仙鹤草水提液对肺癌A549细胞增殖的抑制作用
仙鹤草水提液对肺癌A549细胞增殖的抑制作用
温雅,毕蕾,张义彪,陈卫平
3
(南京中医药大学,江苏 南京 210046)
摘 要:目的:探讨不同浓度的仙鹤草水提液对体外培养的人肺癌细胞系A549细胞增殖及凋亡的影响。方法:采用不同浓度的仙鹤草水提液分别作用于体外培养的人肺癌细胞A549细胞株24、48、72h 后,MTT 法观察药物对肺癌A549细胞生长的抑制效应;作用于细胞48h 后倒置显微镜观察细胞形态学的改变;应用AO /EB 荧光染色法,观察不同浓度仙鹤草水提液对细胞凋亡的影响;Annexin V /P I 双染流式细胞仪检测经药物作用后A549细胞凋亡情况。结果:仙鹤草水提液可剂量依赖性地抑制A549细胞的增殖,以48h 的增殖抑制作用最好;通过倒置显微镜可明显观察到细胞的形态学改变;细胞荧光染色出现了明显的凋亡细胞,表现为细胞皱缩、核染色质浓缩、核碎裂等凋亡特征性的形态学改变;流式细胞仪检测结果显示,仙鹤草水提液可明显诱导细胞凋亡。结论:仙鹤草水提液可对人肺癌细胞系A549细胞具有显著的体外增殖抑制作用,其主要作用可能与诱导细胞凋亡有关。关键词:仙鹤草水提液;A549细胞;细胞凋亡中图分类号:R285.5 文献标识码:A 文章编号:1002-2406(2009)05-0033-03基金项目:国家十一五支撑计划(2006BA I 11B08)
作者简介:温雅(1983-),女,硕士研究生,主要研究方向:临床中药学。通讯作者:3陈卫平(1959-),女,教授,博士研究生导师,主要研究方
向:中药配伍效应与临床应用研究。
收稿日期:2009-06-01修回日期:2009-06-25
中药治疗肿瘤是我国在肿瘤治疗上的一大特色,由于中药的毒副作用小,对人体损害少甚至无害,使得中药抗肿瘤研究备受关注。根据多年临床用药经验,笔者选取了八味临床常用抗肿瘤中药:莪术、三棱、丹参、川芎、羊乳、仙鹤草、附子、半夏,经过预实验发现仙鹤草具有较好的体外肿瘤增殖抑制作用。仙鹤草又名龙芽草、黄牛尾、蛇疙瘩、脱力草、止血草,来源于蔷薇科植物龙芽草(Agri m onia p il osa Ledeb .)的地上部分,性平、味苦涩,入肺、脾经,具有收敛止血、截疟、止痢、解毒等功效,多用于治疗咳血、止血、疟疾、脱力劳伤和
痈肿等症[1]
。近年来关于仙鹤草的药效学研究以抗肿瘤居多,现代中医药研究发现,仙鹤草具有较强的抗
癌功效[2,3]
,肺癌是目前世界上发病率最高的恶性肿瘤,而且全世界肺癌发病率还在每年以0.5%的速度
增长[2]
。国外对仙鹤草水提液对人肺癌细胞的作用鲜有报导,本实验探讨了仙鹤草水提液对人肺腺癌细胞A549增殖和凋亡的影响,为仙鹤草水提液的抗肿瘤作用提供实验依据。1 材料和仪器1.1 仙鹤草供试液的制备 称取干燥仙鹤草(购于
南京中医药大学百草堂门诊部,产地:安徽,批号:
090206,经南京中医药大学王春根教授鉴定为仙鹤草)20g,加水提取2次,加水量分别为药材重量的10倍,先浸0.5h,每次1.5h 、1h,合并提取液,浓缩为40
m l,所得药液为原液,生药含量为0.5g ?m l -1
,高速冷冻离心,4℃冷藏备用。1.2 细胞株 人肺癌细胞株A549,由南京中医药大学分子生物学教研室惠赠。1.3 试剂 噻唑蓝(MTT,Amersco 公司),DMS O (二甲基亚砜,国产分析纯);顺铂(南京制药厂有限公司生产,生产批号:20070405);吖啶橙(AO )与溴化乙啶(EB )(sig ma 公司);DME M 培养基(Gibco 公司);小牛血清(杭州四季青公司);胰蛋白酶(Amersco 公司);96、24孔培养板(Costa )。1.4 仪器 二氧化碳培养箱(日本S ANY O );倒置显微镜(日本O ly mpus 公司);荧光显微镜(日本O ly mpus 公司);酶联免疫检测仪(美国B i o -RAD 公司);超净工作台(苏州净化设备公司);F ACS -Calibur 流式细胞仪(美国BD 公司)。2 实验方法2.1 细胞培养 将人肺癌细胞株A549用DME M 培
养基(含10%小牛血清、青霉素100μg ?L -1、链霉素
100
μg ?L -1)5%CO 2培养箱中培养,以0.25%的胰蛋白酶消化,取对数生长期细胞进行实验。2.2 供试药液制备 取药物原液用DME M 完全培养
液稀释成终浓度为5mg ?m l -1、2.5mg ?m l -1
、1.25
mg ?m l -1、0.625mg ?m l -1
(相当于生药量),用DM 2
S O 助溶(DMS O 终浓度0.5%),用直径为0.22
μm 的?
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微孔滤膜正压过滤除菌备用。
2.3 MTT 法检测抑制率 取对数生长期A549细胞,
消化,计数,调节细胞浓度至1×105?m l -1
,加入96孔
板中,每孔180μl,于37℃和5%CO 2的培养箱中培养
过夜。设空白阴性对照组,实验组和顺铂阳性对照组,
实验组加入各浓度的仙鹤草提取液20
μl ,空白阴性对照组加入等体积的含终浓度为0.5%DMS O 的DME M
完全培养液,阳性对照组加入等体积的终浓度为5
μg ?m l 的顺铂药液,每组均设四个复孔,置培养箱中培养24h 、48h 、72h 后小心吸弃孔内液体,然后每孔分加入
由D ME M 单培配制的终浓度为1mg ?m l -1
的MTT100
μl,继续培养4h 后,小心吸弃孔内液体,加150
μl 二甲基亚砜,充分振荡10m in,溶解结晶物,于酶标仪490n m 处测吸光度值,以空白对照孔调零,在全自动酶标仪上测定各孔光吸收值(OD ),计算各组四个复孔的平均值,计算细胞生长抑制率(I R ),以细胞生长抑制率及药物浓度作曲线,计算I C50,实验重复3次。
细胞生长抑制率=[(空白对照组平均值-实验组平均值)÷空白对照组平均值]×100%
2.4 倒置显微镜观察 在倒置显微镜下观察5
mg ?m l -1、2.5mg ?m l -1、1.25mg ?m l -1
浓度的仙鹤草水提液作用A549细胞48h 后的细胞形态学及细胞数目的改变。2.5 荧光染色观察 取对数生长期的A549细胞,细
胞浓度稀释成1×5?m l -1
,接种于含有无菌盖玻片的6孔培养板中,待细胞贴壁后,加入终浓度为5
mg ?m l -1、2.5mg ?m l -1、1.25mg ?m l -1
的仙鹤草水提
液,设空白阴性对照组,作用48h 后,加入AO /E B 染色
液5
μl (100μg ?m l -1AO ,100μg ?m l -1EB ),封固后置荧光显微镜下观察,并摄影记录。
2.6 AnnexinV -F I T C 流式细胞术检测细胞凋亡 将
处于对数生长期的A549细胞以1×105?m l -1
的浓度接种于6孔细胞培养板中,待细胞贴壁后,加入终浓
度为5mg ?m l -1、2.5mg ?m l -1、1.25mg ?m l -1
的仙鹤草水提液,并设置空白阴性对照组。加药24h 后用0.25%的胰蛋白酶消化细胞,细胞悬液移入洁净试管
中,于4℃条件下1000r ?m in -1
离心8m in 沉淀细胞,弃上清后用冷P BS 洗2次。将收集的细胞重悬于100
μl 结合缓冲液中,加入5μl Annexi V -F I T C 和5
μlP I 轻轻混匀后室温避光反应15m in 反应结束后,再加入400
μl 结合缓冲液,流式细胞仪上机检测。2.7 统计学方法 实验数据采用SPSS 13.0统计软件进行方差分析,实验结果用( x ±s )表示,P <0.05为差异有统计学意义,抑制率以百分率表示。3 结果
3.1 MTT 检测结果 仙鹤草水提液5mg ?m l -1
、2.5
mg ?m l -1
实验组对A549细胞的增殖有抑制作用,与空白对照组相比均有显著性差异(P <0.05),1.25
mg ?m l -1、0.625mg ?m l -1
实验组则抑制作用不明显(表1),说明本药物作用呈现一定的剂量依赖性(图1),但以作用48h 细胞增殖抑制作用最佳,仙鹤草水提液48h 、72h 对A549细胞的半数抑制浓度值分别为
1.54mg ?m l -1、
2.18mg ?m l -1
。
表1 不同浓度、不同时间仙鹤草水提液作用后的吸光度值及其抑制率(顺铂的浓度为5μg ?m l -1)( x ±s ,n =4)
仙鹤草水提液(mg ?m l -1)
24h
OD I R (%)48h
OD I R (%)72h
OD I R (%)空白 1.114±0.0100
0.838±0.0200 1.411±0.0200顺铂
1.167±0.033—
0.423±0.02349.560.842±0.01840.320.625 1.064±0.035 4.49
0.642±0.08023.39 1.173±0.01816.831.25 1.135±0.043—
0.540±0.05035.620.935±0.05333.752.50.967±0.08613.200.187±0.056377.660.526±0.039362.725
0.795±0.0193
3
28.64
0.086±0.0413
3
89.79
0.357±0.0413
74.73
注:与空白对照组相比,3P <0.05,3
3
P <0.01。
3.2 倒置相差显微镜观察结果 阴性对照组A549
细胞在相差显微镜下呈多角形,贴壁生长,轮廓清晰,生长旺盛,仅有少数悬浮细胞(图2A );加药组细胞形态发生明显变化,表现为细胞数明显减少,细胞形态不规则,细胞体积缩小变圆,胞浆固化,部分核碎裂,细胞裂解等细胞凋亡的特征性改变(图2B -图2D )。3.3 荧光染色结果 AO /E B 染色后用荧光显微镜观察,空白阴性对照组A549细胞贴壁生长,细胞呈多边形铺展状态,细胞质被AO 染成绿色,细胞核清晰,呈
黄绿色,近圆形,内有3～5个圆形的黄色核仁,无明显
凋亡细胞(图3A )。仙鹤草水提液5mg ?m l -1
作用A549细胞48h 后,出现晚期凋亡细胞,部分细胞核碎裂,呈致密浓染的橘红色,细胞质芽状突起脱落,形成凋
亡小体(图3B )。仙鹤草水提液2.5mg ?m l -1
、1.25mg ?m l -1
作用细胞48h 后,细胞铺展程度下降,趋于圆形,生长停止,出现凋亡细胞,早期凋亡细胞发生核质核膜反应,呈黄绿色,形态呈固缩状、圆珠状或新月状(图3C,图3D )。
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3.4 流式细胞术检测细胞凋亡结果 P I加Annexin V -F I TC双染法检测A549细胞凋亡结果见图4。仙鹤草水提液5mg?m l-1、2.5mg?m l-1、1.25mg?m l-1作用A549细胞24h后较对照组细胞凋亡率高,差异有统计学意义(P<0.05),主要以中、晚期凋亡为主,说明仙鹤草水提液可诱导A549细胞发生凋亡
。
图4 流式细胞术检测细胞调亡结果
4 讨论
近年来的研究结果表明,肿瘤的发生、发展不仅与肿瘤分化异常及细胞增殖过度有关,而且亦与细胞凋亡减少有关[4]。本实验中MTT法结果表明,仙鹤草水提液对A549人肺腺癌细胞株有良好的抑制增殖作用;细胞形态学观察表明,仙鹤草水提液作用A549细胞后,细胞形态发生明显改变,细胞铺展程度下降,趋于圆形,生长停止;荧光显微镜观察发现,用药组出现凋亡细胞,表现为细胞皱缩、核染色质浓缩、核碎裂、细胞出芽以及凋亡小体形成等凋亡特征性的形态学改变;流式细胞术检测细胞凋亡结果说明,仙鹤草水提液可诱导A549细胞发生凋亡。仙鹤草水提液可对人肺癌细胞系A549细胞具有显著的体外增殖抑制作用,其主要作用可能与诱导细胞凋亡有关。
本实验通过对仙鹤草水提液对A549细胞增殖抑制及凋亡的研究,为仙鹤草水溶性物质的抗癌作用提供了理论依据。肺癌病人病情的加重与肿瘤细胞的侵袭转移有关,仙鹤草作为收敛止血药的一种被广泛应用于肿瘤的治疗,但其是否具有抑制肿瘤侵袭和转移的作用尚不明确,我们将在今后的实验中做进一步的研究。
参考文献:
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[M].广州:羊城晚报出版社,2002:191-192.
[2] 李玉祥,樊华,张劲松,等.中草药抗癌的体外实验[J].中国药科
大学学报,1999,30(1):37.
[3] 高凯民,周玲,陈金英,等.仙鹤草煎剂对HL-60细胞的体外诱
导凋亡作用[J].中药材,2000,23(9):561.
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ABSTRACTS FR OM O R I G I NAL ART I CL ES
Therapeuti c Thought of An ti-hepa ti c F i brosis Ba sed on the Theory of “J i D isea se”
WANG Xu-dong,YU L i-ying,YAN Ting-ting,WU X iao-qun Abstract:Hepatic fibr osis bel ongs t o the“Hepatic J i”in Traditi onal Chinese Medidine and the therapeutic p rinci p les were“p r omoting bl ood circulati on and eli m inating bl ood stasis”and“s oftening hard masses and dis pelling nodes”.Ancient literatures t ook the key pathol ogical mechanis m of“J i D is2 ease”for“insufficiency of Yang-Q i,accumulati on of cold phleg m,stagna2 ti on of Q i and bl ood stasis”.On the basis of data m ining,p rescri p ti on and drug screening,we designed the p rescri p ti on“Hepatic J i Pulvis”,which had the functi ons of“purging cold phleg m,dis persing Q i stasis and p r omoting bl ood circulati on by war m ing”.Further more,the rat experi m ents p r oved that the p rescri p ti on could i m p r ove hepatic inflammati on and fibr osis observed by
hist ol ogical pathol ogy,reduce hyalur onic acid and p r ocollagen III in p las ma deter m ined by radi oi m munoassay.So we got the conclusi on that the p rescri p2 ti on“Hepatic J i Pulvis”based on the theory of“war m ing Yang and dis pelling Ji”had s ome effects on hepatic fibr osis.
Author’s address:Nanjing University of Chinese Medicine,Nanjing210046, china
Key words:Hepatic Ji;Hepatic J i Pulvis;Hepatic Fibr osis;Therapeutic Thought of TC M
(O riginal article on Page 1) I nh i b itory Effects of Agr i m on i a P ilos a L edeb.W a ter Extract on A549 Cells i n V itro
W EN Ya,B I L ei,ZHANG Yi-biao,CHEN W ei-ping
Abstract:Objective:To exp l ore the effects of Agri m onia p il osa Ledeb water extract on p r oliferati on and apop t osis of human lung cancer A549cell line.. Methods:MTT assay was used t o observe the anti-p r oliferati on effect of Agri m onia p il osa Ledeb water extract on A549cells for24h,48h,72h;Mor2 phol ogical changes were observed under op tical m icr oscopy;AO/EB fluores2 cence double-dye technol was perfor med t o observe the apop t osis of cells treated for48h in fluorescence m icr oscope;The apop t osis rates of A549cells treated by Agri m onia p il osa Ledeb.water extract f or24h were deter m ined by fl ow cyt ometry(FC M).Results:Agri m onia p il osa Ledeb.water extract inhibi2 ted the p r oliferati on of A549cells in a dose-dependent manner.And the best p r oliferati on was after being treated for48h;Mor phol ogical changes were dra2 matical under op tical m icr oscopy;I n experi m ent gr oup appeared yell ow or chrys oidine fluorescence,cont racted,beaded and crescent and the number of apop t otic cells increased;FC M analysis showed that cells were induced ap2 op t osis dose-dependently.Conclusi on:Agri m onia p il osa Ledeb.water extract has significant antitumor activity in vitr o.The potential mechanis m of anti-p r oliferati on effect caused by Agri m onia p il osa Ledeb.water extract may be cell apop t osis.
Author’s address:Nanjing University of Chinese Medicine,Naujing 210046,China
Key words:Agri m onia p il osa Ledeb.;A549cells;cell apop t osis
(O riginal article on Page 33) Study on Pharmacok i n eti cs on Colon-spec i f i c D eli very Coa ted Tablet
of Syr i n ga Extract i n Rabb it Body
WANG J ian-m ing1,L IU X in2
Abstract:Objective:T o investigate the phar macokinetics p r operty of Syringo2 p icr osid in the tablets of col on-s pecific delivery coated and naked in vivo s o that t o p r ovide the scientific data f or the target effect.Methods:After orally adm inistrated t o rabbits,the bl ood sa mp les were taken during the different ti m es and the Syringop icr osid concentrati on in bl ood sa mp les were deter m ined with HP LC instrument.And then the phar macokinetics para meterswere taken by aut o-fitting data with DAS2.0phar macokinetics s oft w are.Results:The Syringop icr osid in t w o p reparati ons shows t o be2-compart m ent model after orally adm inistrated t o rabbits.The ti m e appeared drug concentrati on in rabbit p las ma t o col on-s pecific delivery p reparati on was later than common tablet and the Tlag was about4.6.Conclusi on:The phar macokinetics para meters showed that the col on-s pecific delivery tablet has good target effect in col on.Author’s address:1.Heil ongjiang University of Chinese Medicine,Harbin
150040,China; 2.Phar macy College of Zhejiang University,Hangzhou 310058,China
Key words:D ingxiang extract;Syringop icr osid;Phar macokinetics in vivo; Conl on-s pecific delivery coated tablet
(O riginal article on Page 38) Expressi on of Genes Rel a ted to Genom e St ab ility and D NA Repa i r i n
Na sopharyngea l Carc i n oma C luster i n g Fam ili es w ith Vacuous Pa ttern ZHOU X iao-jun1,TIAN D ao-fa2,WANG Shi-zhen3,RUAN Yan3,Q I U B ao-shan3,ZHAN G L i-j uan1,LU B iao-qing1
Abstract:Objective:W e will observe the exp ressi on of genes related t o ge2 nome stability and DNA repair in the members of NPC clustering fa m ilies with vacuous pattern.Methods:I n the Zhongshan city which there is highly inci2 dence rate of NPC,we chose the me mbers of the NPC clustering fa m ilies with vacuous pattern as object,and the me mbers of the NPC clustering fam ilies with nor mal pattern and NPC patient as the contr ol gr oup.The gene chi p and real-ti m e fluorescence quantitative technique was used t o exam ine the ge2 nome stability and DNA repair genes in the nas opharyngeal tissue.Results: More Genome Stability and DNA Repair Genes were up-regulated in the members of the NPC clustering fa m ilies than the NPC patient,and the range of up-regulated is high.Fe wer Genome Stability and DNA Repair Genes were down-regulated in the me mbers of the NPC clustering fa m ilies than the NPC patient,and the range of down-regulated is l ow,and more Genome Stability and DNA Repair Genes were up-regulated in the me mbers of the NPC clustering fam ilies with vacuous pattern than nor mal pattern,the results als o could be confir med by real-ti m e fluorescence quantitative.Conclusi on: There are s pecially exp ressi on character of genome stability and DNA repair genes in the me mbers of NPC clustering fa m ilies,and the vacuous of constitu2 ti onal pattern may affect the Genome Stability and DNA Repair Genes change. Author’s address:1.Zhongshan Chinese Medicine Hos p ital Affiliated t o Guangzhou University of Chinese Medicine,Zhongshan528400,China;2.Hu2 nan University of Chinese Medicine,Changsha,410007,China;3.First Clin2 ical College t o Guangzhou University of Chinese Medicine,Guangzhou 510405,China.
Key words:Nas opharyngeal carcinoma;Cancer clustering fa m ilies;Genome stability and DNA repair gene;Gene chi p;Real-ti m e fluorescence quantita2 tive PCR;Vacuous pattern
(O riginal article on Page 59) Effects of M arrow-supple m en t and Blood-engender i n g Granule I m2
pacti n g on I L-12
WAN G J in-huan1,SUN An-tao2,SUN W ei-zheng1
Abstract:This article observed the difference in exp ressi on level of I L-12in CAA patients before and after treat m ent by marr ow-supp le menting and bl ood -engendering(MS ABE).Method:Sixty-five cases of CAA were divided in2 t o t w o gr oup s random ly-thirty-f our patients of the treated gr oup were treated with MS ABE and thirty-one of the contr ol gr oup were treated with Zaizhang shengxue tablet(ZZS XT).W e exp l ored EL I S A t o detect sixty-five patients the exp ressi on of related adhesi on cyt okines I L-12before and after treat2 ment.Result:The levels of I L-12were all higher than that in the nor mal contr olled gr oup bef ore treat m ent,but after treat m ent it got reduced.The de2 crease in experi m ental gr oup was superi or t o that in contral gr oup.After treat2 ment the comparis on bet w een the Yang deficiency type and Yin deficiency type showed significant difference in I L-12.Conclusi on:The abnomal ex2 p ressi on of I L-12of serum can affect the bone marr ow hae mat opoiesis and are related cl osely t o CAA.MS ABE can i m p r ove bone marr ow hae mat opoiesis by regulating the exp ressi on after treat m ent.
Author’s address:1.First Hos p ital Affiliated t o Heil ongjiang University of Chinese Medicine,Harbin150040,China;2.Heil ongjiang University of Chi2 nese Medicine,Harbin150040,China
Key words:Ane m ia;Ap lastic;Cyt okines;I L-12
(O riginal article on Page 63)