大肠癌 Open Access
Copyright ©The Author(s) 2004. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2004-10-15; 12(10): 2307-2311
在线出版日期: 2004-10-15. doi: 10.11569/wcjd.v12.i10.2307
前药热化疗对转CD基因结肠癌细胞SW480的作用机制
黎成金, 王烈, 涂小煌, 宋京翔
黎成金, 王烈, 涂小煌, 宋京翔, 中国人民解放军南京军区福州总医院普外科 福建省福州市 350025
黎成金, 男, 1964-10-23生, 广西陆川人, 汉族. 2003年第四军医大学博士研究生毕业, 主治医师, 研究方向: 结肠癌的基因治疗.
通讯作者: 黎成金, 350025, 福建省福州市西二环北路156号, 中国人民解放军南京军区福州总医院普外科. licheng_jin@yahoo.com.cn
电话: 0591-2859415 传真: 0591-3796885
收稿日期: 2004-07-05
修回日期: 2004-07-09
接受日期: 2004-07-15
在线出版日期: 2004-10-15

目的: 探讨5-氟胞嘧啶 (5-fluorocytosine, 5-FC)热化疗对转染组织特异性胞嘧啶脱氨基酶(cytosine deaminase, CD)基因的大肠癌细胞SW480的作用及机制.

方法: 脂质体法将CEA基因顺式转录调控序列(TRS)驱动CD基因的组织特异性逆转录病毒载体G1CEACDNa转导入大肠癌细胞SW480, 以G418筛选阳性克隆扩增后, 采用水浴加温法, 43 ℃作用30 min共3次, 同时给予前药5-FC进行敏感试验; RT-PCR法检测目的基因的表达, MTT法检测细胞存活率, 电镜检测细胞的形态学变化, 流式细胞术检测细胞周期变化及凋亡.

结果: SW480-CEACD细胞在43 ℃作用30 min共3次条件下, CD基因能稳定表达; 热疗本身对SW480细胞有一定的杀伤作用(P <0.05), 转CD基因后, SW480细胞对5-FC的敏感性明显提高(P <0.01), 热疗与前药5-FC合用, 对SW480-CEACD的杀伤作用显著大于对未转基因细胞的杀伤作用(P <0.01, t = 4.356, n = 9 ), 亦大于单独应用5-FC时对SW480-CEACD细胞的杀伤作用(P <0.05, t = 2.376, n = 9), 增加了SW480-CEACD细胞对5-FC的敏感性, 电镜及流式细胞术检测显示, 细胞死亡以细胞凋亡为主, 前药热化疗导致结肠癌细胞SW480产生G1期阻滞, 凋亡细胞比例增加.

结论: 热疗与前药5-FC联合应用, 导致结肠癌细胞SW480产生G1期阻滞, 细胞凋亡比例增加, 提高了CEA组织特异性CD/5-FC系统的靶向性杀伤作用.

关键词: N/A
引文著录: 黎成金, 王烈, 涂小煌, 宋京翔. 前药热化疗对转CD基因结肠癌细胞SW480的作用机制. 世界华人消化杂志 2004; 12(10): 2307-2311
Mechanism of thermochemotherapy with 5-fluorocytosine on human colon cancer cell line SW480 transfected cytosine deaminase gene
Cheng-Jin Li, Lie Wang, Xiao-Huang Tu, Jing-Xiang Song
Cheng-Jin Li, Lie Wang, Xiao-Huang Tu, Jing-Xiang Song, Department of General Surgery, Fuzhou General Hospital of Nanjing Military Area, Fuzhou 350025, Fujian Province, China
Correspondence to: Dr. Cheng-Jin Li, Department of General Surgery, Fuzhou General Hospital of Nanjing Military Area, Fuzhou 350025, Fujian Province, China. licheng_jin@yahoo.com.cn
Received: July 5, 2004
Revised: July 9, 2004
Accepted: July 15, 2004
Published online: October 15, 2004

AIM: To investigate the mechanism of thermochemotherapy with 5-fluorocytosine (5-FC) on human colon cancer cell line SW480 transfected carcinoembryonic antigen (CEA) tissue-specific cytosine deaminase (CD) in vitro.

METHODS: Recombinant retroviral vector G1CEACDNa, in which the CD gene was controlled under the CEA promoter, was introduced through liposome technique to human colorectal carcinoma cell line SW480, and then the cells were selectively cultured in G418. The proliferated colonies were treated with the combined therapy of 5-FC and hyperthermia at a temperature of 43 ℃ for 30 min, 3 times. RT-PCR was performed to detect the expression of CD gene in target cells after being heated. The cell survival rate was detected by MTT method. The ultrastructures of cells were observed by electron microscopy and apoptosis was verified by flow cytometry.

RESULTS: The expression of CD genes in target cells was detected after being heated. After transfection, SW480-CEACD cells were more sensitive than their parental cells (P <0.01, t = 5.620, n = 9) to 5-FC, the killing effect of hyperthermia on SW480 cells was observed (P <0.05, t = 2.999, n = 9). Furthermore, after being treated with thermoche-motherapy of 5-FC at a temperature of 43 ℃ for 30 min, the killing effect on SW480-CEACD cells was more significant than that on SW480 cells (P <0.01, t = 4.356, n = 9). Treatment with the combination of 5-FC and hyperthermia displayed a higher anti-tumor effect than that with 5-FC alone on SW480-CEACD cells in vitro (P <0.05, t = 2.376, n = 9). Apoptotic bodies in the field of electron microscope were observed. G1 blockage was confirmed and the increased rate of apoptosis cells was verified after hyperthermia with 5-FC by flow cytometry.

CONCLUSION: The combination of 5-FC and hyperthermia will result in G1 blockage of human colorectal carcinoma cell lines SW480 transfected with the CEA tissue-specific CD genes, which will improve the outcome of the anti-tumor effect on that cell line.

Key Words: N/A
0 引言

在转移性肝癌中, 大肠癌占50%以上, 此类患者80-90%难以外科手术切除, 其5年存活率不到30%, 少数患者即使能切除, 其复发率也达40-70%, 其中位生存期约6-12 mo, 此类患者主要依赖非手术疗法如放疗、化疗及局部治疗来延长生命[1-8], CD/5-FC系统作为前药转换基因系统之一, 近年来对其在肿瘤治疗中的作用进行了广泛研究[9-12], 已进行了少量Ⅰ期临床实验[9,13-14]. 研究表明, 癌胚抗原(CEA)转录调控序列可控制CD基因在CEA阳性的大肠癌组织中高效表达, 在前药5-FC作用下, 产生选择性杀伤肿瘤细胞的作用. 然而, 我们的研究显示, CEA启动子控制的CD基因对低表达CEA的大肠癌细胞SW480的靶向性杀伤作用并不理想[15], 5-FC热化疗能增加对转基因细胞的靶向性杀伤作用探讨如下.

1 材料和方法
1.1 材料

CEA启动子调控CD基因表达的逆转录病毒载体G1CEACDNa由第二军医大学长海医院惠赠; 大肠癌SW480细胞株由第四军医大学动物研究所提供; 感受态菌由第四军医大学遗传与发育教研室制备; 脂质体liopfectamineTM 2000 (LF 2000)为Invitrogen公司产品; G 418, DMEM为Gibco公司产品; 5-FC, MTT为Sigma公司产品; RPMI 1640培养基, 胎牛血清, Access RT-PCR System 为Promega公司产品; 总 RNA提取试剂盒 Trizol Reagent为Life Technologies Inc公司产品; RT-PCR引物由Biotech公司合成, CD基因(扩增产物1.5 kb )为: 正义链 5'-ATA GAA TTC AGG CTA ACA ATG TCG AAT TAA CGC TT-3', 反义链 5'-TAT GGA TCC TCA ACG TTT GTA ATC CAT GGC TT-3'. Tubulin(扩增产物410 bp): 5'-CTC ATC ACA GGC AAG GAA GAT-3', 5'-TTA AGG TAA GTG TAG GTT GGG-3'.

1.2 方法

质粒扩增 、大肠癌细胞的转染见文献[17].分别收集加热前、加热3次24 h后的培养细胞5×106个, 采用 Life Technologies Inc的总 RNA提取试剂盒 Trizol Reagent提取细胞总RNA, 见文献[17]. 用荧光成像系统扫描定量. 将转染G1CEACDNa之SW480细胞及未转基因之SW480细胞以5×104个/孔接种到96孔细胞培养板中, 接种同时加入含各种梯度浓度的前药5-FC, 每种浓度设3个复孔, 同时设置对照孔及调零孔. 在37 ℃, 50 mL/L CO2孵箱中培养, 每天换液1次, 热疗组分别于加入5-FC的同时及第24, 48 h 在43 ℃下热疗30 min, 对照组不热疗, 第8 d去除培养液, 以MTT法测定活细胞比率并计算杀伤率.将1×106个转染G1CEACDNa之SW480细胞分别接种到7 cm的细胞培养皿中, 接种同时加入2 mmol/L 5-FC , 热疗组处理后, 每天换液1次, 培养72 h后分别收集培养上清备用. 24孔培养板中每孔接种5×104个未转基因SW480细胞, 每组各8孔, 实验组1用热疗组培养上清培养, 实验组2用非热疗组培养上清培养, 对照组常规培养, 每2 d换液1次, 第 4, 8 d各取4孔细胞行MTT比色实验.取200 mL玻璃培养瓶接种处于对数生长期的细胞, 以含2.0 mmol/L 5-FC的RPMI 1640完全培养基培养, 在37 ℃, 50 mL/L CO2孵箱中培养, 热疗方法同上, 对照组不热疗, 每天换液1次, 第4 d去除培养液, 铀－铅双染色, 用JEM-2000EX型透射电镜观察细胞超微结构. 取对数生长期的细胞接种于玻璃培养瓶(12瓶), 以含2.0 mmol/L 5-FC的RPMI 1640完全培养基在37 ℃, 50 mL/L CO2孵箱中培养, 热疗组(6瓶)处理方法同上, 非热疗组(6瓶)不热疗, 对照组(6瓶, 培养液不含5-FC)常规培养, 每天换液1次, 第 4 d去除培养液, 处理后用ProfileⅡ型流式细胞仪在488 nm激发波长下测定细胞DNA含量, 并用Multicycle软件进行分析. PI和annexin Ⅴ-FITC染色FCM检测方法: 各组细胞处理后, 取1 ×106 细胞离心去上清后, 重新悬浮于1 mL 的annexin Ⅴ-FITC 染色液, 室温下染色5 min; 在细胞悬液中加入10 mL的PI 保存液, 室温下染色5 min 后上机检测.

统计学处理 数据用均值±标准差 (mean±SD)表示, 在SPLM软件中输入药敏实验中各浓度前药下每组细胞生存率, 作线图并计算IC50, 采用未配对计量资料的t检验进行统计学处理, P <0.05有显著性差异.

2 结果
2.1 目的基因在靶细胞中稳定表达

提取细胞总RNA后, 转G1CEACDNa之SW480细胞RT-PCR产物电泳可见1.5 kb片段(图1). 扫描定量灰度值分别为1.38和 1.35.

图1
图1 RT-PCR检测CD 基因在SW480 细胞的表达. 1: 加热前; 2: 加热3次后24 h; 3: lDNA Hind Ⅲ Marker.
2.2 热疗加前药5-FC对转基因大肠癌细胞SW480的杀伤作用

热疗本身对SW480细胞有一定的杀伤作用(P <0.05, t = 2.999, n = 9), 转CD基因后, SW480细胞对5-FC的敏感性明显提高(P <0.01, t = 5.620, n = 9), 热疗与前药5-FC合用, 对SW480-CEACD的杀伤作用显著大于对未转基因细胞的杀伤作用(P <0.01, t = 4.356, n = 9), 亦大于单独应用5-FC时对SW480-CEACD细胞的杀伤作用(P <0.05, t = 2.376, n = 9 )热疗提高了5-FC对转基因大肠癌细胞SW480对前期药5-FC的敏感性, 其IC50分别为1.2 mmol/L和 0.1 mmol /L(图2).

图2
图2 前药5-FC加热疗对转CD基因SW480细胞的杀伤作用. A: 未转基因SW480细胞单独应用5-FC治疗; B: 未转基因SW480细胞联合应用5-FC及热疗; C: 转基因SW480细胞单独应用5-FC治疗; D: 转基因SW480细胞联合应用5-FC及热疗.
2.3 上清移换实验

实验组较对照组吸光度A值明显减低, 有统计学意义(PG1d4 <0.05, tG1d4 = 2.867, n = 4; PG1d8 <0.05, tG1d8 = 2.751, n = 4; PG2d4 <0.01, tG2d4 = 6.305, n = 4; PG2d8 <0.01, tG2d8 = 6.257, n = 4), 但实验组1与实验组2吸光度A值无明显差异(表1).

表1 上清移换实验结果(mean±SD, A, n = 4).
t/d实验组1实验组2对照组
40.537±0.240a0.586±0.135a0.937±0.232
80.418±0.114b0.430±0.103b1.737±0.451
aP <0.05,
bP <0.01 vs 对照组.
2.4 电镜观察

热疗组及非热疗组均可见明显的凋亡表现: 表现为细胞核皱缩, 染色质聚集在核膜下以及凋亡小体形成(图3).

图3
图3 热疗组与非热疗组细胞超微结构图TEM×6 000. A: 非热疗组; B: 热疗组; C: 对照组.
2.5流式细胞仪检测

热疗组与对照组相比, PI和annexin Ⅴ-FITC染色FCM检测显示凋亡细胞比例(4区)明显增多(图4), DNA含量分析则显示热疗组有明显的G1期阻滞(Pa1<0.01, ta1 = 4.619, n = 6; Pb<0.05, Pb = 2.382, n = 6; Pa2<0.01, ta2 = 3.381, n = 6), 并在细胞周期前出现明显的凋亡峰(表2).

表2 前药热疗对SW480细胞周期的影响(mean±SD, %, n = 6).
分组G1SG2
热疗组69.5±13.4b1c27.4±6.56.7±1.1
非热疗组58.6±9.8c229.3±10.212.1±3.0
对照组41.2±12.343.3±8.515.5±3.1
bP<0.01 vs 对照组;
cP<0.05 vs 非热疗组.
图4
图4 PI-annexin Ⅴ-FITC染色FCM分析图. A;非热疗组,B;热疗组, C;对照组.
3 讨论

胞嘧啶脱氨基酶(cytosine deaminase, CD)基因是来源于某些细菌和真菌的一种自杀基因, 其编码的CD酶能将对真核细胞相对无毒的5-氟胞嘧啶(5-FC)转换成细胞毒性化疗药5-氟尿嘧啶(5-FU), 抑制细胞RNA和DNA的合成而致细胞死亡[16]. 5-FU作为结肠癌的一线化疗药物, 对机体正常组织的毒副作用限制了其在结肠癌化疗的临床应用[17-19], 靶向基因治疗可将治疗基因在肿瘤组织中特异地表达, 使正常组织免受损害. CEA基因属于组织特异性表达基因, 利用CEA基因的转录调控序列来调控CD基因的表达, 可特异性杀死CEA阳性分泌的大肠癌细胞[20], 对结肠癌肝转移也有特异性治疗作用及旁杀伤效应[21-23]. 临床资料显示, 在大肠癌患者中, 近2/3血清CEA正常[24], Dukes A期患者血清CEA水平仅5.3±1.8 mg/L[25], 表明临床有相当一部分大肠癌患者其CEA表达水平较低. 我们所用的SW480是低表达CEA的人大肠癌细胞株, CEA分泌水平为3.1 fg/ cell.

热疗对肿瘤的治疗作用已研究多年, 临床实验表明, 术中或术后行局部热化疗, 可明显提高患者的生活质量和生存期[26], 对结肠癌肝转移, 热疗安全有效, 与放疗或化疗联合应用, 可使不能手术者获得手术机会[27-30], 热疗对肿瘤的治疗作用机制有: (1)乏氧的肿瘤细胞比正常的组织细胞对热更敏感, 热疗可在不引起正常组织细胞功能损害时就导致肿瘤细胞蛋白质和DNA热变性; (2)热疗可使肿瘤细胞超微结构破坏; (3)加热促进了肿瘤细胞对化疗药物的吸收作用, 使细胞内药物浓度增大, 从而提高化疗药的细胞毒性; (4)加热启动了细胞凋亡机制, 促进细胞凋亡[31-37]; (5)在转CD基因肿瘤细胞中, 加热可提高目的基因在靶细胞的表达[38].

我们用前药5-FC对转染G1CEACDNa的SW480细胞进行热化疗实验研究, 结果显示, 转G1CEACDNa基因SW480细胞在43 ℃作用30 min共3次条件下, CD基因能稳定表达, 用前药5-FC热化疗增加了SW480-CEACD细胞对5-FC的敏感性, 使其IC50提高了12倍, 我们对其作用机制进行了初步探讨, RT-PCR结果及上清移换实验提示, 热疗本身并不增加目的基因的表达, 亦不增加含5-FC代谢产物的细胞培养上清的细胞毒性, 电镜观察显示, 热化疗组细胞死亡机制与单独应用前药的细胞死亡机制相似, 均以细胞凋亡为主, 流式细胞检测实验提示, 用前药5-FC热化疗可导致细胞G1期阻滞, 明显增加细胞的凋亡比例, 从而增加前药5-FC的杀肿瘤细胞作用. 本实验表明, 热疗与前药5-FC共同应用, 提高了转基因SW480细胞对5-FC的敏感性, 从而提高CEA组织特异性CD/5-FC系统对肿瘤细胞的靶向性杀伤作用. 由于热疗副作用小, 可以反复进行, 并能明显增加化疗的效果, 减少化疗药物的用量, 从而降低化疗药物的细胞毒性, 这将为低表达CEA大肠癌的基因治疗, 特别是结肠癌肝转移的治疗提供一新途径.

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