全 文 ：D evelopm en t of a screen ing m ethod for poten tia l an ti-SARS
com pounds based on Vero-E6 cells
W AN G L in1, 2, L I Rong2song2, GUO H ai2yan2, ZHAN G H ai2qing2, L I Sh i2you1,
CH EN Cong2, X IAO Pei2gen3, TAN Xue2hai2Ξ
(1. Schoo l of Ch inese M ateria M edica, Beijing U niversity of Ch inese M edicine, Beijing 100102, Ch ina;
2. Beijing Genom ic Inst itu te, Beijing 101300, Ch ina; 3. Inst itu te of M edicinal P lan ts D evelopm ent,
Ch inese A cadem y of M edical Science and Pek ing U nion of M edical Co llege, Beijing 100094, Ch ina)
　　Abstract: Object　To develop a V ero2E6 cell line based assay fo r screen ing po ten t ia l an t i2SA R S
compounds. M ethods　Condit ion s of the V ero2E6 cell line based M T S assay w ere op t im ized. V iru s t iter
cu rve had been genera ted. To screen an t i2SA R S viru s compounds, V ero2E6 cells w ere p la ted in 962w ell
p la tes, test compounds w ere added and imm edia tely fo llow ed w ith BJ 201 SA R S viru s. Cell mo rpho logy
w as ob served by m icro scope after 48 h of incubat ion. M T S and PM S m ix tu re w as added in 96 h and
ab so rbance values w ere m easu red at 490 nm. Results　M o re than 4 000 compounds had been screened and
ten po ten t ia l an t i2SA R S compounds had been found. Conclusion 　T he V ero2E6 cell based M T S assay
offers a rap id, safe, and conven ien t m ethod fo r screen ing po ten t ia l an t i2SA R S compounds.
Key words: severe acu te resp ira to ry syndrom e (SA R S) ; V ero2E6 cells; 32(4, 52dim en thylth iazo l222
yl) 252(32carboxym ethoxyphenyl) 222(42su lfophenyl) 22H 2tet razo lium (M T S)
基于 Vero E6 细胞的抗 SARS 药物筛选方法的构建
王　琳1, 2, 黎荣松2, 郭海燕2, 张海清2, 栗世铀1, 陈　聪2, 肖培根3, 谈学海23
(11 北京中医药大学中药学院, 北京　100102; 21 北京华大基因研究中心, 北京　101300;
31 中国医学科学院 中国协和医科大学药用植物研究所, 北京　100094)
摘　要: 目的　构建基于 V ero2E6 细胞的抗 SA R S 药物筛选模型。方法　运用四唑氮化合物 (M T S) 方法, 对由
SA R S 病毒 (BJ 201) 引起的对V ero2E6 细胞的细胞毒性作用进行检测。首先优化了实验条件 (检测了M T S 不同孵
育时间和不同接种细胞数对测试结果的影响, 并绘制了病毒滴度曲线) ; 在检测化合物的抗病毒作用时, 首先将细
胞接种于 96 孔板中, 加入待测药物和 BJ 201 病毒, 48 h 后用显微镜观察细胞形态变化, 96 h 后加入M T S 和电子
耦联剂 (PM S) 的混合溶液, 在 490 nm 检测其吸光度。结果　对 4 000 多种化合物的抗 SA R S 病毒作用进行了检
测。获得 10 种可能有抗 SA R S 病毒作用的药物。结论　基于V ero2E6 细胞的M T S 检测方法提供了一种快速、安
全和方便的抗 SA R S 药物筛选方法。
关键词: 重急性呼吸综合征; V ero2E6 细胞; 四唑氮化合物
中图分类号: R 96511　　　文献标识码: A 　　　文章编号: 0253 2670 (2004) 09 1019 05
In troduction
　　SA R S ( severe acu te resp ira to ry syndrom e) is
a very con tagiou s disease w ith h igh mo rta lity. A
new co ronaviru s ( SA R S2associa ted co ronaviru s,
SA R S2CoV ) w as iden t if ied as its pathogen [1 ]. L ast
year the global ou tb reak of SA R S cau sed w ide2
ranging disrup t ion and increased the dem and to
search fo r effect ive therap ies fo r th is d isease.
　　N ow a rap id, safe, and conven ien t m ethod—
the V ero2E6 cell based 32(4, 52dim ethylth iazo l222
yl) 252( 32carboxym ethoxyphenyl ) 222( 42su lfophen2
yl) 22H 2tet razo lium (M T S ) assay fo r screen ing
po ten t ia l an t i2SA R S compounds has been
developed. U sing th is assay, mo re than 4 000
compounds have been screened, and ten po ten t ia l
an t i2SA R S compounds have been found.
·9101·中草药　Ch inese T radit ional and H erbal D rugs　第 35 卷第 9 期 2004 年 9 月
Ξ 收稿日期: 2004202206
基金项目: 国家高技术研究发展计划 (863 计划) 资助项目 (2003AA 208217)
作者简介: 王　琳 (1973—) , 女, 北京中医药大学和北京华大基因研究中心联合培养的博士研究生, 研究方向为中药基因组学。
E2m ail: w anglin@genom ics. o rg. cn
1　M a ter ia ls and m ethods
1. 1　V iru s and cells: BJ 201, a st ra in of SA R S2
CoV iso la ted from the lung of a dead SA R S pat ien t
in Beijing w as u sed. T he cell line V ero2E6 derived
from the A frican green monkey k idney w as
pu rchased from Am erican T ype Cu ltu re Co llect ion
(A TCC ). Cells w ere p la ted in 962w ell t issue
cu ltu re p la tes u sing D u lbecco’s M odified Eagle
M edia (DM EM , Gibco , U SA ) w ith 10% heat2
inact iva ted feta l bovine serum ( FBS, H yclone,
U SA ) , 200 mmo löL L 2glu tam ine (Gibco , U SA ) ,
1% Pen icillin ( Sigm a, U SA ) and 1%
Strep tom ycin (Gibco , U SA ).
1. 2　O ther m ateria ls: M T S w as pu rchased from
P rom ega (U SA ). Phenazine m etho su lfa te (PM S)
w as the p roduct of Sigm a (U SA ). O ther reagen ts
and so lven ts u sed in the experim en t are of analyt ic
grade.
1. 3 　 Compounds p repara t ion: T he test
compounds w ere from bo th natu ra l compounds and
chem ical systhesis. T he natu ra l compounds w ere
ex tracted and iso la ted from tradit ional Ch inese
herb s including single and comp lex compounds.
A ll compounds w ere disso lved in DM SO o r w ater
acco rd ing to their d ifferen t po larity.
1. 4　T im e cou rse of M T S assay on V ero2E6 cells:
In m etabo lica lly act ive cells, M T S is reduced by
dehydrogenase enzym es in to an aqueou s so lub le
fo rm azan p roduct. T he ab so rbance can be
m easu red direct ly a t 490 nm from 962w ell assay
p la tes and the quan t ity of fo rm azan p roduct w as
con si2dered to be direct ly p ropo rt ional to the
num ber of viab le cells in cu ltu re [2, 3 ].
　　 In o rder to determ ine the op t im al M T SöPM S
incubat ion t im e ( suff icien t co lo r developm en t of
fo rm azan dependen ts on cell lines) , the ab so rbance
of fo rm azan at d ifferen tM T SöPM S incubat ion t im e
w as m easu red. V ero2E6 cells w ere p la ted in to a
Co rn ing○R 962w ell t issue cu ltu re p la te in final
vo lum e of 100 ΛL ( 4 000 cellsöw ell). A fter 24
hou rs of incubat ion (37 ℃, 5% CO 2) , cells w ere
trea ted w ith 0. 001% and 0. 1% triton X2100 ( to
k ill cells). T hen 20 ΛL öw ell of com b ined M T Sö
hou rs a t 37 ℃ in a hum idif ied, 5% CO 2
atmo sphere, 50 ΛL of 10% sodium dodecyl su lfa te
(SD S) w as added to each w ell to stop the react ion.
T he op t ica l den sity w as analyzed on a M u lt iscan
A scen t reader (L yb system s, H elsink i) a t 490 nm.
A b so rbance values w ere the m ean ± standard
devia t ion ( SD ) of th ree rep lica tes fo r each
trea tm en t and Z’facto r w as calcu la ted.
1. 5　Cell num ber cou rse of M T S assay on V ero2
E6 cells: To determ ine the effect of V ero2E6 cell
num ber on the ab so rbance at 490 nm m easu red in
th is assay, d ifferen t num bers of V ero2E6 cells
(from 1 000 to 35 000 cellsöw ell) w ere p la ted in a
962w ell p la te. A fter 24 hou rs of incubat ion, M T Sö
PM S m ix tu re w as added in each w ell. A fter
incubat ing 3 hou rs a t 37 ℃ in a hum idif ied, 5%
CO 2 atmo sphere, ab so rbance at 490 nm w as
reco rded.
1. 6　V iru s t iter assay: Seria l vo lum es (10- 5—10ΛL ) of BJ 201 SA R S vira l stock s w ere added, 24
hou rs after seeding V ero2E6 cells in 962w ell
p la tes. Cell mo rpho logy w as ob served after 482
hou r po st infected, and 96 hou rs la ter, cytopath ic
effects (CPE ) w ere m easu red w ith M T S assay.
Cell con tro ls and m edium con tro ls w ere included.
1. 7　V isual and M T S inh ib it ion of vira l cytopath ic
effect assay: To screen fo r an t i2SA R S viru s
compounds, V ero2E6 cells w ere p la ted in 962w ell
p la tes. A fter 24 hou rs of incubat ion, test
compounds w ere added at 10 Λmo löL and
imm edia tely fo llow ed w ith 2 ΛL of BJ 201 SA R S
vira l stock s. Cell mo rpho logy w as ob served after
482hou r po st2infect ion. CPE w as m easu red w ith
M T S assay after 96 hou rs of compound addit ion.
Con tro ls included cells on ly and cells w ith viru ses.
2　Results
2. 1　T im e cou rse of M T S assay on V ero2E6 cells:
T im e cou rse cu rve after M T SöPM S so lu t ion
incubat ion of 1 - 4 hou rs w as determ ined by
ab so rbance at 490 nm. T he ab so rbance values w ere
the m ean± SD of th ree rep lica tes fo r each
trea tm en t. A s show n in F ig. 1, the ab so rbance
values increased w ith the incubat ion t im e. A linear
im e cou rse of
·0201· 中草药　Ch inese T radit ional and H erbal D rugs　第 35 卷第 9 期 2004 年 9 月
M T S assay and the co rrela t ion coeff icien t of line
w as over 0. 993. Z′2facto r w as calcu la ted to assess
the assay reliab ility. In genera l, assays w ith Z′2
facto r values greater than 0. 5 are con sidered good
assays. In ou r assay Z′2facto r value w as p roduced
greater than 0. 6 at 1 hou r incubat ion and mo re
than 0. 8 after 2 hou rs incubat ion. It ind ica ted th is
assay w o rked very w ell.
12cell　220. 001% triton X2100　320. 1% triton X2100
F ig. 1　Time course curve of M TS assay
2. 2　Cell num ber cou rse of M T S assay: Effect
of V ero2E6 cell num ber on ab so rbance at 490 nm
w as m easu red (F ig. 2). Each po in t rep resen ts the
m ean± SD of fou r rep lica tes. T he ab so rbance
increased w ith the cell num ber. A nd in the range
from 1 000 to 15 000 cellsöw ell (10 000 to 150 000
cellsömL ) there w as a linear respon se betw een cell
num ber and ab so rbance at 490 nm ( r = 0. 997).
T he background ab so rbance show n at zero cellö
w ell w as sub tracted from these data.
2. 3　V iru s t iter assay: Effect of d ilu t ion s of BJ 201
SA R S vira l stock s on V ero2E6 cells w as m easu red
(F ig. 3). A b so rbance values are the m ean ± SD of
dup lica te. T he CC ID (50% cell cu ltu re infect iou s
do se) w as genera ted (0. 007 ΛL ).
F ig. 2　Cell number course of M TS assay
F ig. 3　BJ-01 SARS v irus titer curve
2. 4　P rim ary screen ing of compounds by visual
and M T S inh ib it ion of vira l cytopath ic effect
assay: T he p la te layou t fo r p rim ary test ing of
an t ivira l compounds w as show n in F ig. 4. Cell
con tro ls determ ined the ab so rbance of un infected
cells and gave values equal to 100% viab ility.
V iru s con tro ls determ ined the ab so rbance of cells
k illed by BJ 201 viru s t rea ted and equal to 0%
viab ility. M edium con tro ls determ ined the
background ab so rbance due to M T SöPM S so lu t ion
in DM EM w ith 10% FBS and these values w ere
sub tracted from co rre2sponding eff icacy values.
　 　 Som e resu lts w ere show n in F ig. 5. T he
ab so rbance values of compounds w ithou t po ten t ia l
an t i2SA R S funct ion w ere sim ilar to the viru s
con tro ls and percen ts of CPE reduct ion w ere abou t
0%. Som e compounds show ed w eak inh ib it ion of
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
samp le samp le samp le samp le samp le samp le samp le samp le samp le samp le samp le
samp le samp le samp le samp le samp le samp le samp le samp le samp le samp le samp le
samp le samp le samp le samp le samp le samp le samp le samp le samp le samp le samp le
virus con tro ls
samp le samp le samp le samp le samp le samp le samp le samp le samp le samp le samp le
samp le samp le samp le samp le samp le samp le samp le samp le samp le samp le samp le
samp le samp le samp le samp le samp le samp le samp le samp le samp le samp le samp le
cell con tro ls
samp le samp le samp le samp le samp le samp le samp le samp le samp le samp le samp le
samp le samp le samp le samp le samp le samp le samp le samp le samp le samp le samp le
m edium
contro ls
F ig. 4　Plate layout for pr imary screen ing of an ti-SARS compounds
·1201·中草药　Ch inese T radit ional and H erbal D rugs　第 35 卷第 9 期 2004 年 9 月
BJ 201 SA R S2CoV w ith 20% - 50% CPE
reduct ion. In the test, d ilu t ion s of vira l stock s
w ith 200 CC ID w ere added to the cells. A few
compounds w ith over 60% of CPE reduct ion ( i. e.
A 3, B 4, G3 in the sheet below , F ig. 5 ) w ere
con sidered as st rong inh ib ito rs of SA R S2CoV and
as h its. Examp les of mo rpho logy ob servat ion w ere
show n in F ig. 6.
F ig. 5　Result of one typ ica l 96-well pla te shown as
va lues of percen t of CPE reduction
3　D iscussion and conclusion
　　D u ring rep lica t ion, m any viru ses ( i. e. H IV 21,
SA R S2CoV ) destroy no t on ly the ho st cells tha t
they infect bu t a lso neighbo ring un infected cells by
CPE. So the CPE inh ib ito ry assays are w idely u sed
in the field of iden t ifying po ten t ia l an t ivira l agen ts
by evaluat ing the inh ib it ion of th is viru s induced
cell death [4 ]. T radit ional CPE assays p rovide visual
cell mo rpho logy and credib le evidence of an t iviru s
of compounds. How ever, they cou ld no t offer
quan t ita t ive resu lts and h igh eff iciency w hen great
num ber of samp les need to be tested.
　　N eu ral red (N R ) vita l sta in and M T T assay
u sed in m any labo ra to ries including the Cen ters fo r
D isease Con tro l and P reven t ion (CDC ) in U SA.
and Ch ina offer mo re quan t ita t ive resu lts[5 ].
Bu t du ring these tests free neu tra l red dyes m u st be
w ashed o r vo la t ile o rgan ic so lven t is requ ired to
so lub ilize the fo rm azan p roduct. It increases no t
A 2compound T Y2AD 297 (po sit ion B4 in F ig. 4 and 5) w ith 99. 2% of CPE reduction　B2compound T Y2AD 290
(po sit ion G3 in F ig. 4 and 5) w ith 62. 9% of CPE reduction　C2un infected V ero2E6 cells (cell con tro l)
D 2infected V ero2E6 cells w ithou t treatm en t (virus con tro l)
F ig. 6　Effects of two positive compounds on morphology of BJ-01 infected Vero-E6 cells
on ly the step s of the screen ing bu t a lso the danger
of the test becau se of SA R S2Cov in the
supernatan ts of w ells. Besides that these assays
are no t su itab le fo r every cell line [4 ].
　　 In the test a V ero2E6 cell based M T S CPE
assay w as developed fo r screen ing po ten t ia l an t i2
SA R S compounds. It p rovides a faster, safer, and
mo re conven ien t screen ing m ethod as compared to
the trad it ional visual CPE assays, N R vita l sta in
and M T T assay. U n like M T T fo rm azan, the M T S
fo rm azan p roduct is so lub le in t issue cu ltu re
m edium. So the step of decan t ing supernatan ts and
adding vo la t ile o rgan ic so lven t w as no t needed.
T he ab so rbance spectrum genera ted by cu ltu re
m edium con ta in ing M T S is 382 nm and the
b io reduct ion is 490 nm. So there w as no w ash ing
o r cell harvest ing requ ired in the test. Besides
that, SD S w as added to each w ell af ter M T SöPM S
so lu t ion incubat ion. T he step w as no t ju st to stop
react ion, bu t mo re impo rtan t ly to k ill the viru s in
supernatan ts in o rder to reduce the sp reading of
viru ses.
In the w ho le, a V ero2E6 cell based M T S CPE
assay fo r screen ing po ten t ia l an t i2SA R S
compounds w as developed. T h is aqueou s so lub le
fo rm azan assay elim inates the need to remove
cu ltu re m edia o r perfo rm o ther samp le
m an ipu la t ion s. It resu lts in h igh eff iciency and
th roughpu t as w ell as decreased w ell2to2w ell
varia t ion. So th is assay can be con sidered as a
A R S efficacy
·2201· 中草药　Ch inese T radit ional and H erbal D rugs　第 35 卷第 9 期 2004 年 9 月
of test compounds in a h igh2th roughpu t fo rm at.
U sing th is assay, mo re than 4 000 compounds have
been tested in the p rim ary screen ing and ten
compounds show ed po ten t ia l an t i2SA R S
inh ib it ion.
References:
[ 1 ]　Ksiazed T G, E rdm an D , Go ldsm ith C S, et a l. A novel
co ronavirus associated w ith severe acu te resp irato ry
syndrom e [J ]1 N E ng l J M ed , 2003, 348 (20) : 1953219661
[ 2 ]　Sutherland M W , L earmonth B A 1 T he tetrazo lium dyes
M T S and XT T p rovide new quan titative assays fo r
superox ide and superox ide dism utase [J ]1 F re R ad R es, 1997, 27 (3) : 28322891[ 3 ]　Goodw in C J , Ho lt S J , Dow nes S, et a l. M icrocu ltu retetrazo lium assays: a comparison betw een two newtetrazo lium salts, XT T and M T S [J ]1 J Imm unol M ethod s,1995, 179 (1) : 9521031[ 4 ]　M o rrey J D , Sm ee D F, Sidw ell R W , et a l. Iden tification ofactive an tiviral compounds against a N ew Yo rk iso late ofW est N il virus [J ]1 A ntiv ira l R es, 2002, 55: 10721161[ 5 ]　Gien i R S, L i Y, H ayGlass K T 1 Comparison of [ 3H ]thym idine inco rpo ration w ith M T T 2 and M T S2base b ioassaysfo r hum an and m urine IL 22 and IL 24 analysis. T etrazo liumassays p rovide m arkedly enhanced sensit ivity [J ]. J Imm unolM ethod s, 1995, 187 (1) : 852931
山茱萸鞣质活性部位对佐剂性关节炎大鼠免疫功能的影响
吕晓东, 杨　胜, 齐春会, 张永祥, 茹祥斌, 周文霞, 吴　禾, 赵毅民Ξ
(军事医学科学院毒物药物研究所, 北京　100850)
摘　要: 目的　研究山茱萸免疫抑制活性部位 (鞣质活性部位, F21C) 对佐剂性关节炎 (AA ) 大鼠免疫功能的影
响。方法　运用淋巴细胞增殖反应、流式细胞技术和酶联免疫吸附试验 (EL ISA ) 观察 F21C 体内和体外对AA 大
鼠免疫功能的影响, 并与环孢素 A (C sA ) 和雷公藤多苷片 (TW ) 进行比较。结果　ig 给予 F21C (30 m gökg) 对
AA 大鼠原发性足肿胀具有明显的治疗作用, 该作用较 TW 强, 较 C sA 弱; F21C 对 AA 大鼠低下的脾细胞增殖反
应具有改善作用; 对亢进的胸腺细胞增殖反应具有抑制作用。F21C 体外对AA 大鼠低下的脾细胞产生 IgG 水平具
有明显的促进作用, 并能抑制亢进的胸腺细胞增殖反应。结论　F21C 能够治疗 AA 大鼠原发足肿胀, 该作用可能
与纠正 AA 大鼠的异常的免疫反应有关。
关键词: 山茱萸; 免疫抑制; 佐剂性关节炎; F21C
中图分类号: R 28515　　　文献标识码: A 　　　文章编号: 0253 2670 (2004) 09 1023 04
Effect of tann in from Cornus of f icina lis on imm une function of adjuvan t arthr it is ra ts
LU
　 ¨X iao2dong, YAN G Sheng, Q I Chun2hu i, ZHAN G Yong2x iang, RU X iang2b in,
ZHOU W en2x ia, W U H e, ZHAO Y i2m in
( Inst itu te of Pharm aco logy and Toxico logy, A cadem y of M ilitary M edical Science, Beijing 100850, Ch ina)
Abstract: Object　To study the therapeu t ic effect and the po ssib le m echan ism of F21C, a tann in
fract ion w ith imm uno supp ressive effect iso la ted from Cornus of f icina lis Sieb. et Zucc. , in the trea tm en t of
adjuvan t arth rit is (AA ) ra ts. M ethods　AA rat model w as rep roduced to ob serve the effect on imm une
funct ion bo th in v ivo and in v itro and compare w ith Cyclo spo rine (C sA ) and T rip tery g ium w ilf ord ii
( TW ). T he effect of F21C on jo in t sw elling and the imm une respones of AA rats w ere evaluated by
lymphocyto t ic react ion, f low cytom etry, and EL ISA. Results　F21C by ig adm in ist ra t ion to AA rats
decreased the p rim ary jo in t sw elling of AA rats, st ronger than TW and w eaker than C sA , p romo ted the
decreased sp lenocyte p ro lifera t ion induced by Con A , and inh ib ited the augm en ted thymocyte p ro lifera t ion
induced by Con A in AA rats. T he evaluat ion in v itro show ed the F21C inh ib ited the augm en ted thymocyte
p ro lifera t ion from AA rats and p romo ted the deficien t IgG p roduced by sp lenocyte from AA rats.
Conclusion　F21C has therapeu t ic effect on AA rats and the modu la t ing imm une funct ion is one po ssib le
underlying m echan ism.
Key words: Cornus of f icina lis Sieb. et Zucc. ; imm uno supp ressive; ad juvan t arth rit is (AA ) ; F21C
·3201·中草药　Ch inese T radit ional and H erbal D rugs　第 35 卷第 9 期 2004 年 9 月
收稿日期: 2003212214
基金项目: 国家重点基础研究发展规划项目资助 (G1999054401)
作者简介: 吕晓东 (1966—) , 男, 山东人, 医学硕士, 现为解放军 305 医院药剂科主任, 副主任药师, 主要从事中药复方研究。