临床研究 Open Access
Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2003-08-15; 11(8): 1223-1226
在线出版日期: 2003-08-15. doi: 10.11569/wcjd.v11.i8.1223
汉防己甲素抑制肝癌细胞增生的作用
荆绪斌, 李涛, 杨绮华, 郭光华, 胡辉, 陈素钻
荆绪斌, 李涛, 杨绮华, 郭光华, 胡辉, 陈素钻, 汕头大学医学院第一附属医院消化内科 广东省汕头市 515041
荆绪斌, 男, 1961-11-18生, 山东省平度市人, 汉族, 1996年上海第二医科大学博士研究生毕业, 2000年留学日本, 副教授, 主要从事肝硬化及肝癌防治的研究, 发表论文12篇.
通讯作者: 荆绪斌, 515041, 广东省汕头市长平路57号, 汕头大学医学院第一附属医院消化内科. xbjing@stu.edu.cn
电话: 0754-8520164
收稿日期: 2002-03-14
修回日期: 2002-04-20
接受日期: 2002-05-24
在线出版日期: 2003-08-15

目的

研究汉防己甲素(tetrandrine, TTD)对肝癌细胞的作用及其机制.

方法

采用MTT比色法观察了TTD对肝癌细胞增生的影响; 采用流式细胞仪法观察了TTD对肝癌细胞内活性氧的影响; 采用琼脂凝胶电泳法观察了DNA片断化的梯形条带.

结果

10 and 20 μmol/L TTD作用于肝癌细胞系24, 48, 72 h后其增生率分别为90.1±1.0% and 77.5±2.0%, 70.2±2.9% and 56.6±1.6%, 61.6±2.0% and 47.2±1.9% (F = 40.025, P<0.001); 0、10和20 μmol/L TTD作用于肝癌细胞系2 h后O2- 为35%、36.6%、63.2%; H2O2.为24.5%、40.5%、84.6%. 48 h后20 μmol/L TTD组肝癌细胞出现典型的凋亡表现.

结论

TTD能够通过诱导肝癌细胞内活性氧的产生, 而引起细胞凋亡, 呈明显的剂量依赖性.

关键词: N/A
引文著录: 荆绪斌, 李涛, 杨绮华, 郭光华, 胡辉, 陈素钻. 汉防己甲素抑制肝癌细胞增生的作用. 世界华人消化杂志 2003; 11(8): 1223-1226
Inhibitory mechanism on proliferation of hepatocellular carcinoma cell line by tetrandrine
Xu-Bin Jing, Tao Li, Qi-Hua Yang, Guang-Hua Guo, Hui Hu, Su-Zhuan Chen
Xu-Bin Jing, Tao Li, Qi-Hua Yang, Guang-Hua Guo, Hui Hu, Su-Zhuan Chen, Department of Gastroenterology, the First Affiliated Hospital, Shantou University Medical College, Shantou 515041, Guangdong Province, China
Correspondence to: Dr. Xu-Bin Jing, Department of Gastroenterology, the First Affiliated Hospital, Shantou University Medical College, Shantou 515041, Guangdong Province, China. xbjing@stu.edu.cn
Received: March 14, 2002
Revised: April 20, 2002
Accepted: May 24, 2002
Published online: August 15, 2003

AIM

To study the mechanism of tetrandrine (TTD) on proliferation and apoptosis of hepatic cell carcinoma (HCC) cell line.

METHODS

MTT colorimetry was used to evaluate the influence of TTD on proliferation of cells. Flow cytometry was used to test reactive oxygen species (ROS) levels in cells. Electrophoresis in 1.5% agarose gels was used to assess the DNA laddering.

RESULTS

Proliferation rate of HCC cell line stimulated by 10 and 20 μmol/L TTD at 24 h, 48 h, and 72 h was 90.1±1.0%, 77.5±2.0%, 70.2±2.9% and 56.6±1.6%, 61.6±2.0% and 47.2±1.9%, respectively (F = 40.025, P<0.001). O2- and H2O2. generated by HCC cell lines incubated with 0, 10 and 20 μmol/L TTD for 2 hours were 35 and 24.5%, 36.6 and 40.5%, 63.2 and 84.6%, respectively. Typical DNA ladder was observed after the cell line was incubated in 20 μmol/L TTD for 48 hours.

CONCLUSION

TTD can induce the apoptosis of ROS of hepatic cell carcinoma cell line by generation of ROS in a dose dependent manner.

Key Words: N/A
0 引言

原发性肝癌是我国常见的恶性肿瘤之一[1-9], 发病率在恶性肿瘤中居第二位. 因为起病隐匿, 病情发展迅速, 临床诊断时多为中晚期, 化学治疗仍是重要的治疗手段之一 , 许多化疗药物如5-FU、阿霉素、顺铂、长春新碱等通过细胞凋亡的途径杀死肿瘤细胞[10-25], 但其毒副作用及多药耐药限制了其临床应用.恶性肿瘤细胞凋亡的研究不仅对认识肿瘤的发病机制有积极意义, 而且在肿瘤治疗及预后方面具有重要意义. 汉防己甲素(tetrandrine, TTD)是传统中药汉防己中的主要成分, 近年研究证实, 其具有抑制肿瘤细胞增生的作用[26-31]. 我们探讨了TTD对肝癌细胞作用的可能机制, 为防治肝癌探索新的途径.

1 材料和方法
1.1 材料

汉防己甲素和噻唑蓝(MTT)购于Sigma公司; DMEM培养基购于Gibco公司; 核酸抽提试剂盒购于日本Sepa Gene公司; 2', 7'-dichlorofluorescein diacetate (DCFH-DA)和Dihydroethidium (HE)分子探针 购于Egene, OR公司. 人的肝癌细胞系Mahlavu 被培养在含100 mL/L的胎牛血清(FCS; Gibco)、100 kU/L 青霉素 (Gibco)和100 mg/L 链霉素(Gibco)的DMEM培养液中; 培养条件为37 °C、50 mL/L CO2 / 950 mL/L空气.

1.2 方法

培养细胞长满单层后, 用胰蛋白酶消化并混悬于含100 mL/L FCS的DMEM培养液中, 调整细胞数为2×107/L, 以每孔100 μL加入96孔培养板中, 置于37 °C、50 mL/L CO2 / 950 mL/L 空气的培养箱, 24 h后加入不同浓度的TTD, 并设对照组加入等量DMEM(每组设5个复孔), 继续分别孵育24、48、72 h后, 加入2 g/ L的MTT溶液50 μL, 振荡后继续培养4 h, 于每孔内加入DMSO 200 μL, 使形成的甲月赞Formazan充分溶解, 用酶标仪(NJ-2300, Japan)双波长测定其A值, 测定波长为570 nm, 参考波长为630 nm.酶标仪所示A值为570减去630 nm, 以消除非特异性光吸收效应A值换算成细胞增生率: 实验组A值/对照组A值×100%[24]. HE是一个特异性的O2- 染料[33]; DCFH-DA常用于检测细胞内H2O2[34] . HE和DCFH-DA溶解于DMSO, 用PBS稀释成5 μmol/L备用. 细胞被接种在6孔培养板中, 生长至90%融合时, 加入不同浓度的TTD, 1 h后细胞被胰蛋白酶消化、PBS洗涤2次, 用流式细胞仪(becton dickinson, san jose, CA)检测细胞荧光强度. 在检测30 min前分别加入5 μmol/L的HE和DCFH-DA. 激发波长488 nm, 发射波长530 nm. 将细胞数调整为2×109/L接种到100 mm培养板中, 生长至90%融合时, 加入不同浓度的TTD, 48 h后贴壁和黏附细胞被收集, 采用硫氰酸胍酸/酚/氯仿法抽提核酸, 按说明书进行.抽提的DNA 被悬浮在70 mL/L乙醇中, 于-20 °C 过夜后, 高速离心2 500 g, 5 min除去乙醇, 再悬浮DNA于含RNA酶(Rnase, 100 mg/L, Sigma)的Tris/EDTA缓冲液30 μl, 37 °C 2 h 孵育后, 使用15 g/L 凝胶电泳, 1 h后在紫外灯下观察细胞凋亡的DNA梯形条带.

2 结果

经TTD处理的肝癌细胞, 在24、48、72 h后, 其抑制效应与TTD剂量有良好的相关关系(图1). 与对照组(图2A)比较, TTD10 μmol/L组(图2B)和20 μmol/L组(图2C) O2- 和HH2O2阳性细胞百分比增加, 20 μmol/L组增加明显. TTD处理的肝癌细胞 20 μmol/L组48 h后引起了DNA片断化, 出现了典型的梯形条带(图3), 这是细胞凋亡的晚期表现.

图1
图1 采用MTT比色法观察了TTD对肝癌细胞增生的影响, 从5组实验数据中可知TTD对Mahlavu 增生抑制呈时间和剂量依赖关系.
图2
图2 用HE和DCFH-DA染色检测细胞内O2- , H2O2 . 细胞被接种在6孔培养板中, 生长至90%融合时, 加入不同浓度的TTD作用2 h, (a为对照, b为10 μmol/L, c为20 μmol/L), PBS洗涤2次, 用流式细胞仪检测细胞荧光强度, 在检测30 min前分别加入5 μmol/L的HE和DCFH-DA.
图3
图3 TTD处理的肝癌细胞48h后引起了DNA片断化, 在15g/L凝胶电泳中出现了典型的梯形条带. M为100单位碱基对标记组, 带1为对照组, 带2, 3为TTD ( 10 μmol/L and 20μmol/L)处理组.
3 讨论

细胞凋亡是真核生物细胞最基本的生命过程之一, 肿瘤生长是由于异常的细胞分裂和凋亡的失衡, 肿瘤诱导分化中的细胞凋亡、肿瘤治疗中的细胞凋亡、肿瘤浸润中的细胞凋亡是近年研究的热点[24,25]. TTD对艾氏腹水癌细胞、小鼠淋巴细胞白血病细胞、小鼠肉瘤细胞、Burkitt淋巴瘤细胞等增生具有抑制作用[35], 其对肝癌细胞的作用及机制, 尚未见报道. 我们发现, TTD呈明显的剂量依赖性抑制肝癌细胞的增生、引起肝癌细胞DNA片断化, 提示TTD可以诱导肝癌细胞的调亡, 从而抑制肝癌细胞的生长、增生. ROS是指氧的某些代谢产物和一些反应的含氧产物, 其中含O2-、O、OH、H2O2等. 生理状态下, 机体产生的自由基与抗氧化防御系统处于相对平衡; 在各种病理因子作用下, 机体产生大量自由基, 或机体抗氧化防御系统受到破坏, 造成细胞结构和功能的破坏, 诱导细胞调亡的发生[34]. ROS可能通过引起细胞脂质过氧化、损伤DNA分子或调节细胞凋亡相关基因而诱导凋亡[35]. TTD作用于肝癌细胞后, 在2 h内, 迅速引起细胞内ROS的增加, 48 h后20 μmol/L TTD组出现DNA片断化, 具有典型的梯形条带; 10 μmol/L TTD组未见明显梯形条带变化, 具有明显的量效关系, 提示TTD可能通过干扰细胞线粒体的功能而产生活性氧, 进一步诱导肝癌细胞凋亡.

总之, 本研究提示, TTD能够通过诱导细胞内活性氧的产生, 而引起细胞凋亡, 为临床应用TTD治疗肝细胞肝癌提供了理论依据, 也为进一步寻找更有效的治疗药物提供了新的思路.

致 谢

感谢日本兵库医科大学第三内科波田寿一教授、上木升先生的指导.

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