大肠癌 Open Access
Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2003-03-15; 11(3): 294-297
在线出版日期: 2003-03-15. doi: 10.11569/wcjd.v11.i3.294
IFN-γ基因转染抗大肠癌细胞的作用机制
吴文溪, 丁强, 沈历宗, 华一兵, 许德华, 刘新垣
吴文溪, 丁强, 沈历宗, 华一兵, 南京医科大学第一附属医院普外科 江苏省南京市 210029
许德华, 刘新垣, 中国科学院上海生物化学研究所 上海市 200031
吴文溪, 男, 1947-1生, 江苏省无锡人, 汉族, 硕士, 教授, 主任医师.
通讯作者: 吴文溪, 210029, 江苏省南京市, 南京医科大学第一附属医院普外科. wuwenxi@yahoo.com
电话: 025-6619960
收稿日期: 2002-07-01
修回日期: 2002-07-15
接受日期: 2002-07-30
在线出版日期: 2003-03-15

目的: 通过检测IFN-γ基因转染对大肠癌细胞表面抗原的表达情况、对细胞的生长抑制、细胞周期的影响情况, 初步探讨IFN-γ基因治疗抗肿瘤作用的机制.

方法: 制备含人IFN-γ基因的真核表达质粒pcDNA-3-IFN-γ, 利用阳离子脂质体将其转染进入人大肠癌细胞株LOVO、SW62O、HCT116BG及人宫颈癌细胞株Hela, 检测基因转染后IFN-γ基因的表达情况, 同时检测基因转染对大肠癌细胞CEA、HLA-DR抗原表达的诱导作用和细胞凋亡及细胞周期的变化.

结果: 基因转染后, 原来高表达CEA的细胞株(LOVO、 HCT116BG)其上清中CEA含量增加明显(从21.25±6.76 m/l增加到34.96±7.07 mg/L P<0.05), 而原来低表达甚至不表达CEA的细胞株(Hela、SW620)其上清CEA含量则无明显增加 (P>0.05). 各细胞株表面的HLA-DR的表达量在导入IFN-γ基因后明显增强(平均表达率从3.91%±3.61% 增加到11.67±7.20% P<0.01). 通过对细胞的凋亡情况和细胞周期的变化显示: 转染IFN-γ基因后促进了LOVO细胞的凋亡(各时段平均凋亡率对照组与治疗组分别为8.27±5.62%与15.32±11.41% P<0.05), 细胞的S期比例随作用时间延长而呈逐渐降低趋势, 显示了基因转染后DNA的合成代谢受到抑制.

结论: IFN-γ基因转染后可有效增强大肠癌细胞表面抗原的表达, 诱导机体产生抗肿瘤免疫应答; 并可能通过抑制细胞DNA的合成, 促进细胞的凋亡, 抑制肿瘤细胞的增生等机制发挥抗肿瘤作用.

关键词: N/A
引文著录: 吴文溪, 丁强, 沈历宗, 华一兵, 许德华, 刘新垣. IFN-γ基因转染抗大肠癌细胞的作用机制. 世界华人消化杂志 2003; 11(3): 294-297
Effect of expression of interferon-g gene in inhibition of growth of colon cancer cell
Wen-Xi Wu, Qiang Ding, Li-Zong Shen, Yi-Bing Hua, De-Hua Xu, Xin-Yuan Liu
Wen-Xi Wu, Qiang Ding, Li-Zong Shen, Yi-Bing Hua, Department of General Surgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
De-Hua Xu, Xin-Yuan Liu, Shanghai Institute of Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China
Correspondence to: Wen-Xi Wu, Department of General Surgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China. wuwenxi@yahoo.com
Received: July 1, 2002
Revised: July 15, 2002
Accepted: July 30, 2002
Published online: March 15, 2003

AIM: To investigate the effect of expression of interferon-γ(IFN-γ) in tumor cell and its inhibitory effect on the growth of tumor cell.

METHODS: pcDNA3-IFN-γ vector containing IFN- γ gene was constructed and transfected into LOVO, SW620, HCT116BG and Hela cell lines by lipofectamine, respectively. The expression of IFN-γ, CEA and HLA-DR in transfected cells were tested. Both the number of apoptosis of and the proportion of cell cycles of tumor cells were measured to investigate the anti-tumor effect of IFN-γ gene therapy.

RESULTS: LOVO and HCT116BG transfected cell lines had high expression of CEA, the average level of CEA was significantly increased from 26.02±6.76 to 38.85±7.07 mg/L (P < 0.05). However, there was no detectable increase in the supernatants of Hela, SW620 cell lines that naturally expressed little of CEA. Flow cytometry analysis showed that HLA-DR expression rate (11.67±7.20) was significantly higher than that prior gene transfection (3.91±3.61) (P < 0.01), and the IFN-γ gene transfer effectively induced the apoptosis of tumor cells, the proportion of DNA synthesis phase was deceased gradually after IFN-γ gene transfer, which indicated that the synthesis of DNA and growth of tumor cells were repressed.

CONCLUSION: IFN-γ gene therapy enhanced the expression of antigens on cell surface and thus induced powerful antitumor immunity. Repressing of synthesis of DNA, inducing the apoptosis of tumor cells and inhibiting the proliferation of tumor cells might be anti-tumor mechanisms of IFN-γ.

Key Words: N/A
0 引言

肿瘤细胞靶向的细胞因子基因治疗是肿瘤治疗领域的研究热点[1-5]. γ-干扰素在肿瘤的治疗中起着独特的作用[6-8], 将其基因导入肿瘤细胞内使之表达, 以提高患者自身抗肿瘤免疫能力, 在当前肿瘤基因治疗中占有重要的地位[9-11]. 大肠癌是国内外常见的恶性肿瘤, 随着生活水平的提高, 其发病率呈逐年上升趋势[12-16]. 我们构建了含IFN-γ基因的真核表达质粒pcDNA3-IFN-γ, 并利用阳离子脂质体Lipofectamine将其转导进入大肠癌细胞株, 通过检测IFN-γ基因的转染、表达情况, 对细胞表面抗原表达的影响及肿瘤细胞的凋亡和细胞周期的变化, 探讨其抗肿瘤作用的机制.

1 材料和方法
1.1 材料

携带人IFN-γ基因的质粒pLXSN-IFN-γ, 克隆用质粒pBluescript, 腺病毒穿梭质粒pAdE1CMV, 真核表达质粒pcDNA-3由中科院上海生物化学研究所提供. 人大肠癌细胞株LOVO, SW620, HCT116BG及人宫颈癌细胞株Hela由上海生化所传代保存, 各细胞株经检测无支原体污染. 阳离子脂质体Lipofectamine购自Gibco公司; 人IFN-γ ELISA试剂盒购自晶美生物公司; RNA抽提试剂盒(TRIZOL)、cDNA第一条链合成试剂盒(superscript preamplification system for first strand cDNA synthesis)、PCR试剂盒及内切酶均购自Sangon公司; IFN-γ基因引物由上海生工公司合成, 序列为: 5'引物: 5'-CTGCCAGGAC CCATATGTACA-3' 3'引物: 5'-GATGCTCTTCG ACCTCGAAACA-3.

1.2 方法

构建携带人γ-干扰素的真核表达质粒pc-DNA3-IFN-γ, 经酶切鉴定后, 大量扩增和纯化, 质粒的制备、扩增、纯化均按《分子克隆实验指南》所示方法进行. 分别取生长旺盛的LOVO, SW62O, HCT116BG细胞接种至6孔板中, 每孔2×105个细胞, 37℃, 50 mL/L CO2孵育12 h, 至细胞长至80%融合时进行转染, 利用空白载体pcDNA-3质粒进行对照. 转染方法按照lipofectamine操作手册进行. 收集基因转染后24 h的大肠癌细胞, 按试剂盒所示方法用PCR, RT-PCR对huIFN-γ基因的整合和表达进行鉴定. PCR反应条件: 94℃ 1 min, 56℃ 1 min, 72℃ 1 min, 30个循环. 利用双抗体夹心ELISA法测定基因转染后4, 12, 24, 48, 72, 96, 120 h的细胞培养上清中IFN-γ的活性, 测定方法按照ELISA试剂盒说明书进行. 分别取生长旺盛的LOVO, SW62O, HCT116BG, Hela细胞接种至6孔板中, 每孔2×105个细胞, 37℃、50 mL/L孵育12 h, 至细胞80%融合时利用阳离子脂质体转染IFN-γ基因, 分别收集基因转染后48 h的细胞和细胞培养上清, 利用相同培养条件下未进行基因转染的细胞和细胞培养上清作对照, 放射免疫法测定细胞培养上清中CEA含量; 流式细胞仪检测细胞表面HLA-DR的表达情况. 放免测定由复旦大学附属中山医院核医学科完成; 流式细胞仪检测HLA-DR由中科院上海细胞所流式细胞室完成. 取生长旺盛的LOVO细胞接种至6孔板中, 每孔2×105个细胞, 按照上述细胞培养条件, 至细胞80%成片时利用阳离子脂质体转染IFN-γ基因, 分别收集基因转染后24, 48, 72, 96, 120 h的细胞, 利用相同培养条件下未进行基因转染的细胞作对照, 利用流式细胞仪检测细胞凋亡情况和细胞周期的变化. 流式细胞仪检测细胞凋亡由中科院上海细胞所流式细胞仪室完成.

统计学处理 采用配对资料t检验、方差分析和秩和检验.

2 结果

pcDNA3- IFN-γ载体的结构示意图如图1所示. 经HindⅢ和XbaI双酶切后可得到501 bp的IFN-γ片段(图2). 基因转染后4 h细胞上清内即可检测到IFN-γ活性, 24 h达到最大值, 上清中IFN-γ活性持续6 d仍未见明显下降(图3). 转染空白质粒载体和单独受lipofectamine作用的细胞株, 其培养上清中均未检测到IFN-γ活性. 不同的细胞株所分泌的IFN-γ的量不同, 显示了细胞株之间生物学特性的差异.

图1
图1 pcDNA3- IFN-γ载体的结构示意图
图2
图2 pcDNA3- IFN-γ载体酶切鉴定图. M: DNA Marker DL2000; 1: 未经酶切的质粒pcDNA3- IFN-γ; 2, 3: 经HindⅢ和XbaI双酶切后的电泳图谱.
图3
图3 基因转染后细胞上清中的IFN-γ浓度.
2.1 基因转染后细胞CEA的变化

利用放射免疫法测定细胞上清中CEA的含量, 由(图4)可以看出, 经基因转染后, 原来高表达CEA的细胞株(LOVO, HCT116BG)其上清中CEA含量增加明显(P<0.05), 而原来低表达甚至不表达CEA的细胞株(Hela, SW620)其上清CEA含量则无明显增加(P>0.05).

图4
图4 基因转染前后细胞分泌CEA的含量变化.
2.2 基因转染后HLA-DR的表达情况

导入IFN-γ基因后, 四株细胞HLA-DR平均表达率从3.91±3.61% 增加到11.67±7.20% (P<0.01, 图5).

图5
图5 基因转染前后细胞表面HLA-DR的表达情况.
2.3 IFN-γ基因转染后细胞的凋亡与周期

转染IFN-γ基因后促进了LOVO细胞的凋亡(图6). 转染基因后5 d各时段平均凋亡率对照组与治疗组分别为8.27%±5.62%与15.32%±11.41%(P<0.05). 转染IFN-γ基因后细胞的S期比例随作用时间而呈逐渐降低趋势(表1), 显示DNA的合成代谢受到抑制, 从而抑制了肿瘤细胞的增生.

表1 基因转染对LOVO 细胞周期的影响.
作用时间(h)细胞周期比例(%)
G0-G1SG2-M
2458.7424.1817.08
4861.8621.8816.25
7259.7418.0922.16
9664.4415.2220.34
图6
图6 基因转染后Lovo细胞凋亡情况检测.
3 讨论

我们将IFN-γ基因导入大肠癌细胞中, 观察和检测基因的转染、表达及对肿瘤细胞的作用. 结果表明, IFN-γ基因可以被有效地转染进入大肠癌细胞内并顺利得到表达, 不同的细胞株其分泌的IFN-γ的量不同, 显示了细胞株之间生物学特性的差异; 一次有效的基因转染可以使细胞持续分泌IFN-γ达1 wk之久而滴度未见明显下降.

IFN-γ在肿瘤免疫中起扩大作用[17-26], 是干扰素家族中唯一能调节MHC-Ⅱ抗原表达的, 有研究表明, 人类大肠癌细胞的HLA抗原异常表达与肿瘤细胞DNA含量有关, HLA-DR异常表达的分化程度愈高, 预后愈好. HLA抗原的表达率愈高, 则肿瘤的转移率愈低[27-29]. MHC-Ⅱ类分子表达与否及其表达水平, 直接决定免疫应答的发生及其强度. IFN-γ通过受体激活细胞内信号传导途径, 作用于HLA基因的转录水平, 加速HLA的转录过程, 引起HLA表达增高[30-35]. 本实验中, IFN-γ基因被导入不同的大肠癌细胞株中, 各细胞株表面HLA-DR的表达量均明显增加, 以HCT116BG和LOVO细胞株增加最为显著. IFN-γ可以增加许多肿瘤表面抗原的表达, 人类CEA抗原为一180KD糖蛋白, 在许多肿瘤中均有表达, 构建表达CEA蛋白的瘤苗作为肿瘤基因治疗的方法之一已屡见报道[36-41], 本实验中, 原先表达CEA抗原的LOVO、HCT116BG细胞株在导入IFN-γ基因后, 细胞所分泌的CEA的量明显增加. 而原先不表达或低表达CEA抗原的SW620、Hela细胞株在导入治疗基因后, CEA的表达未见改变.

我们还发现, 经过IFN-γ基因作用后的肿瘤细胞凋亡明显, 在对细胞周期变化的分析中, 可以看出经过基因转染后的肿瘤细胞, 其S期呈逐渐减少趋势, 表明肿瘤细胞的DNA合成受到了抑制, 从而影响了肿瘤细胞的分裂和增生. IFN-γ是直接引起细胞凋亡, 还是通过其他的机制(激活其他细胞因子, 激活凋亡基因等)尚有待研究, 有报道IFN-γ可直接增加抑癌基因如IRF-1和PKB基因的表达而抑制肿瘤的产生[42-45].

编辑: N/A

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