GM-SCF，IL-21和Rae-1基因联合治疗对肝癌小鼠模型的影响

吕一峰，程明荣，吴衍

(上海市浦东新区周浦医院 普通外科，上海 201318)



GM-SCF，IL-21和Rae-1基因联合治疗对肝癌小鼠模型的影响

吕一峰，程明荣Δ，吴衍

(上海市浦东新区周浦医院 普通外科，上海 201318)

目的 将基因巨噬细胞集落刺激因子(GM-SCF)，白介素-21(IL-21)和视黄酸早期转录因子-1(Rae-1)构建成重组质粒，观察重组质粒对小鼠肝癌皮下模型的疗效。方法 利用RT-PCR的方法将GM-SCF，IL-21和Rae-1构建成重组质粒，将小鼠皮下植模型分6组，分别为control组，IRES/GFP，IRES/IL21，IRES/GM-SCF，IRES/GM-SCF-IL21和IRES/combination组，每组15只，分别予以相应的基因治疗，观察各组60 d的生存率，并观察小鼠机体干扰素-γ(IFN-γ)和白介素-2(IL-2)水平的变化。结果 pGM-CSF-GFP-IRES-Rae-1-IL-21已经成功构建，control组到第14天小鼠全部死亡；IRES/GFP组到第16天小鼠全部死亡；治疗60 d后，IRES/GM-SCF组剩2只模型小鼠存活，其存活率为13.33%；IRES/IL21组的模型存活仅剩1只，其存活率为6.67%；IRES/GM-SCF-IL21模型存活11只模型小鼠，其存活率最高为73.33%。IRES/GM-SCF-IL21组的存活率明显高于其他各组(P<0.05或<0.01)。治疗后1～6 d IRES/combination，IRES/GM-SCF-IL21，IRES/GM-SCF和IRES/IL21组的IL-2和INF-γ水平出现逐渐升高，以IRES/combination水平最高，IRES/GM-SCF-IL21次之，IRES/GM-SCF和IRES/IL21组水平相对较低(P<0.01)，6～10 d，IRES/combination组的IL-2和INF-γ水平仍为稳步增长，而IRES/GM-SCF-IL21，IRES/GM-SCF和IRES/IL21组出现逐渐下降。治疗结束时，IRES/GM-SCF-IL21组的IL-2和INF-γ水平较IRES/GM-SCF和IRES/IL21组明显增高(P<0.01)，而IRES/GM-SCF和IRES/IL21组的IL-2和INF-γ水平较IRES/GFP和control组明显增高(P<0.01)，以IRES/combination在的IL-2和INF-γ水平最高(P<0.01)。结论 联合GM-SCF，IL-21和Rae-1基因具有明显抑制小鼠肝癌的作用，其机理与机体的免疫激活有关。

巨噬细胞集落刺激因子；白介素-21；视黄酸早期转录因子-1；肝癌

肿瘤细胞能够通过多种机制逃避机体免疫系统识别和攻击，从而得以在体内生存和增殖。提高机体免疫细胞活性，或者促使肿瘤细胞表达能被免疫细胞识别的抗体或者配体，就能够促进机体自身识别和杀灭肿瘤细胞，从而达到肿瘤治疗的目的。前期实验及文献均发现巨噬细胞集落刺激因子(GM-SCF)和白介素-21(IL-21)基因融合质粒能明显抑制肿瘤的生长，并能促进NK细胞及CTL细胞的免疫活性[1-4]。体外实验发现高表达视黄酸早期转录因子-1(Rae-1)的肿瘤细胞更易被免疫细胞所识别[5]。本研究在前期实验的基础上，将Rae-1基因和绿色荧光蛋白(GFP)基因与融合质粒GM-CSF-IL-21通过基因拼接技术拼接成多功能融合质粒，其中GM-CSF和IL-21基因的功能是促进机体免疫系统的激活，尤其是NK细胞和CTL细胞的激活。Rae-1基因转染到肿瘤细胞后，主要促进肿瘤细胞表达Rae-1，后者促进肿瘤细胞被NK细胞和CTL细胞的识别。GFP基因主要用于监测基因体内外的转染情况。采用GM-SCF、IL-21和Rae-1三基因联合治疗小鼠原位肝癌模型，现报道如下。

1 材料与方法

1.1 材料及仪器 Balb/c小鼠购自上海复旦大学动物中心[SCXK(沪)2007-0002]，雌性，7周龄，体质量约20 g左右。所有小鼠饲养条件为SPFB级，小鼠肝癌细胞(H22)购自 the China Center for Type Culture Collection (CCTCC,武汉, 中国)。RPMI 1640培养基(四季青生物工程材料有限公司产品，杭州)。实验得到上海市周浦医院和复旦大学医学院伦理委员会审查批准。四甲基偶氮唑蓝(MTT，Sigma有限公司, 上海)；大剂量质粒抽提试剂盒购自Promega公司；干扰素(IFN-γ)和白细胞介素2(IL-2)、酶联免疫吸附(ELISA)测定试剂盒(圣克鲁斯生物工学, 美国)，酶标仪(美国Bio-rad公司)。

1.2 方法

1.2.1 重组质粒pGM-CSF-GFP -IRES-IL-21-Rae-1的构建：从小鼠脾脏中调取GM-CSF、IL-21基因，化学合成Rae-1，GFP基因。针对GM-CSF和IL-21基因的CDS区序列设计并合成PCR 引物(见表1)，在GM-CSF基因的5’端添加XhoI酶切位点，3’端添加EcoRI酶切位点。在GFP基因的5’端添加EcoRI酶切位点，3’端添加MluI酶切位点。在Rae-1基因的5’端添加Xba1酶切位点，3’端添加SalI酶切位点。在IL-21基因的5’端添加SalI酶切位点，3’端添加Not1酶切位点。GFP片段与目的载体pIRES的连接，获得pGFP-IRES；GM-CSF基因与目的载体pGFP-IRES的连接，获得pGM-CSF-GFP-IRES载体；将Rae-1基因PCR产物插入pGM-CSF-GFP-IRES载体，获得pGM-CSF-GFP-IRES-Rae-1载体；将IL21片段插入pGM-CSF-GFP-IRES-Rae-1载体，获得pGM-CSF-GFP-IRES-Rae-1-IL-21真核表达载体。MCS A处是GM-CSF-GFP，MCS B处是Rae-1-IL-21。

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

加粗字体分别为酶切位点XhoI和EcoRI；EcoRI和MluI；XbaI和SalI；SalI和NotI

1.3 动物模型建立 用小鼠肝癌细胞株(H22)建立小鼠肝癌皮下模型[3]。将小鼠处死后解剖出肿瘤组织，挑选生长旺盛新鲜的肿瘤组织，制成6×107个/mL 的肿瘤细胞悬液。用1 mL注射器注射肿瘤细胞悬液50 μL注入右前肢腋窝皮下。

1.3.1 重组质粒治疗小鼠皮下肝癌模型：待小鼠肝癌皮下模型成模后第5天，肝癌组织约4～6 mm，按随机数字法，将小鼠分为6组，分别为control组，IRES/GFP，IRES/GM-SCF，IRES/IL21，IRES/GM-SCF-IL21和IRES/combination组，每组15只。control组：肿瘤内注射PBS 200 μL；IRES/GFP，IRES/GM-SCF，IRES/IL21，IRES/GM-SCF-IL21和IRES/combination组分为在肿瘤内注射200 μL(分别含100 μg相应的质粒)，每天1次，连续5天，到治疗第10天，60天后，观察并记录各组小鼠的生存率。

1.4 IL-2和INF-γ的ELISA检测 各组分别于治疗0、2、4、6、8、10 d检测模型IL-2和INF-γ的水平，其具体方法为：小鼠中分离各组小鼠的血清标本，-20 ℃保存，取血清标本于37 ℃恒温箱解冻，双蒸馏水稀释至500 mL,倍比稀释标准品至8000 μg/L,每孔加入测定稀释液150 μL，继加入标准品或测定样品50 μL，15 min内完成，充分振荡摇匀后室温孵育2 h；取尽液体后，加入400 μL洗涤液重复。洗涤4次，加入辣根过氧化物酶标记IL-2和INF-γ多抗200 μL用上述方法振荡摇匀，室温孵育2 h;再取尽液体后，加入400 μL洗涤液重复洗涤4次;将显色剂A,B等量混匀后加入200 μL酶标抗体，室温避光孵育30 min；加50 μL终止液终止酶反应；立即置酶标仪中测定，以450 nm波长读光密度值，根据标准曲线计算样品的含量。

2 结果

2.1 pGM-CSF-GFP-IRES-Rae-1-IL21重组载体酶切鉴定 从图1可知，采用EcoRI和MluI双酶切pGM-CSF-GFP-IRES-Rae-1-IL21得目的条带(红色箭头)与目的基因GFP大小一致的片段；采用XhoI和EcoRI双酶切pGM-CSF-GFP-IRES-Rae-1-IL21得目的条带(红色箭头)与目的基因GM-CSF大小一致的片段；采用Xba1和Sal1双酶切pGM-CSF-GFP-IRES-Rae-1-IL21得目的条带(红色箭头)与目的Rae-1一致的片段；采用Sal1和Not1双酶切pGM-CSF-GFP-IRES-Rae-1-IL21得目的条带(红色箭头)与目的基因IL21一致的片段，得到的基因GFP，GM-CSF，Rae-1和IL21与Genbank的原序列完全一致，证明载体构建成功。

图1 pGM-CSF-GFP-IRES-Rae-1-IL21重组载体酶切鉴定泳道M：Marker 5000(从上到下分别为5000,3000,2000,1500,1000,750,500,250,100bp)；泳道1：EcoRI和MluI酶切pGM-CSF-GFP-IRES-Rae-1-IL21(目的条带GFP：720bp)；泳道2：XhoI和EcoRI酶切pGM-CSF-GFP-IRES-Rae-1-IL21(目的条带GM-CSF：425bp)；泳道3：Xba1和Sal1酶切pGM-CSF-GFP-IRES-Rae-1-IL21(目的条带Rae-1：1106bp)；泳道4：Sal1和Not1酶切pGM-CSF-GFP-IRES-Rae-1-IL21(目的条带IL21：441bp)；泳道5：XhoI和Not1酶切pGM-CSF-GFP-IRES-Rae-1-IL21(目的条带GM-CSF-GFP-IRES-Rae-1-IL21：3.3kb)；泳道6：XhoI和Not1酶切pIRES空载(目的条带：5.5kb +673bp(IRES))；泳道7：pGM-CSF-GFP-IRES-Rae-1-IL21质粒注：因IL21的381位有EcoRI酶切位点所以用EcoRI和MluI酶切时有条2160bp条带(泳道1)，用XhoI和EcoRI酶切时有条2880的条带(泳道2)，见上图中蓝色箭头指示。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重组质粒对小鼠肝癌模型的抑制作用 将原位肝癌移植模型，随机分为6组，每组15只，按照前述的方法予以治疗，并用Kaplan-Meier生存分析进行统计分析。control组从第6天开始有小鼠死亡，到第14天小鼠全部死亡，其中位生存时间为11 d；IRES/GFP组从第5天开始死亡，到第16 d小鼠全部死亡，中位生存时间为12 d；IRES/GM-SCF组小鼠第17天开始死亡，到观察结束第60天剩2只模型小鼠存活，中位生存时间为35 d，其存活率为13.33%；IRES/IL21组的模型小鼠从第15天开始出现小鼠死亡，到第60天小鼠存活仅剩1只，中位生存时间为37 d，其存活率为6.67%；IRES/GM-CSF-IL-21模型小鼠从20 d开始死亡，到第60天存活4只小鼠模型，中位生存时间39 d，其存活率为26.67%；IRES/combination模型小鼠从30 d开始死亡，到第60天存活11只模型小鼠，未超过半数小鼠死亡，其存活率最高为73.33%。IRES/GM-SCF-IL21组的存活率明显高于其他各组(P<0.05，P<0.01)。

图2 重组质粒治疗后对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重组质粒对小鼠IL-2和INF-γ水平的影响 从图3，表2～3可知，治疗后1～6 d IRES/combination，IRES/GM-SCF-IL21，IRES/GM-SCF和IRES/IL21组的IL-2和INF-γ水平出现逐渐升高，以IRES/combination水平最高，IRES/GM-SCF-IL21次之，IRES/GM-SCF和IRES/IL21组水平相对较低(P<0.01)，6～10 d，IRES/combination在的IL-2和INF-γ水平仍为稳步增长，而IRES/GM-SCF-IL21，IRES/GM-SCF和IRES/IL21组出现逐渐下降。治疗结束时，IRES/GM-SCF-IL21组的IL-2和INF-γ水平较IRES/GM-SCF和IRES/IL21组明显增高(P<0.01)，而IRES/GM-SCF和IRES/IL21组的IL-2和INF-γ水平较IRES/GFP和control组明显增高(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组，IRES/GFP和control组之间的IL-2和INF-γ水平差异无统计学意义。在1～10 d，IRES/GFP和control组小鼠的IL-2和INF-γ水平变化不明显。

图3 各组对模型小鼠血清IL-2和INF-γ水平的影响(n=3)**P<0.01，与control组比较，##P<0.01，与IRES/GM-SCF或IRES/IL-21组比较，△△P<0.01，与IRES/GM-SCF-IL21组比较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

组别治疗天数(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 各组治疗后对模型小鼠机体INF-γ水平的动态变化(pg/mL)Tab.3 Dynamic change of INF-γlevels in model mice after different treatment(pg / mL)

3 讨论

肿瘤的发生发展与机体的免疫具有明显的相关性[6]，肝癌与其他肿瘤一样出现了机体的免疫功能的抑制，由于肝癌细胞表面表达的分子的改变，可以使肝癌细胞逃避免疫监视的识别[7]，导致肝癌细胞不能被NK细胞和CTL细胞识别并清除，同时肿瘤细胞表达被CTL和NK细胞识别的分子(如配体MHC-Ⅰ类相关分子，小鼠中为Rae-1)明显减少或者消失，导致肝癌细胞能在机体内长期存活[8-9]。现在研究较多的是用GM-SCF和(或)IL21基因治疗肿瘤，以提高CTL和NK细胞活性[10-11]，没有同时提高肿瘤组织表达又能被NK和CTL细胞识别的基因，或者仅有提高肿瘤组织表达但不能被NK和CTL细胞识别的基因[12]，而没有同时提高能促进机体NK和CTL细胞活性的基因治疗。为了提高机体的免疫功能，同时又增强肿瘤组织表达被免疫细胞识别的蛋白，本实验成功构建了“免疫逃逸抑制系统”即重组质粒pGM-CSF-GFP-IRES-Rae-1-IL21，且通过RT-PCR和基因测序证实。前期实验[3]已经证实GM-SCF和IL-21能够明显抑制小鼠肝癌模型，通过激活机体的NK和CTL细胞活性达到抑制小鼠模型的作用。本组实验在前期基础上构建了重组质粒，对小鼠肝癌皮下模型治疗发现，通过60d的观察，IRES/combination的生存期最长，其存活率高达73.33%，明显优于IRES/GM-SCF-IL21，IRES/GM-SCF和IRES/IL21组。而双基因的IRES/GM-SCF-IL21组的疗效明显优于单基因治疗组(IRES/GM-SCF和IRES/IL21组)，说明IRES/combination的联合治疗抑制模型小鼠生长作用最强，联合GM-SCF，IL-21和Rae-1基因治疗小鼠肝癌模型的疗效确切。

细胞因子是免疫细胞产生的一类小分子活性物质，具有明显的免疫调节作用，并在肿瘤的免疫中具有重要作用[13-14]。现已知GM-SCF和IL-21可以促进机体的INF-γ和IL-2水平的表达，提高机体CTL细胞和NK细胞的活性，从而达到抑制肿瘤生长的作用[15-16]。本组研究表明GM-CSF，IL-21和Rae-1联合治疗后，小鼠体内的INF-γ和IL-2水平呈持续升高，并且呈高水平的表达，较其他基因治疗明显增强。同时发现IRES/combination组的CTL和NK细胞的数量和活性均明显增强，较IRES/GM-SCF-IL-21，IRES/GM-SCF和IRES/IL-21组明显增强，说明GM-SCF,IL-21和Rea-1三种基因具有协同促进INF-γ和IL-2水平，及CTL和NK细胞的数量及活性。INF-γ是一族糖蛋白，具有明显的抗病毒、免疫调节和抗增殖等生物特性，其主要由CTL和NK细胞分泌[17-18]，对肿瘤细胞具有明显的抑制作用，同时具有一定的免疫调节活性，能激活和促进单核巨噬细胞和NK细胞的吞噬作用，促进B细胞和T细胞的分化成熟，增强CTL细胞的细胞毒作用，并可以诱导肿瘤细胞表达MHC-I类抗原的表达，增强免疫细胞如CTL和NK细胞的识别[19]。IL-2作为T淋巴细胞的生长因子，对T淋巴细胞的激活、增殖，对B细胞和巨噬细胞的活化过程具有重要作用，同时活化的T细胞可以分泌IL-2[20]。故说明GM-SCF,IL-21和Rea-1 3种基因促进肝癌小鼠皮下模型机体的免疫系统的激活，促进机体NK细胞和CTL数量和细胞毒性增强，激活的CTL细胞和NK 细胞分泌INF-γ水平明显增加，而高水平INF-γ又促进NK细胞和CTL细胞的激活和细胞毒性；同样激活的CTL细胞分泌的IL-2水平明显增加，而IL-2又促进机体CTL细胞的激活，形成良性循环，从而达到抑制肿瘤生长作用。

综上所述，联合GM-SCF，IL-21和Rae-1基因治疗明显抑制小鼠肝癌生长的作用，其机理与机体的免疫激活有关。

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(编校：谭玲)

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导类基因(114119a4700)；上海市科委纳米专项(12nm0502202)

吕一峰，男，硕士，主治医师，研究方向：肝癌的靶向治疗，E-mail：ssmu_lyf@163.com；程明荣，通讯作者， 男，硕士，副主任医师，研究方向：肝癌的靶向治疗，E-mail：15921300758@126.com。

R574.62；R574.63

A

1005-1678(2015)06-0017-05