全 文 ：高内涵法探讨土槿皮乙酸对
MCF-7细胞抑制作用的机制 *
段绍维 徐 波 陈云利 李 敏 傅宏征 ** 崔景荣 **
(北京大学天然药物及仿生药物国家重点实验室，北京 100191)
摘要 土槿皮乙酸(pseudolaric acid B，PAB)是土槿皮(金钱松根皮)的主要生物活性成分，对多种人肿瘤细胞有细胞毒性
作用．高内涵分析(high content analysis，HCA)是一种基于荧光显微技术的新技术，它以细胞为研究对象，可以同时对多个
荧光靶点的荧光强度、分布，以及细胞形态进行自动化定量分析．利用高内涵分析、流式细胞术研究 PAB 对人乳腺癌
MCF-7细胞抑制作用的机制．磺酰罗丹明实验显示，PAB抑制MCF-7细胞增殖，且呈现出剂量和时间依赖性，72 h IC50为
(1.80±0.33) μmol/L．流式细胞术碘化丙锭(PI)单染显示 PAB 作用 24 h，可致 MCF-7 细胞 G2/M期比例增至 93%以上，
annexin V-FITC和 PI双染显示 PAB促进MCF-7细胞凋亡．高内涵分析显示：PAB作用 16 h，MCF-7细胞有丝分裂指数可
达 40%左右，伴有 cyclin B1含量增加；PAB促进微管解聚，干扰有丝分裂二极纺锤体形成；PAB引起线粒体增生；PAB
导致“葡萄串样”细胞核形成，提示有丝分裂滑脱．结果表明，PAB抑制MCF-7细胞增殖、促进MCF-7细胞凋亡，这些作
用可能与其促进微管蛋白解聚、干扰二极纺锤体形成、阻滞有丝分裂、促进线粒体增生有关．
关键词 高内涵分析，土槿皮乙酸，MCF-7细胞，微管，有丝分裂阻滞，凋亡
学科分类号 R961，R965.1，R979.1 DOI: 10.3724/SP.J.1206.2010.00335
生物化学与生物物理进展
Progress in Biochemistry and Biophysics
2010, 37(12): 1313~1322
www.pibb.ac.cn
研究报告Research Papers
* 国家“十一五”攻关项目,重大新药创制资助项目(2009ZX09301-
010).
**通讯联系人.
崔景荣. Tel: 010-82802467, E-mail: jrcui@bjmu.edu.cn
傅宏征. Tel: 010-82805212, E-mail: drhzfu@yahoo.com.cn
收稿日期：2010-06-25，接受日期：2010-09-30
土槿皮为松科植物金钱松(Pseudolarix kaempferi
Gordon)的根皮，是一种治疗皮肤真菌感染的传统
中药．从土槿皮中分离得到的二萜类化合物土槿皮
乙酸(pseudolaric acid B，PAB；图 1)，是土槿皮抗
真菌的主要成分[1]．体外实验显示，PAB对多种人
肿瘤细胞系的增殖有抑制作用[2]，而且相对于人正
常细胞表现出选择性(PAB作用于肿瘤细胞的 IC50
约为正常细胞的 1/100)[3-4]，诱导人白血病、胃癌、
肝癌、结肠癌、乳腺癌、宫颈癌和黑色素瘤等恶性
肿瘤细胞凋亡[3, 5-10]，抑制血管生成[11-12]，促进微管
解聚[4, 11]．高内涵分析(high content analysis)是一种
基于荧光显微技术的自动化技术，它以结构完整的
细胞作为研究对象，可以同时对多个荧光靶点的荧
光强度、分布，以及细胞形态进行自动化的定量分
析，可用于药物机制研究或者大规模药物筛选[13-14].
乳腺癌是妇女最多发的恶性肿瘤，而且其发病率呈
逐渐上升趋势．本文利用高内涵分析等技术研究
PAB对人乳腺癌MCF-7细胞抑制作用的机制．
1 材料与方法
1．1 试剂和药品
RPMI-1640细胞培养基购自 Gibco公司；胎牛
Fig. 1 Chemical structure of pseudolaric acid B (PAB)
HOOC
COOCH3
OCOCH3H
O
O
生物化学与生物物理进展 Prog. Biochem. Biophys. 2010; 37 (12)
血清购自杭州四季青生物工程材料有限公司；磺酰
罗丹明 B(sulforhodamine B，SRB)、核糖核酸酶 A
(RNase A)、碘化丙锭 (propidium iodide，PI)购自
Sigma-Aldrich公司；annexinⅤ凋亡检测试剂盒由
北京大学人类疾病基因研究中心提供．高内涵分析
试剂盒 Mitotic Index Kit (K0500011)、 Cyclin B1
Activation Kit(8404402)、Cytoskeletal Rearrangement
Kit(8402402)、Multiparameter Cytotoxicity 2 Multiplex
Kit(8400002)购自 Cellomics 公司 (Pittsburgh，PA，
USA)．兔抗 pHH3 IgG、Alex Fluor 488羊抗兔 IgG
取自 Mitotic Index Kit(K0500011)；鼠抗 cyclin B1
抗体、 DyLightTM 549 羊抗鼠 IgG、 DAPI 取自
Cyclin B1 Activation Kit(8404402)；DY554-鬼笔环
肽、鼠抗微管蛋白抗体、DyLightTM 649羊抗鼠 IgG
取自 Cytoskeletal Rearrangement Kit(8402402)；线
粒体染液取自MultiparameterCytotoxicity 2 Multiplex
Kit(8400002)．高内涵分析使用的打孔液、封闭液
取自前述各试剂盒．
PAB由北京大学天然药物及仿生药物国家重
点实验室傅宏征教授制备，溶于 DMSO，储存于
-20℃，储备液用 RPMI-1640完全培养基稀释为不
同浓度．作用于细胞时 PAB终溶液的 DMSO含量
为 0.5%(体积比)．紫杉醇注射液购自海口市制药
厂；长春碱取自Mitotic Index Kits(K0500011)．
1．2 细胞培养
人乳腺癌细胞 MCF-7 由本室保存，以含有
10%灭活胎牛血清、100 U/ml 青霉素、100 mg/L
链霉素的 RPMI-1640培养基于 37℃、5%CO2、饱
和湿度培养箱中培养．
1．3 磺酰罗丹明实验
取对数生长期MCF-7细胞 5000个 /孔接种于
96孔板(Costar3599)．培养箱中培养 24 h后吸去培
养基，加入不同浓度 PAB 溶液 200 μl/ 孔．含
0.5%DMSO的 RPMI-1640完全培养基和 1 μmol/L
紫杉醇分别作为阴性和阳性对照．每种处理方式设
6个平行孔．加药后 0、48、72 h，参考 Skehan的
方法 [15]，在培养基液面上加 50 μl 50%三氯乙酸
(trichloroacetic acid，TCA)，4℃固定 1 h，自来水
冲洗，0.4%SRB染色，1%乙酸冲洗，10 mmol/L
Tris溶解细胞结合的 SRB．最后用酶联免疫检测仪
在 540 nm波长测定吸光度(A)值，计算抑制率、
IC50和 GI50[16]．抑制率=(C-T)/C×100%，IC50为抑制
率等于 50%时的药物浓度，GI50为(T-T0)/(C-T0)＝
50%时的药物浓度．T、C、T0分别为药物作用后
的 A值、阴性对照的 A值、药物作用 0 h的 A值.
1．4 流式细胞术
取对数生长期MCF-7细胞 1×105个 /ml接种于
培养瓶．24 h后加入不同浓度 PAB溶液，含 0.5%
DMSO的 RPMI-1640 完全培养基和 2.5 μmol/L紫
杉醇溶液(终浓度)分别作为阴性和阳性对照．
1．4．1 细胞周期(PI单染)．收集细胞，经 PBS洗涤
后取约 1×106个细胞，70%冷乙醇固定过夜．固定
后的细胞经 PBS洗涤，RNase A(终浓度 100 mg/L)
37℃孵育 30 min，再次 PBS洗涤，筛网过滤，PI
染色 (PI 终浓度 50 mg/L)，上流式细胞仪用
CellQuest软件分析细胞周期．
1．4．2 细胞凋亡(annexin V-FITC+PI). 用 annexinⅤ
凋亡检测试剂盒处理细胞．收集细胞，经 PBS洗
涤后取约 1 ×106 个细胞，在结合缓冲液中用
annexinⅤ-FITC室温孵育 15 min，筛网过滤，加
入 PI，上流式细胞仪用 CellQuest软件分析凋亡细
胞比例．
1．5 高内涵分析(high content analysis，HCA)
取对数生长期 MCF-7细胞，稀释细胞悬液至
适宜密度(多参数细胞周期分析、细胞核和线粒体
分析 5×104个 /ml，微管和微丝分析 3×104个 /ml )，
每孔 100 μl加入 96孔板(Costar 3599)，96孔板事
先用 0.1%明胶包被．24 h后加入不同浓度 PAB溶
液 50 μl/ 孔，含 0.5% DMSO的 RPMI-1640 完全
培养基为阴性对照，1 μmol/L紫杉醇(终浓度)、
1 μmol/L长春碱(终浓度)为阳性对照．
1．5．1 多参数细胞周期分析．加药后 24 h，加入
37℃预热的 16%甲醛溶液 50 μl/孔(终浓度 4%)室
温固定 30 min，再用打孔液、封闭液分别作用
15 min．鼠抗 cyclin B1(1∶500)、兔抗磷酸化组蛋
白 H3(phospho-histone H3, pHH3, 1∶400)50 μl/孔
室温孵育 1 h．PBS 洗 2 次．细胞核染料 DAPI
(1∶2000)、DyLightTM 549羊抗鼠 IgG(1∶500)、Alex
Fluor 488羊抗兔 IgG(1∶250) 50 μl/孔室温避光孵
育 30 min．PBS 洗 2 次．使用高内涵分析设备
KineticScan Reader(Cellomics, Pittsburgh, PA, USA)
采集荧光图像，配套软件 Cell Cycle BioApplication
计算 cyclin B1 荧光强度和有丝分裂指数 (mitotic
index，MI)．因为 pHH3阳性可视为M期细胞的标
志[17]，因此 MI = 100% × M期细胞数 /总细胞数=
100% × pHH3阳性细胞数 /DAPI着色细胞数．
1．5．2 微管和微丝．加药后 8 h，MCF-7细胞经固
定、打孔、封闭(同 1．5．1)，室温下用鼠抗微管蛋
1314· ·
段绍维等：高内涵法探讨土槿皮乙酸对MCF-7细胞抑制作用的机制2010; 37 (12)
白抗体(1∶1 000)孵育 1 h， PBS 洗 2 次， DAPI
(1∶2 000)和 DyLightTM 649羊抗鼠 IgG(1∶1 000)室
温避光孵育 30 min，PBS 洗 2 次．Compartmental
Analysis BioApplication计算微管蛋白荧光强度．
1．5．3 细胞核和线粒体．加药后 23.5、47.5 h，加
入线粒体染液 60 μl/孔作用 30 min，加入 37℃预
热的 16%甲醛 70 μl/ 孔 (终浓度 4%)室温固定
30 min，PBS洗 1次，DAPI(1∶2 000) 50 μl/孔室
温避光孵育 30 min，PBS 洗 2 次．Compartmental
Analysis BioApplication分析细胞核形态和线粒体
荧光强度．
1．6 统计学分析
计量资料用 x ± s表示，加药组和阴性对照之
间的比较分析采用 Students t检验．
2 实验结果
2．1 PAB 对细胞增殖的抑制作用
PAB作用MCF-7细胞 24 h后，倒置显微镜下
见大量细胞变圆，作用 48 h或 72 h后，见细胞数
目少于阴性对照孔，细胞碎片增多，可见体积变大
的细胞．SRB实验结果显示，PAB对MCF-7细胞
增殖的抑制作用表现出剂量和时间依赖性(图 2)．
PAB 作用 72 h的 IC50为(1.80±0.33) μmol/L．PAB
作用 48 h和 72 h的 GI50分别为(1.85±0.30) μmol/L
和(1.43±0.26) μmol/L．
2．2 PAB 导致细胞 G2/M 期阻滞、有丝分裂指数
升高、cyclin B1 含量升高
2．2．1 PAB 导致 G2/M 期阻滞．不同浓度 PAB
作用于 MCF-7 细胞 24 h，PI染色后流式细胞术
分析细胞周期．结果显示随着 PAB浓度升高(0～
1.25 μmol/L)，S 期细胞比例逐渐增大．PAB 浓
度≥2.5 μmol/L时，S期细胞比例减小，G2/M期
比例明显增大,达 93%以上(图 3a). 2.5 μmol/L PAB
作用于 MCF-7 细胞 12、24 h，G2/M期细胞比例
增大，表现出时间依赖性 (图 3b)．高内涵法通
过 DAPI荧光强度分析细胞周期，5 μmol/L PAB、
1 μmol/L紫杉醇作用于MCF-7细胞 16 h后，细胞
4 N峰升高(图 4a)，与流式细胞术结果类似．
2．2．2 PAB导致 MI升高．0.5%DMSO、不同浓度
的 PAB或 1 μmol/L紫杉醇作用于MCF-7细胞 16 h.
20
0
0.31 0.63 1.25 2.5 5 PTX
c(PAB)/(μmol·L-1)
40
60
80
In
hi
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/%
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*
** **
**
**
**
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Fig. 2 Effect of PAB on the proliferation of MCF-7 cells
MCF-7 cells were exposed to increasing concentrations of PAB for 48 h
or 72 h, and 1 μmol/L paclitaxel (PTX) was used as positive control.
Inhibition rates were determined by SRB assay. Each data point
represents the x ± s deviation of 3 independent experiments．*P < 0.05,
**P < 0.01, vs solvent control. : 48 h; : 72 h.
20
0
0
c(PAB)/(μmol·L-1)
40
60
80
100
Ce
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%
0.31
*
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0.63 1.25 2.5 5
(a)
20
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40
60
80
100
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%
(b)
12 24
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Fig. 3 Effect of PAB on cell cycle distribution
of MCF-7 cells
MCF-7 cells were stained with PI, and cell cycle distribution was
determined by flow cytometry analysis of DNA content. (a) MCF-7 cells
were exposed to increasing concentrations of PAB for 24 h. Data were
given as x ± s, n=2. (b) MCF-7 cells were exposed to 2.5 μmol/L PAB
for 12 h or 24 h. Data were given as x ± s, n=3. *P < 0.05, **P < 0.01,
vs solvent control. : G0/G1; : S; : G2/M.
1315· ·
生物化学与生物物理进展 Prog. Biochem. Biophys. 2010; 37 (12)
PAB作用后高表达 pHH3、cyclin B1 的细胞增多，
高表达 pHH3 者可见染色质凝集(图 4a)．定量分
析见 PAB 导致 MI 升高，且表现出剂量依赖性
(图 4b)．PAB导致细胞 cyclin B1 含量增高，且表
现出剂量依赖性(图 4c)．另外，从图 4a可见 pHH3
阳性细胞常伴随着较亮的 cyclin B1荧光．为了比
较M期细胞(pHH3阳性)和间期细胞(pHH3阴性)的
cyclin B1含量，选择适宜的 pHH3荧光强度作为临
界值，根据此临界值区分 M期细胞和间期细胞，
计算其各自的 cyclin B1荧光强度．结果显示，在
溶剂对照组、PAB 组、紫杉醇组中，M期细胞
cyclin B1含量都要高于间期细胞(图 4d)．
Fig． 4 Multiparametric analysis of cell cycle by high content analysis
MCF-7 cells were treated for 16 h with 0.5%(v/v) DMSO, indicated concentrations of PAB or 1 μmol/L paclitaxel (PTX). (a) Representative images of
MCF-7 cells treated with 0.5%DMSO, 5 μmol/L PAB or 1 μmol/L paclitaxel. Cells were simultaneously stained for nucleus, phospho-histone H3 and
cyclin B1. PAB and paclitaxel induced increases of phospho-histone H3-positive cells (mitotic cells) and cyclin B1- highly expressed cells. Right panels
represent DNA content distribution obtained from DAPI staining. Images were captured with KineticScan Reader (Cellomics, Pittsburgh, PA, USA)
with a 10× objective. The background of some images of the same fluorescence target appears different because images were displayed in contrast
stretched mode to emphasize weak fluorescence, while quantitative data were not stretched. (b) Quantitation of mitotic index of MCF-7 cells. PAB
induced an increase in mitotic index in a dose-dependent manner. (c) PAB induced an increase in cyclin B1 fluorescence intensity in a dose-dependent
manner. (d) Cyclin B1 fluorescence intensity of mitotic cells was compared with that of interphase cells for each treatment. Mitotic cells showed higher
cyclin B1 fluorescence intensity than interphase cells. Quantitation in (b), (c) and (d) was performed with the Cell Cycle BioApplication (Cellomics,
Pittsburgh, PA, USA), and at least 600 cells per well were analyzed. Data were given as x ± s, n=3. *P < 0.05, **P < 0.01, ***P < 0.001, vs solvent
control. in (d): Interphase cells; in (d):Mitotic cells.
(a)
(b)
10
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20
30
40
50
(c)
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0.63 1.25 2.5 5 PTX10
DNA contentCyclin B1Phospho-histone H3Nuclei
0.5%DMSO
1 μmol/LPTX
5 μmol/LPAB
2N
4N
2N
2N
4N
4N
20 μm
1316· ·
段绍维等：高内涵法探讨土槿皮乙酸对MCF-7细胞抑制作用的机制2010; 37 (12)
Fig. 6 Effect of PAB, paclitaxel and vinblastine on
interphase microtubule networks in MCF-7 cells
MCF-7 cells treated for 8 h with 0.5% (v/v) DMSO, PAB, paclitaxel
(PTX) orvinblastine (VLB) were stained fornucleus (blue), F-actin (red)
and tubulin (green). (a) Compared with solvent control, microtubules in
cells were slightly disrupted after treatment with 2.5 μmol/L PAB and
more completely with 5 μmol/L PAB or 1 μmol/L vinblasinte. On the
other hand, cells treated with 1 μmol/L paclitaxel have stabilized
microtubule bundles around the nuclei. Images were captured with
KineticScan Reader (Cellomics, Pittsburgh, PA, USA) with a 20 ×
objective. (b) Microtubule fluorescence intensity was quantified using
Compartmental Analysis BioApplication (Cellomics, Pittsburgh, PA,
USA), and at least 120 cells per well were analyzed. Data were given as
x ± s, n=3. * P < 0.05, vs solvent control.
Fig. 5 PAB and paclitaxel induced spindle
abnormalities in MCF-7 cells
MCF-7 cells were treated for 8 h with 0.5% (v/v) DMSO, indicated
concentrations of PAB or 1 μmol/L paclitaxel. Cells were then
counterstained for nucleus (blue) and tubulin (red). Representative
images of mitotic cells were shown (arrows). Metaphase cells in control
wells have normal bipolar spindles, and their chromosomes congregate
to the equator forming the equatorial plate (left upper panel). Both PAB
and paclitaxel arrested cells in prometaphase and induced spindle
abnormalities. Images were captured with KineticScan Reader
(Cellomics, Pittsburgh, PA, USA) with a 20 × objective. Images were
displayed in contrast stretchedmode to emphasize weak fluorescence.
2．3 PAB 干扰两极纺锤体形成
高内涵分析采集的荧光图片显示，溶剂对照孔
可见正常的有丝分裂中期细胞(图 5)，染色体排列
成赤道板(equatorial plate)，微管形成两极纺锤体．
2.5、5 μmol/L PAB 作用于 MCF-7 细胞后 8 h，
M期细胞染色体凝集但是分布紊乱，未见赤道板，
微管染色淡，未见两极纺锤体形态．荧光图片中
(2.5、5、10 μmol/L PAB 共 90 个视野约 1 500 个
细胞 )未发现一个正常的有丝分裂中期细胞．
1 μmol/L紫杉醇作用 8 h 后也未能见到两极纺
锤体．
2．4 PAB 促进间期细胞微管解聚
PAB 作用于 MCF-7 细胞 8 h，纤维状肌动蛋
白(F-actin)和微管蛋白(tubulin)行免疫荧光染色后作
高内涵分析．从图 6a可见 2.5 μmol/L PAB作用后
细胞微管纤维连续性较溶剂对照差，出现更多
中断，5 μmol/L PAB 作用后微管蛋白荧光强度
明显减弱，细胞淡染，核周残余较亮的荧光团块．
1 μmol/L长春碱作用后细胞淡染、核周较亮，但
是荧光分布较 PAB作用后均匀．1 μmol/L紫杉醇
作用后微管蛋白荧光强度明显增强，在核周形成高
亮的微管束．定量分析表明(图 6b) 5、10 μmol/L
PAB导致细胞微管蛋白荧光强度减弱，紫杉醇与
长春碱分别导致微管蛋白荧光强度增强和减弱．
0.5%DMSO
1 μmol/LPTX5 μmol/LPAB
2.5 μmol/LPAB
Merge
0.5%DMSO
1 μmol/LPTX
5 μmol/LPAB
(a) F-actin Tubulin
2.5 μmol/LPAB
1 μmol/LVBL
0
20
40
(b)
0 1.25 2.5 5 PTX
c(PAB)/(μmol·L-1)
10 VLB
M
ic
ro
tu
bu
le
flu
or
es
ce
nc
e
in
te
ns
ity
(%
of
so
lv
en
tc
on
tro
l)
60
80
100
120
140
160
180
200
**
*
*
20 μm
20 μm
1317· ·
生物化学与生物物理进展 Prog. Biochem. Biophys. 2010; 37 (12)
2．5 PAB 引起细胞核形态改变、线粒体增生
PAB作用后，MCF-7细胞形成“葡萄串”样
细胞核．1 μmol/L紫杉醇也引起类似改变(图 7a)．
定量分析见 PAB作用 24、48 h后，细胞核增大、
形状变得不规则，尤以 PAB 浓度≥2.5 μmol/L时
明显(图 7b、7c)．PAB还导致线粒体荧光强度明显
增强，提示线粒体增生．
2．6 PAB 促进细胞凋亡
不同浓度 PAB作用于 MCF-7细胞一定时间，
用 annexin V-FITC和 PI染色，流式细胞术分析凋
亡细胞比例．结果显示，PAB作用后 72 h, annexin
Fig. 7 PAB and paclitaxel induced nuclear morphology and mitochondrial changes in MCF-7 cells
(a) Representative images of MCF-7 cells treated for48 h with 0.5%(v/v) DMSO, PAB orpaclitaxel (PTX) were stained fornucleus andmitochondrion.
PAB (≥2.5 μmol/L) and 1 μmol/L paclitaxel induced giant and grape-like nuclei and increase of mitochondrial fluorescence intensity. Images were
captured with KineticScan Reader (Cellomics, Pittsburgh, PA, USA) with a 20× objective. (b) and (c) Quantitation of nuclear size and roundness (P2A).
Nuclear size increased while roundness decreased after treatment for24 h or48 hwith PAB (≥2.5 μmol/L). NuclearP2A = (perimeter)2 ÷ (4π × area of
nucleus). P2A value is 1 for circle, and irregularly shaped objects have values greater than 1. (d) PAB and paclitaxel induced increase of mitochondrial
fluorescence intensity. Quantitation in (b), (c) and (d) was performed with the Compartmental Analysis BioApplication (Cellomics, Pittsburgh, PA,
USA), and at least 100 cells perwell were analyzed (mitotic cellss were excluded when nuclear size and roundness were measured). Data were given as
x ± s, n=3. *P < 0.05, **P < 0.01, ***P < 0.001, vs solvent control. : 24 h; : 48 h.
1.0
0
c(PAB)/(μmol·L-1)
1.2
0.63 1.25 2.5 5 PTX10
N
uc
le
ar
P2
A
1.4
1.6
1.8
*
* *
**
*
*
(b)
20
0
c(PAB)/(μmol·L-1)
25
0.63 1.25 2.5 5 PTX10
N
uc
le
ar
siz
e/
pi
xe
ls
30
35
40(c)
******
**
** **
** ****
**
***
***
(d)
0
0
c(PAB)/(μmol·L-1)
200
0.63 1.25 2.5 5 PTX10
*
*
M
ito
ch
on
dr
ia
lm
as
s
(%
of
so
lv
en
tc
on
tro
l)
400
600
**
**
**
******
0.5%DMSO
1 μmol/LPTX
5 μmol/LPAB
2.5 μmol/LPAB
Nuclei Mitochondria(a)
20 μm
1318· ·
段绍维等：高内涵法探讨土槿皮乙酸对MCF-7细胞抑制作用的机制2010; 37 (12)
V-FITC阳性细胞(早期凋亡细胞 + 晚期凋亡和坏
死细胞)比例高于溶剂对照，且表现出剂量依赖性
(2.5～10 μmol/L)(图 8a)．PAB 5 μmol/L作用后，
annexin V-FITC阳性细胞比例升高，且表现出时间
依赖性(图 8b)．
3 讨 论
实验结果显示，PAB 抑制 MCF-7 细胞增殖，
使细胞有丝分裂指数增高、cyclin B1 含量增加，
干扰有丝分裂期两极纺锤体形成，促进细胞凋亡．
由此可见，PAB对MCF-7细胞的抑制作用可来自
两个方面，一是增殖抑制作用，二是包括凋亡在内
的细胞毒性作用．
从流式细胞术 PI单染的实验结果可见，PAB
将 MCF-7 细胞阻滞在 G2/M 期．高内涵法通过
DAPI染色也可得到与流式细胞术类似的结果．不
过，PI或者 DAPI单染只是通过 DNA含量对细胞
进行分期，不能区分 G2期和 M期细胞．高内涵
法直接量化有丝分裂指数，结果显示 PAB导致有
丝分裂指数明显增高．由此推测 PAB对MCF-7细
胞的周期阻滞主要是在 M期，而非通过激活 G2
期周期检验点把细胞阻滞在 G2/M期转换处．有文
献报道 PAB作用于 HepG2细胞后 G2期检验点相
关蛋白的表达并无明显变化[18]，这与直接测量有丝
分裂指数的结果是一致的．另外流式细胞术显示，
浓度≤1.25 μmol/L的 PAB作用MCF-7细胞 24 h，
或者 2.5 μmol/L PAB 作用 12 h后，S期细胞比例
较阴性对照增加．考虑出现该现象的原因是，G1
期细胞不断进入 S期，而有丝分裂阻滞导致新形成
的 G1 期细胞减少，所以 S 期比例相应增大．当
然，不排除 PAB可能导致暂时的 S期阻滞．DNA
拓扑异构酶Ⅰ抑制是 S期阻滞的常见原因[19]，不过
有文献报道 PAB对 HL-60细胞的拓扑异构酶Ⅰ没
有抑制作用[3]．
细胞有丝分裂受到 cyclin B1/Cdk1复合物的调
节，cyclin B1 是该复合物的催化亚基．Cyclin B1
开始出现于 S期末，在 G2期逐渐增加，M期起始
时从胞质进入胞核[17]．Cyclin B1与 Cdk1结合后使
后者激活，促发有丝分裂起始[20]．在有丝分裂早中
期末、中期初始，cyclin B1 开始降解，到晚期开
始时绝大多数 cyclin B1 已经被降解 [21]．Cyclin B1
降解导致 cyclin B1/Cdk1复合物失活，细胞退出有
丝分裂[22]．实验结果显示，PAB作用后 MCF-7细
胞的 cyclin B1 含量增加，这一点也提示 G2 和 M
期细胞比例有所增加．通过 pHH3荧光强度把细胞
分成两群——M期细胞和间期细胞，分别检测二
者的 cyclin B1含量，结果显示无论是阴性对照还
是 PAB作用后，M期细胞的 cyclin B1含量都要高
于间期细胞．同时，从高内涵分析获取的荧光图片
Fig. 8 PAB induced MCF-7 cells apoptosis
MCF-7 cells were stained with annexinⅤ-FITC and PI, and percent of
apoptotic cells were determined by flow cytometry. (a) MCF-7 cells
were exposed to increasing concentrations of PAB for 72 h. (b) MCF-7
cells were exposed to 5 μmol/L PAB for 0 h, 24 h, 48 h and 72 h. Data
were given as x ± s, n =2. **P < 0.01, vs solvent control. : Early
apoptotic cells; : Late apoptotic ornecrotic cells.
0
5
(b)
0
10
15
t/h
24 48 72
20
25
30
35
40
A
po
pt
os
ic
ce
lls
(%
of
to
ta
lc
el
ls)
**
10 μmol/LPAB5 μmol/LPAB
2.5 μmol/LPAB
2.5 μmol/L paclitaxel
100
101
102
103
104
PI
100 101 102 103 104
AnnexinⅤ
0.5%DMSO
6.46%
3.80%
100
101
102
103
104
PI
100 101 102 103 104
AnnexinⅤ
8.79%
16.76%
21.36%
9.48%
100
101
102
103
104
PI
100 101 102 103 104
AnnexinⅤ
100
101
102
103
104
PI
100 101 102 103 104
AnnexinⅤ
10.59%
21.58%
100
101
102
103
104
PI
100 101 102 103 104
AnnexinⅤ
4.75%
6.91%
(a)
1319· ·
生物化学与生物物理进展 Prog. Biochem. Biophys. 2010; 37 (12)
可见阴性对照孔有典型的M期中期细胞(形成赤道
板和两极纺锤体)，较高浓度(≥2.5 μmol/L)的 PAB
作用后，M期细胞染色体凝集却未见赤道板和二
极纺锤体，呈现出早中期有丝分裂细胞的表现．
5、10 μmol/L PAB 可使微管染色变淡，定量结果
显示细胞微管蛋白荧光强度减弱．因为细胞打孔之
后游离状态的微管蛋白会透出细胞膜，最后荧光染
色所见的主要是聚合成微管的微管蛋白，所以微管
蛋白荧光强度减弱提示 PAB促进微管解聚这与其
他文献微管蛋白聚合实验的结果是一致的[11, 18]．由
此推测，PAB可能通过促进微管蛋白解聚干扰正
常纺锤体形成，使染色体不能与纺锤体正确结合，
激活有丝分裂检验点，导致 cyclin B1 等调节有丝
分裂中、后期转换的蛋白质不能降解，引起有丝分
裂阻滞[23]．
PAB 作用后的 MCF-7 细胞出现“葡萄串样”
的巨型细胞核．高内涵法定量结果显示细胞核的平
均大小和边缘不规则程度都有增加．推测部分
MCF-7细胞在被阻滞于M期之后没有保持阻滞状
态，而是在未发生细胞分裂的情况下退出 M期重
新进入 G1期，即发生了有丝分裂滑脱[24]．有丝分
裂滑脱的机制尚未阐明，有文献认为可能与药物干
扰细胞的微管组装状态有关，也与 cyclin B持续缓
慢的降解有关[25-26]．另外，流式细胞术 annexinⅤ-
FITC和 PI双染显示 PAB 可导致 MCF-7 细胞凋
亡，高内涵分析显示 PAB可导致线粒体增生，而
线粒体增生可见于细胞凋亡等情况 [27]．据文献报
道，抗有丝分裂药物作用之后，细胞可能有多种表
现——维持有丝分裂阻滞状态直至药物被移除，直
接在有丝分裂阻滞时死亡，发生有丝分裂滑脱重新
进入 G1期[28]．而细胞发生有丝分裂滑脱之后，可
能继续细胞周期循环或者发生死亡[28]．PAB导致的
细胞凋亡是发生在 M期还是发生在有丝分裂滑脱
之后，尚需进一步研究．
高内涵分析是一种综合性的方法，具备荧光显
微术、免疫印迹技术、流式细胞术的部分功能，既
能提供细胞形态学信息，也能对特定荧光靶点进行
定量和定位，还可以根据特定标准把细胞分成相应
亚群进行单独分析．高内涵分析主要是在微量培养
板(96孔、384孔)上进行的，对细胞、试剂的需求
量较少，而细胞染色之后的荧光激发、图片采集和
分析过程高度自动化，可以减小工作量、减少实验
人员主观判断失误对实验结果的影响(比如计算有
丝分裂指数时)．高内涵分析有助于研究以有丝分
裂为靶的药物作用机制．
综上所述，PAB抑制MCF-7细胞增殖，该作
用可能与其促进微管蛋白解聚、干扰二极纺锤体形
成、阻滞有丝分裂有关，PAB促进MCF-7细胞凋
亡，该作用可能与其促进线粒体增生有关．而
PAB导致的有丝分裂阻滞与有丝分裂滑脱、凋亡
之间的具体关系，以及除了凋亡之外细胞有没有其
他死亡方式(比如有丝分裂灾变 [29])尚需要进一步
研究．
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1320· ·
段绍维等：高内涵法探讨土槿皮乙酸对MCF-7细胞抑制作用的机制2010; 37 (12)
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1321· ·
生物化学与生物物理进展 Prog. Biochem. Biophys. 2010; 37 (12)
Explore The Mechanism of Inhibitory Effects of Pseudolaric Acid B
on MCF-7 Cells by High Content Analysis*
DUAN Shao-Wei, XU Bo, CHEN Yun-Li, LIMin, FU Hong-Zheng**, CUI Jing-Rong**
(State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China)
Abstract Pseudolaric acid B (PAB), a major biologically active component of TuJinPi (the root bark of
Pseudolarix kaemferi Gordon), exhibited cytotoxicity in many human tumor cell lines. High content analysis
(HCA) is a fluorescence microscopy-based automated technology used for quantitative analysis of multiple targets
in cells. HCA could yield rich information about the temporal-spatial dynamics of the fluorescence-labeled cell
constituents. The mechanism of inhibitory effects of pseudolaric acid B on human breast cancerMCF-7 cells was
explored by high content analysis and flow cytometry. As shown by sulforhodamine B assay, PAB inhibited the
proliferation of MCF-7 cells in a dose-dependent and time-dependent manner, and the 50% inhibition
concentration (IC50) for 72 h was (1.80±0.33) μmol/L. Flow cytometry (propidium iodide staining) showed that,
after treatment with PAB for24 h, the proportion of MCF-7 cells at G2/M phase could increase to about 93%. Flow
cytometry (annexin V-FITC and propidium iodide staining) showed that, PAB induced apoptosis of MCF-7 cells.
High content analysis showed that: after treatment with PAB for16 h, the mitotic index of MCF-7 could increase to
about 40%, and cyclin B1 was upregulated; PAB caused dose-dependent disassembly of microtubules and inhibited
the formation of mitotic bipolar spindles; PAB induced increase of mitochondrial mass; PAB induced grape-like
giant nuclei indicating mitotic slippage in MCF-7 cells. These results suggest that PAB inhibits MCF-7 cell
proliferation and induces apoptosis, these inhibitory effects may be related to disassembly of microtubules, spindle
abnormalities, mitotic arrest and increase of mitochondrial mass.
Key words high content analysis, pseudolaric acid B, MCF-7 cells, microtubule, mitotic arrest, apoptosis
DOI: 10.3724/SP.J.1206.2010.00335
*This work was supported by a grant from The 11th Five Years Key Programs for Science and Technology Development of China, Chinese National
Significant Project of New Drugs Creation (2009ZX09301-010).
**Corresponding author.
CUI Jing-Rong. Tel: 86-10-82802467, E-mail: jrcui@bjmu.edu.cn
FU Hong-Zheng. Tel: 86-10-82805212, E-mail: drhzfu@yahoo.com.cn
Received: June 25, 2010 Accepted: September30, 2010
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