肝癌 Open Access
Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2003-09-15; 11(9): 1369-1371
在线出版日期: 2003-09-15. doi: 10.11569/wcjd.v11.i9.1369
TRAIL诱导肝癌细胞系SMMC-7721的凋亡作用
李小安, 房殿春, 司佩任, 张汝刚, 杨柳芹, 秦建平
李小安, 秦建平, 中国人民解放军成都军区总医院消化内科 四川省成都市 610083
李小安, 男, 1968-09-01生, 安徽省东至县人, 汉族, 2002年第三军医大学博士毕业. 现工作单位为成都军区总医院消化内科. 发表文章10篇, 研究方向为消化道肿瘤.
房殿春, 司佩任, 张汝刚, 杨柳芹, 中国人民解放军第三军医大学西南医院全军消化专科中心 重庆市 400038
基金项目: 全军"十五"科研基金资助项目, No. 01MA172
通讯作者: 房殿春, 400038, 重庆市高滩岩, 中国人民解放军第三军医大学西南医院全军消化专科中心.
电话: 028-86570349
收稿日期: 2002-08-24
修回日期: 2002-09-20
接受日期: 2002-10-12
在线出版日期: 2003-09-15

目的

观察TRAIL诱导肝癌SMMC-7721细胞凋亡的作用.

方法

采用MTT 法检测细胞存活分数; TUNEL 法检测细胞凋亡率; 流式细胞仪检测细胞凋亡和细胞周期; 电镜观察凋亡细胞超微结构.

结果

TRAIL对SMMC-7721细胞的存活分数和凋亡率的影响呈典型的量效关系, 经TRAIL作用后的细胞, 流式细胞仪检测呈标准的亚二倍峰, 电镜观察发现经TRAIL作用的部分细胞具有凋亡细胞的典型形态特征.

结论

TRAIL可诱导SMMC-7721细胞凋亡.

关键词: N/A
引文著录: 李小安, 房殿春, 司佩任, 张汝刚, 杨柳芹, 秦建平. TRAIL诱导肝癌细胞系SMMC-7721的凋亡作用. 世界华人消化杂志 2003; 11(9): 1369-1371
Role of TRAIL in inducing apoptosis of SMMC-7721 cells
Xiao-An Li, Dian-Chun Fang, Pei-Ren Si, Ru-Gang Zhang, Liu-Qin Yang, Jian-Ping Qing
Xiao-An Li, Jian-Ping Qing, Department of Gastroenterology, Chinese PLA, General Hospital of Chengdu Military Command, Chengdu 610083, Sichuan Province, China
Dian-Chun Fang, Pei-Ren Si, Ru-Gang Zhang, Liu-Qin Yang, Department of Gastroenterology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China
Supported by the Scientific Research Programs Foundation during the Tenth Five-Year Plan of PLA, No. 01MA172
Correspondence to: Dian-Chun Fang, Department of Gastroenterology,Southwest Hospital, Third Military Medical University, Chongqing 400038, China
Received: August 24, 2002
Revised: September 20, 2002
Accepted: October 12, 2002
Published online: September 15, 2003

AIM

To observe the role of TRAIL in inducing apoptosis of SMMC-7721 cells.

METHODS

The survival fraction of SMMC-7721 cells was measured by MTT assay; Apoptosis rate was determined by TUNEL method and the ultramicrostructure of apoptosic cells induced by TRAIL was observed by electron-microscopy.

RESULTS

The role of TRAIL in survival fraction and apoptosis rate demonstrated a good relationship and the typic structure of apoptosic cells was foud in some cells treated by TRAIL.

CONCLUSION

TRAIL can induce apoptosis in SMMC-7721 cells.

Key Words: N/A
0 引言

肿瘤坏死因子相关的凋亡诱导配体(TNF-related apoptosis-inducing ligand, TRAIL)基因是Wiley小组于1995年最早克隆和命名的, 为TNF家族成员[1]. 近年来, 人们发现其可以诱导肿瘤细胞凋亡, 对正常细胞的影响却很少, 因而受到了国内外学者的广泛重视[1,2]. 在本文中, 我们研究了TRAIL诱导肝癌SMMC-7721细胞凋亡的作用.

1 材料和方法
1.1 材料

可溶性TRAIL蛋白(氨基酸114-281, 带六聚组氨酸尾)由本实验室生产; MTT购自上海生物工程技术公司; TUNEL原位凋亡检测试剂盒购自罗氏公司; RPMI1640培养基购自Sigma公司; 肝癌细胞株SMMC-7721由本实验室保种.

1.2 方法

细胞培养: SMMC-7721细胞培养于含100 mL/L灭活的小牛血清、100 kU/L青霉素和链霉素的RPMI1640的培养液中, 培养条件为37 °C, 50 mL/L CO2, 饱和湿度, 每2-3 d用2.5 g/L胰酶消化, 以1:3-1:5传代. MTT法测细胞存活分数[3] : 以2.5 g/L胰蛋白酶消化细胞, 用含50 mL/L 小牛血清的RPMI1640配成单个细胞悬液, 按每孔3×103 个细胞接种于96孔板, 每孔终体积为200 mL, CO2孵箱内培养2-3 d后按50 mg/L, 150 mg/L, 500 mg/L, 1 500 mg/L, 5 000 mg/L的剂量分别给予TRAIL, 对照组给予同体积的PBS, 药物作用24 h. 测值前4 h每孔加20 mL 5 g/L MTT, 孵育后吸去孔内上清, 每孔加150 mL DMSO, 振荡10 min, 酶联免疫检测仪测A570值. 细胞存活分数(survival fraction)=实验组A570/对照组A570×100 %. 实验重复3次, 取平均值. TUNEL法检测TRAIL对SMMC-7721细胞凋亡率的影响: 将无菌的盖玻片置于六孔板中, 每孔一片. 取对数生长期细胞以2.5 g/L胰蛋白酶消化细胞, 用含100 mL/L 小牛血清的RPMI1640细胞培养液配成单个细胞悬液, 稀释成5108/L. 取0.5 mL滴于盖玻片上, 37 °C、50 mL/L CO2、饱和湿度下孵育2 h后, 每孔加培养液2 mL, 次日, 每孔加入不同浓度(剂量同MTT法)的TRAIL. 24 h后, 按TUNEL试剂盒说明操作, DAB显色后, 高倍镜(400)下随机数200个细胞, 记下凋亡细胞数和未凋亡细胞数, 共数5个视野. 细胞凋亡率(apoptosis rate)= 凋亡细胞数/(凋亡细胞数+未凋亡细胞数)100 %. 流式细胞仪测定TRAIL对SMMC-7721细胞的凋亡率和细胞周期的影响: 按0 mg/L(对照组)、200 mg/L(T1组)、400 mg/L(T2组)给予TRAIL, 24 h后收集不同浓度药物处理组的细胞, PBS漂洗2次, 750 mL/L冷乙醇固定24 h, PI染色后, 用流式细胞仪检测细胞周期和凋亡率. 电镜: 待SMMC-7721细胞长至对数生长期, 取两瓶细胞, 分别加100 mg/L的TRAIL和同体积的PBS, 作用24 h后, 常规胰酶消化细胞, PBS洗2次, 30 g/L的戊二醛固定后送检.

2 结果
2.1 TRAIL对肝癌SMMC-7721细胞存活分数的影响

TRAIL作用于肝癌SMMC-7721细胞24 h, 细胞的存活分数从50 mg/L 的89.1 %降至5 000 mg/L 27.2 %, 存在较好的量效关系.

2. 2 TRAIL对肝癌SMMC-7721细胞凋亡率的影响

TRAIL能明显诱导SMMC-7721细胞凋亡, 存在较好的量效关系, 见图1.

图1
图1 TRAIL对肝癌SMMC-7721细胞凋亡率的影响. A 未处理细胞, 胞膜微绒毛和伪足多见, 核大、核内有多个核仁; B 经TRAIL作用后的细胞线粒体空泡化; C 经TRAIL作用后的细胞, 胞膜微绒毛和伪足减少, 核浓缩、碎裂; D 经TRAIL作用后的细胞, 胞膜微绒毛和伪足减少, 凋亡小体形成.
2.3 TRAIL对SMMC-7721细胞的凋亡率和细胞周期的影响

400 mg/L的TRAIL作用于SMMC-7721细胞, 可见明显的亚二倍峰形成, TRAIL还可使S期细胞的比例增加, G2M期细胞的比例减少, 结果见表1.

表1 TRAIL对SMMC-7721细胞细胞周期和凋亡率的影响.
分组TRAIL剂量(mg/L)细胞周期
凋亡率(%)
G0/G1SG2M
对照组00.390.080.530.72
T1组2000.470.280.2519.31
T2组4000.300.450.2523.83
2.4 电镜

未经处理的SMMC-7721细胞, 胞膜微绒毛和伪足多见, 核大、核内有多个核仁(图1A). 经TRAIL作用后的细胞, 胞膜微绒毛和伪足减少, 线粒体空泡化(图1B), 核浓缩、碎裂(图1C), 凋亡小体形成(图1D).

3 讨论

TRAIL是TNF家族成员, 有五种受体即DR4, DR5, DcR1, DcR2和OPG. TRAIL与DcR1, DcR2和OPG结合不能诱导细胞凋亡, 但与DR4, DR5结合可导致细胞凋亡[4-20]. 我们以前曾证实TRAIL对结肠癌细胞系SW480有杀伤作用[21]. 本研究我们发现TRAIL 可以诱导SMMC-7721细胞凋亡. 经TRAIL作用后的SMMC-7721细胞经流式细胞仪检测, 出现典型的亚二倍峰, 该峰的出现是凋亡的特征之一[22]. 此外, 我们的电镜结果发现, 经TRAIL作用后的细胞, 胞膜微绒毛和伪足减少, 核浓缩、碎裂, 凋亡小体形成, 具有凋亡细胞的典型形态特征. 因此, 我们有充足的证据表明TRAIL是通过诱导细胞凋亡的方式起抗肿瘤作用的. TRAIL诱导的细胞凋亡涉及线粒体膜电位的改变, 线粒体释放细胞色素C [8-10,23-26], 而我们用电镜观察到经TRAIL作用后的SMMC-7721细胞线粒体空泡化, 这种功能和形态的变化有可能存在一定的联系. 流式细胞仪的结果表明TRAIL可使 SMMC-7721细胞G2M期细胞的比例减少, 证实TRAIL可能有抑制细胞增生的作用, S期为细胞的DNA合成期, TRAIL为什么使SMMC-7721细胞S期细胞的比例增加, 值得进一步研究.

早期的研究发现TRAIL诱导细胞凋亡有很高的选择性, 即仅诱导被病毒感染的细胞、转化细胞和肿瘤细胞凋亡, 对正常细胞的影响很小[1,2]. 最近人们发现TRAIL可诱导正常肝细胞凋亡, 但caspase-9的抑制剂Z-LEHD-FMK可以保护正常人的肝细胞, 而不影响TRAIL对一些肿瘤细胞的杀伤作用, 可能与TRAIL诱导不同的细胞凋亡存在不同的传导通路有关[27]. 我们发现TRAIL可诱导肝癌细胞凋亡, 并提示可能与线粒体通路有关.

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