭頭)與目的基因GM-CSF大小一致的片段；采用Xba1和Sal1雙酶切pGM-CSF-GFP-IRES-Rae-1-IL21得目的條帶(紅色箭頭)與目的Rae-1一致的片段；采用Sal1和Not1雙酶切pGM-CSF-GFP-IRES-Rae-1-IL21得目的條帶(紅色箭頭)與目的基因IL21一致的片段，得到的基因GFP，GM-CSF，Rae-1和IL21與Genbank的原序列完全一致，證明載體構(gòu)建成功。

圖1 pGM-CSF-GFP-IRES-Rae-1-IL21重組載體酶切鑒定泳道M：Marker 5000(從上到下分別為5000,3000,2000,1500,1000,750,500,250,100bp)；泳道1：EcoRI和MluI酶切pGM-CSF-GFP-IRES-Rae-1-IL21(目的條帶GFP：720bp)；泳道2：XhoI和EcoRI酶切pGM-CSF-GFP-IRES-Rae-1-IL21(目的條帶GM-CSF：425bp)；泳道3：Xba1和Sal1酶切pGM-CSF-GFP-IRES-Rae-1-IL21(目的條帶Rae-1：1106bp)；泳道4：Sal1和Not1酶切pGM-CSF-GFP-IRES-Rae-1-IL21(目的條帶IL21：441bp)；泳道5：XhoI和Not1酶切pGM-CSF-GFP-IRES-Rae-1-IL21(目的條帶GM-CSF-GFP-IRES-Rae-1-IL21：3.3kb)；泳道6：XhoI和Not1酶切pIRES空載(目的條帶：5.5kb +673bp(IRES))；泳道7：pGM-CSF-GFP-IRES-Rae-1-IL21質(zhì)粒注：因IL21的381位有EcoRI酶切位點所以用EcoRI和MluI酶切時有條2160bp條帶(泳道1)，用XhoI和EcoRI酶切時有條2880的條帶(泳道2)，見上圖中藍色箭頭指示。Fig.1 Design and construction of the new expression vector pGM-CSF-GFP- IRES-Rae-1-IL21 and their enzyme cleavage products.Lane M： Marker 5000 (5000, 3000, 2000, 1500, 1000, 750, 500, 250, and 100 bp ordered reading top to bottom).Lane 1, EcoRI and MluI cleavage of pGM-CSF-GFP-IRES- Rae-1-IL21 (target band GFP, 720 bp).Lane 2： XhoI and EcoRI cleavage of pGM-CSF-GFP- IRES-Rae-1-IL21 (target band GM-CSF, 425 bp).Lane 3： Xba1 and Sal1 cleavage of pGM-CSF-GFP-IRES-Rae-1-IL21 (target band Rae-1, 1106 bp).Lane 4： Sal1 and Not1 cleavage of pGM-CSF-GFP-IRES-Rae-1-IL21 (target band IL21, 441 bp).Lane 5： XhoI and Not1 cleavage of pGM-CSF-GFP-IRES-Rae-1-IL21 (target band GM-CSF-GFP-IRES- Rae-1-IL21, 3.3 kb).Lane 6： XhoI and Not1 cleavage of pIRES (target band IRES, 5.5kb +673bp);Lane 7： pGM-CSF-GFP-IRES-Rae-1-IL21 plasmidNote: EcoRI cleavage site was the 381 of IL21 sequence.So, a band of 2160 bp is present in EcoRI and MluI cleavage band (Lane 1) and a band of 2880 bp is present in EcoRI and XhoI cleavage band (Lane 2), indicated by blue arrow.

2.2 pGM-CSF-GFP-IRES-Rae-1-IL21重組質(zhì)粒對小鼠肝癌模型的抑制作用 將原位肝癌移植模型，隨機分為6組，每組15只，按照前述的方法予以治療，并用Kaplan-Meier生存分析進行統(tǒng)計分析。control組從第6天開始有小鼠死亡，到第14天小鼠全部死亡，其中位生存時間為11 d；IRES/GFP組從第5天開始死亡，到第16 d小鼠全部死亡，中位生存時間為12 d；IRES/GM-SCF組小鼠第17天開始死亡，到觀察結(jié)束第60天剩2只模型小鼠存活，中位生存時間為35 d，其存活率為13.33%；IRES/IL21組的模型小鼠從第15天開始出現(xiàn)小鼠死亡，到第60天小鼠存活僅剩1只，中位生存時間為37 d，其存活率為6.67%；IRES/GM-CSF-IL-21模型小鼠從20 d開始死亡，到第60天存活4只小鼠模型，中位生存時間39 d，其存活率為26.67%；IRES/combination模型小鼠從30 d開始死亡，到第60天存活11只模型小鼠，未超過半數(shù)小鼠死亡，其存活率最高為73.33%。IRES/GM-SCF-IL21組的存活率明顯高于其他各組(P<0.05，P<0.01)。

圖2 重組質(zhì)粒治療后對小鼠荷肝癌模型的抑制作用(n=15)Fig.2 Inhibitory effects of different plasmids on liver cancer of tumor-bearing mice (n=15)

2.3 pGM-CSF-GFP-IRES-Rae-1-IL21重組質(zhì)粒對小鼠IL-2和INF-γ水平的影響 從圖3，表2～3可知，治療后1～6 d IRES/combination，IRES/GM-SCF-IL21，IRES/GM-SCF和IRES/IL21組的IL-2和INF-γ水平出現(xiàn)逐漸升高，以IRES/combination水平最高，IRES/GM-SCF-IL21次之，IRES/GM-SCF和IRES/IL21組水平相對較低(P<0.01)，6～10 d，IRES/combination在的IL-2和INF-γ水平仍為穩(wěn)步增長，而IRES/GM-SCF-IL21，IRES/GM-SCF和IRES/IL21組出現(xiàn)逐漸下降。治療結(jié)束時，IRES/GM-SCF-IL21組的IL-2和INF-γ水平較IRES/GM-SCF和IRES/IL21組明顯增高(P<0.01)，而IRES/GM-SCF和IRES/IL21組的IL-2和INF-γ水平較IRES/GFP和control組明顯增高(P<0.01)，以IRES/combination在的IL-2和INF-γ水平最高(P<0.01)，而IRES/GM-SCF和IRES/IL21組，IRES/GFP和control組之間的IL-2和INF-γ水平差異無統(tǒng)計學(xué)意義。在1～10 d，IRES/GFP和control組小鼠的IL-2和INF-γ水平變化不明顯。

圖3 各組對模型小鼠血清IL-2和INF-γ水平的影響(n=3)**P<0.01，與control組比較，##P<0.01，與IRES/GM-SCF或IRES/IL-21組比較，△△P<0.01，與IRES/GM-SCF-IL21組比較Fig.3 IL-2 and INF-γ levels in mouse serum and expression of Rae-1 in liver cancer tissue in each treatment group(n=3)*P<0.05 , ** P<0.01, compared with control group; #P<0.05 ,##P<0.01, compared with IRES/GM-SCF and IRES/IL-21 group;△P<0.05, △△P<0.01, compared with IRES/GM-SCF-IL21

組別治療天數(shù)(d)0246810Control693.2±20.4703.4±43.2698.3±68.5672±46.3689.3±43.2657.8±44.4IRES/GFP727.1±21.3698.3±41.2719.3±72.1649.3±56.4693.2±46.3696.1±65.3IRES/GM-CSF735.6±22.35996.3±42.31243.5±76.51562.4±68.51446.5±56.31196.5±75.2IRES/IL-21716.5±22.3986.5±56.41165.2±84.21364.3±75.61265.4±68.71062.4±89.6IRES/GM-CSF-IL-21698.3±26.21273.0±46.21732.0±75.351969.0±67.22039.0±69.51925.5±76.5IRES/combination756.3±24.31703.0±52.32140.0±91.22256.0±63.52270.3±71.32291.8±84.2

表3 各組治療后對模型小鼠機體INF-γ水平的動態(tài)變化(pg/mL)Tab.3 Dynamic change of INF-γlevels in model mice after different treatment(pg / mL)

3 討論

腫瘤的發(fā)生發(fā)展與機體的免疫具有明顯的相關(guān)性[6]，肝癌與其他腫瘤一樣出現(xiàn)了機體的免疫功能的抑制，由于肝癌細胞表面表達的分子的改變，可以使肝癌細胞逃避免疫監(jiān)視的識別[7]，導(dǎo)致肝癌細胞不能被NK細胞和CTL細胞識別并清除，同時腫瘤細胞表達被CTL和NK細胞識別的分子(如配體MHC-Ⅰ類相關(guān)分子，小鼠中為Rae-1)明顯減少或者消失，導(dǎo)致肝癌細胞能在機體內(nèi)長期存活[8-9]?，F(xiàn)在研究較多的是用GM-SCF和(或)IL21基因治療腫瘤，以提高CTL和NK細胞活性[10-11]，沒有同時提高腫瘤組織表達又能被NK和CTL細胞識別的基因，或者僅有提高腫瘤組織表達但不能被NK和CTL細胞識別的基因[12]，而沒有同時提高能促進機體NK和CTL細胞活性的基因治療。為了提高機體的免疫功能，同時又增強腫瘤組織表達被免疫細胞識別的蛋白，本實驗成功構(gòu)建了“免疫逃逸抑制系統(tǒng)”即重組質(zhì)粒pGM-CSF-GFP-IRES-Rae-1-IL21，且通過RT-PCR和基因測序證實。前期實驗[3]已經(jīng)證實GM-SCF和IL-21能夠明顯抑制小鼠肝癌模型，通過激活機體的NK和CTL細胞活性達到抑制小鼠模型的作用。本組實驗在前期基礎(chǔ)上構(gòu)建了重組質(zhì)粒，對小鼠肝癌皮下模型治療發(fā)現(xiàn)，通過60d的觀察，IRES/combination的生存期最長，其存活率高達73.33%，明顯優(yōu)于IRES/GM-SCF-IL21，IRES/GM-SCF和IRES/IL21組。而雙基因的IRES/GM-SCF-IL21組的療效明顯優(yōu)于單基因治療組(IRES/GM-SCF和IRES/IL21組)，說明IRES/combination的聯(lián)合治療抑制模型小鼠生長作用最強，聯(lián)合GM-SCF，IL-21和Rae-1基因治療小鼠肝癌模型的療效確切。

細胞因子是免疫細胞產(chǎn)生的一類小分子活性物質(zhì)，具有明顯的免疫調(diào)節(jié)作用，并在腫瘤的免疫中具有重要作用[13-14]?，F(xiàn)已知GM-SCF和IL-21可以促進機體的INF-γ和IL-2水平的表達，提高機體CTL細胞和NK細胞的活性，從而達到抑制腫瘤生長的作用[15-16]。本組研究表明GM-CSF，IL-21和Rae-1聯(lián)合治療后，小鼠體內(nèi)的INF-γ和IL-2水平呈持續(xù)升高，并且呈高水平的表達，較其他基因治療明顯增強。同時發(fā)現(xiàn)IRES/combination組的CTL和NK細胞的數(shù)量和活性均明顯增強，較IRES/GM-SCF-IL-21，IRES/GM-SCF和IRES/IL-21組明顯增強，說明GM-SCF,IL-21和Rea-1三種基因具有協(xié)同促進INF-γ和IL-2水平，及CTL和NK細胞的數(shù)量及活性。INF-γ是一族糖蛋白，具有明顯的抗病毒、免疫調(diào)節(jié)和抗增殖等生物特性，其主要由CTL和NK細胞分泌[17-18]，對腫瘤細胞具有明顯的抑制作用，同時具有一定的免疫調(diào)節(jié)活性，能激活和促進單核巨噬細胞和NK細胞的吞噬作用，促進B細胞和T細胞的分化成熟，增強CTL細胞的細胞毒作用，并可以誘導(dǎo)腫瘤細胞表達MHC-I類抗原的表達，增強免疫細胞如CTL和NK細胞的識別[19]。IL-2作為T淋巴細胞的生長因子，對T淋巴細胞的激活、增殖，對B細胞和巨噬細胞的活化過程具有重要作用，同時活化的T細胞可以分泌IL-2[20]。故說明GM-SCF,IL-21和Rea-1 3種基因促進肝癌小鼠皮下模型機體的免疫系統(tǒng)的激活，促進機體NK細胞和CTL數(shù)量和細胞毒性增強，激活的CTL細胞和NK 細胞分泌INF-γ水平明顯增加，而高水平INF-γ又促進NK細胞和CTL細胞的激活和細胞毒性；同樣激活的CTL細胞分泌的IL-2水平明顯增加，而IL-2又促進機體CTL細胞的激活，形成良性循環(huán)，從而達到抑制腫瘤生長作用。

綜上所述，聯(lián)合GM-SCF，IL-21和Rae-1基因治療明顯抑制小鼠肝癌生長的作用，其機理與機體的免疫激活有關(guān)。

[1] Dou J, Hong X, Zhao F, et al.Investigation of GM-CSF immune accessory effects in tumor-bearing mice by direct gene immunization[J].Immunol Invest,2006,35(2):227-237.

[2] Zhao F, Dou J, Wang J, et al.Investigation on the anti-tumor efficacy by expression of GPI-anchored mIL-21 on the surface of B16F10 cells in C57BL/6 mice[J].Immunobiology,2010,215(2):89-100.

[3] Cheng M, Li Q, Wan T, et al.Synthesis and efficient hepatocyte targeting of galactosylated chitosan as a gene carrier in vitro and in vivo[J].J Biomed Mater Res B Appl Biomater,2011,99(1):70-80.

[4] Pan X C, Li L, Mao J J, et al.Synergistic effects of soluble PD-1 and IL-21 on antitumor immunity against H22 murine hepatocellular carcinoma[J].Oncol Lett,2013,5(1):90-96.

[5] Routes J M,Ryan S, Morris K, et al.Adenovirus serotype 5 E1A sensitizes tumor cells to NKG2D-dependent NK cell lysis and tumor rejection[J].J Exp Med,2005,202(11):1477-1482.

[6] Yigit R, Massuger L F, Figdor C G, et al.Ovarian cancer creates a suppressive microenvironment to escape immune elimination[J].Gynecol Oncol,2010,117(2):366-372.

[7] Nagao M, Nakajima Y, Hisanaga M, et al.The alteration of Fas receptor and ligand system in hepatocellular carcinomas: how do hepatoma cells escape from the host immune surveillance in vivo?[J].Hepatology,1999,30(2):413-421.

[8] Siddle H V, Kreiss A, Tovar C, et al.Reversible epigenetic down-regulation of MHC molecules by devil facial tumour disease illustrates immune escape by a contagious cancer[J].Proc Natl Acad Sci U S A,2013,110(13):5103-5108.

[9] Li M S, Ma Q L, Chen Q, et al.Alpha-fetoprotein triggers hepatoma cells escaping from immune surveillance through altering the expression of Fas/FasL and tumor necrosis factor related apoptosis-inducing ligand and its receptor of lymphocytes and liver cancer cells[J].World J Gastroenterol,2005,11(17):2564-2569.

[10] Zhang P, Wang J, Wang D, et al.Dendritic cell vaccine modified by Ag85A gene enhances anti-tumor immunity against bladder cancer[J].Int Immunopharmacol,2012,14(3):252-260.

[11] Onishi H, Koya N, Kiyota A, et al.A new method for rapid cytotoxic T-lymphocyte induction using a multiple cytokine cocktail[J].Anticancer Res,2012,32(6):2385-2390.

[12] Baba T,Iwasaki S, Maruoka T, et al.Rat CD4+CD8+ macrophages kill tumor cells through an NKG2D- and granzyme/perforin-dependent mechanism[J].J Immunol,2008,180(5):2999-3006.

[13] Barber A, Sentman C L.Chimeric NKG2D T cells require both T cell- and host-derived cytokine secretion and perforin expression to increase tumor antigen presentation and systemic immunity[J].J Immunol,2009,183(4):2365-2372.

[14] Pellegrini M, Mak T W, Ohashi P S.Fighting cancers from within: augmenting tumor immunity with cytokine therapy[J].Trends Pharmacol Sci,2010,31(8):356-363.

[15] Maeda M, Yanagawa Y, Iwabuchi K, et al.IL-21 enhances dendritic cell ability to induce interferon-gamma production by natural killer T cells[J].Immunobiology,2007,212(7):537-547.

[16] Penafuerte C, Bautista-Lopez N, Boulassel M R, et al.The human ortholog of granulocyte macrophage colony-stimulating factor and interleukin-2 fusion protein induces potent ex vivo natural killer cell activation and maturation[J].Cancer Res,2009,69(23):9020-9028.

[17] Ramstead A G, Schepetkin I A, Quinn M T, et al.Oenothein B, a cyclic dimeric ellagitannin isolated from Epilobium angustifolium, enhances IFNgamma production by lymphocytes[J].PLoS One,2012,7(11):e50546.

[18] Ramstead A G, Jutila M A.Complex role of gammadelta T-cell-derived cytokines and growth factors in cancer[J].J Interferon Cytokine Res,2012,32(12):563-569.

[19] Zanon R G, Cartarozzi L P, Victorio S C, et al.Interferon (IFN) beta treatment induces major histocompatibility complex (MHC) class I expression in the spinal cord and enhances axonal growth and motor function recovery following sciatic nerve crush in mice[J].Neuropathol Appl Neurobiol,2010,36(6):515-534.

[20] Zhao M F, Qu X J, Qu J L, et al.The role of E3 ubiquitin ligase Cbl proteins in interleukin-2-induced Jurkat T-cell activation[J].Biomed Res Int,2013,2013:430861.

(編校：譚玲)

Inhibitory effect of joint gene expression of GM-SCF, IL-21, and Rae-1 in treatment of liver cancer model mouse

LV Yi-feng, CHENG Ming-rongΔ, WU Yan

(Department of General Surgery, Pudong New Area Zhoupu Hospital, Shanghai 201318,China)

ObjectiveTo observe recombinant plasmids were constructed with the macrophage colony-stimulating factor (GM-SCF), interleukin -21 (IL-21) and retinoic acid early transcription factor -1 (Rae-1), and observe the inhibitory effects in subcutaneous liver cancer model in mice with the recombinant plasmids.MethodsThe recombinant plasmids of GM-SCF, IL-21 and Rae-1 were constructed with RT-PCR method, mouse model was constructed, the model mice were randomly divided into six groups including control, IRES/GFP, IRES/IL21, IRES/GM-SCF, IRES/GM-SCF-IL21 and IRES/combination with 10 mice included in each group, each groups (15 mice) were treated with the corresponding gene therapy.The survival rate were observed after 60 days.The blood levels of interferon -γ (IFN- γ) and interleukin -2 (IL-2) were detected in each group.ResultsThe pGM-CSF-GFP-IRES-Rae-1-IL-21 has been successfully constructed.All mice had demised 14 and 16 days after treatment in the control and IRES/GFP groups, respectively.There were 2, 1, 11 mice remaining after 60 days of treatment in the IRES/GM-SCF, IRES/IL21 and IRES/GM-SCF-IL21 groups respectively.The survival rate of mice at 60 days of treatment was 73.33%, 13.33%, and 6.67% for groups IRES/GM-SCF-IL21, IRES/GM-SCF and IRES/IL21, respectively.The survival rate of the mice was significantly higher in IRES/GM-SCF-IL21 than the other groups.The levels of IL-2 and INF-γ of mice 1-6 days after treatment gradually increased in the IRES/combination groups, including IRES/GM-SCF-IL21, IRES/GM-SCF and IRES/IL21.They were highest in the IRES/combination group and lowest (P< 0.01) in the IRES/GM-SCF and IRES/IL21 groups, with the IRES/GM-SCF-IL21 group showing intermediate levels.By 6-10 days after treatment, IL-2 and INF-γ levels had stably increased in the IRES/combination groups, but had gradually decreased in the IRES/GM-SCF-IL21, IRES/GM-SCF and IRES/IL21 groups.At the end of treatment, IL-2 and INF-γ levels were significantly (P<0.01) higher in the IRES/GM-SCF-IL21 than were found in either the IRES/GM-SCF group or IRES/IL21 group, which were also significantly (P<0.01) higher than either the IRES/GFP or control groups.The levels of IL-2 and INF-γ were highest in the IRES/combination group (P<0.01) and not significantly different among the IRES/GM-SCF, IRES/IL21, IRES/GFP, and control groups.ConclusionThe inhibitory effects in subcutaneous liver cancer model in mice were obvious significantly, and its mechanism maybe be related to the activation of the body’s immune.

macrophage colony-stimulating factor; interleukin-21; retinoic acid early transcription factor-1; liver cancer

上海市自然基金生物引導(dǎo)類基因(114119a4700)；上海市科委納米專項(12nm0502202)

呂一峰，男，碩士，主治醫(yī)師，研究方向：肝癌的靶向治療，E-mail：ssmu_lyf@163.com；程明榮，通訊作者， 男，碩士，副主任醫(yī)師，研究方向：肝癌的靶向治療，E-mail：15921300758@126.com。

R574.62；R574.63

A

1005-1678(2015)06-0017-05