中国科技论文统计源期刊 中文核心期刊  
美国《化学文摘》《国际药学文摘》
《乌利希期刊指南》
WHO《西太平洋地区医学索引》来源期刊  
日本科学技术振兴机构数据库(JST)
第七届湖北十大名刊提名奖  
医药导报, 2017, 36(1): 32-36
doi: 10.3870/j.issn.1004-0781.2017.01.007
珊瑚姜油对宫颈癌HeLa细胞增殖及凋亡的影响*
Effect of Zingiber corallinum Oil on Proliferation and Apoptosis of Cervical Carcinoma Cell Line HeLa
吴春维1,, 涂云华2, 李明娥2, 叶振源2, 薛月萃2, 曹煜2,

摘要:

目的 探讨珊瑚姜油(ZCO)对宫颈癌HeLa细胞增殖及凋亡的影响及其作用机制。方法 5~80 mg·L-1 ZCO体外作用于HeLa细胞。CCK-8检测细胞毒性;吖啶橙/溴化乙啶(AO/EB)染色,倒置显微镜观察细胞形态变化;比色法检测caspase-3酶活性;Western blotting检测hsp-70表达;流式细胞仪检测细胞凋亡及周期。结果 ZCO以时间-剂量依赖性方式抑制HeLa细胞株增殖,镜下见典型细胞凋亡形态,caspase-3酶活性亦呈剂量依赖性增加。随药物浓度增加,hsp-70蛋白表达量逐渐减少,G2期细胞逐渐增加,细胞阻滞在G2/M期。结论 ZCO对HeLa细胞株具有抑制增殖及促凋亡作用,其机制是激活凋亡关键酶,使hsp-70蛋白表达量逐渐下降,使细胞周期阻滞在G2/M期。

关键词: 珊瑚姜油 ; ; 宫颈 ; HeLa细胞 ; 细胞周期

Abstract:

Objective To investigate the effect of Zingiber corallinum oil(ZCO)on apoptosis and proliferation of cervical carcinoma cell line HeLa. Methods HeLa cells were treated with different concentrations of ZCO(5-80 mg·L-1)in vitro. Cytotoxicity rate was determined by CCK-8 assay. The morphological changes was observed using inverted microscope after AO/EB staining. Caspase-3 activities were measured with a colorimetric method. Protein level of hsp-70 were detected by Western blotting. Cell cycle and apoptosis were analyzed by flow cytometer (FCM).Results ZCO exhibited effect of proliferation inhibition and apoptosis-inducing on the growth of HeLa cells in a dose-dependent manner. Caspase-3 activities increased in a dose-dependent manner while the expression of hsp-70 decreased. Cell cycle was arrested in G2/M phase. Conclusion ZCO exhibites a marked effect of proliferation inhibition and apoptosis-inducing on HeLa cells. The mechanism of ZCO might be activating the key enzyme in apoptotic pathway, so that the expression of hsp-70 is down-regulated, and cell cycle is arrested in G2/M phase.

Key words: oil ; Carcinoma ; cervical ; HeLa cell ; Cell cycle

宫颈癌是最常见的妇科恶性肿瘤。原位癌高发年龄30~35岁,浸润癌高发年龄45~55岁,近年来其发病有年轻化的趋势,且发病率呈上升趋势[1-3] ,因此,寻找对宫颈癌细胞有抑制作用的药物成为当前的研究热点。珊瑚姜是中国的传统苗药之一,通过超临界二氧化碳法从珊瑚姜中提取的珊瑚姜油(Zingiber corallinum oil,ZCO),其主要成分为三戊并烯1,4-二甲氧基(22.01%)、松油稀-4-醇(16.26%)、香桧烯(16.19%)[4-5]。近年来发现,珊瑚姜油具有抗菌、抗真菌、杀虫、抗氧化等活性[6-9]。但抗肿瘤作用笔者还未见报道。本实验主要研究ZCO对HeLa细胞增殖抑制及凋亡的影响并探讨作用机制。

1 材料与方法
1.1 试剂与材料

ZCO[贵阳舒美达药厂,批号:20150801,含量:99%,二甲亚砜(DMSO)溶解后,培养液稀释成1 mg·L-1,滤过除菌备用],HeLa细胞(普诺赛生物科技有限公司),CCK-8(日本同仁化学研究所,产品编号:A311-01/02/03),胎牛血清(杭州四季青有限公司,产品编号:110913),AnnexinⅤ-FITC/PI凋亡试剂盒(上海贝博生物科技有限公司,产品编号:401003)及周期试剂盒(上海贝博生物科技有限公司,产品编号:401278),caspase-3检测试剂盒(南京建成生物工程研究所,产品编号:G015),hsp-70蛋白抗体(GeneTex ,产品编号:AH728-1),RIPA裂解液(产品编号:P0013B)、苯甲基磺酰氟(产品编号:ST505)、蛋白质聚丙烯酰胺凝胶配制试剂盒(产品编号:P0012A)、Western二抗稀释液(产品编号:P0023D)、一抗稀释液(产品编号:P0023A)、封闭液(产品编号:D3308B)、二辛可宁酸蛋白浓度测定试剂盒(产品编号:P0009)、一抗二抗去除液(产品编号:P0025)、聚偏氟乙烯膜(产品编号:FFP36)均购自江苏碧云天生物技术研究所,Goat Anti-Rabbit IgG-HRP(Abmart,产品编号:sc-2004)及Actin Mouse mAb(Abmart,产品编号:AC004),Chemiluminescent HRP Substrate(ImmobilonTM Western,产品编号:WBKLS0050)。

1.2 仪器

奥林巴斯倒置荧光显微镜(奥林巴斯IX51),ELX800酶联免疫检测仪(美国宝特公司),BDFACSCalibur流式细胞仪(美国BD FACSCalibur)。

1.3 方法

1.3.1 细胞培养 HeLa细胞用达尔伯克必需基本培养液(DMEM,含10%胎牛血清+1%青链霉素双抗), 置于37 ℃、5%二氧化碳(CO2)培养箱中培养,0.25%胰酶消化传代,取对数生长期细胞进行实验。

1.3.2 细胞毒性检测 离心收集对数生长期细胞,参考涂云华等[10]介绍的方法,调整细胞密度为1.5×105个·mL-1后接种于96孔板,每孔0.05 mL,培养24 h后,实验组加入ZCO,每孔补足培养液至0.1 mL,使药物终浓度分别为5,10,20,40,80 mg·L-1,共5组,另设正常对照组,即正常细胞,无药物处理;空白对照组无细胞。每组均设6个复孔,CO2培养箱中继续培养24,48及72 h,培养结束的1.5 h前,每孔加入CCK-8溶液10 μL,继续培养,酶标仪测定波长为405 nm时吸光度(A)值。细胞增殖抑制率(%)=(正常对照组A-给药组A/正常对照组A-空白对照组A)×100%。

1.3.3 细胞形态观察 调整实验细胞密度为1.2×104个·mL-1,接种至6孔板,培养24 h后,加入ZCO,使其终浓度为5,20,80 mg·L-1,设空白对照组(无细胞),培养48 h后,经吖啶橙/溴化乙啶(acridine orange/ethidium bromide, AO/EB)染色,倒置显微镜观察细胞形态。

1.3.4 Caspase-3酶活性细胞培养 方法同“1.3.3”项,参考涂云华等[10]介绍的方法离心收集细胞,按照每两百万细胞加入裂解液50 μL的比例加入裂解液,12 000×g离心10~15 min,吸取上清液,按照caspase-3酶活性检测试剂盒步骤加入试剂,37 ℃孵育4 h,酶标仪检测波长为405 nm时吸光度值。caspase-3活化程度=给药组A/正常对照组A

1.3.5 Western blotting检测hsp-70蛋白表达 调整细胞密度为1.4×105个·mL-1,接种于6孔板中,加入ZCO,使其终浓度分别为0,10,40 μmol·L-1,设置不加药物细胞为正常对照组,培养2,48及72 h后,胰酶消化贴壁细胞,参考HWANG等[11]介绍的方法根据RIPA裂解试剂盒,按100∶1比例向RIPA裂解液中加入苯甲基磺酰氟,每孔加入裂解液200 μL进行裂解,根据二辛可宁酸蛋白浓度测定试剂盒检测样品蛋白的浓度,上样前,于96 ℃热水中变性5 min,每孔上样体积为20 μL,上样量为20 μg,进行垂直电泳,浓缩胶80 V,60 min,分离胶120 V,100 min,切胶后,200 mmA电转膜100 min,用封闭液封闭2 h,TBST洗涤3次,每次10 min,一抗孵育过夜(hsp-70用一抗稀释液稀释为1∶1 000),清洗聚偏氟乙烯膜同上,二抗孵育60 min,TBST洗涤3次,每次10 min,按Chemiluminescent HRP Substrate试剂盒加入发光剂,行胶片曝光,用一抗、二抗去除液清洗PVDF膜后,继续检测其他抗体。

1.3.6 细胞凋亡及周期检测 取对数生长期细胞,调整细胞密度为1.6×105个·mL-1,药物浓度设置同“1.2.3”项,培养48 h后,调整细胞密度为1.3×106个·mL-1,参考吴江群等[12]介绍的实验方法,行细胞固定,AnnexinV-FITC/PI染色及按周期试剂盒染色后,BDFACSCalibur流式细胞仪测定细胞凋亡及其周期。

1.4 统计学方法

采用SPSS19.0版统计学软件进行统计分析,计量资料以均数±标准差( x ̅ ±s)表示,组间均数比较采用单因素方差分析,以P<0.05为差异有统计学意义。药效-剂量关系运用曲线估计,用R2及F值来表示。

2 结果
2.1 ZCO组对HeLa细胞的抑制作用

表1,ZCO对HeLa细胞的抑制作用方式呈时间-剂量依赖性,尤以48 h作用较为明显(F=31.25,P<0.05)。

2.2 细胞形态

正常HeLa细胞贴壁生长,呈多角形及不规则形,生长迅速(图1)。ZCO处理后,细胞生长变慢,胞质间隙增大,可见数目不等的漂浮细胞及碎片,AO/EB染色后,从低浓度至高浓度,凋亡细胞及坏死细胞逐渐增多。

2.3 Caspase-3酶活性

表2,随着药物浓度增加,caspase-3酶活性随之增强(F=31.15, P<0.05)。

2.4 hsp-70蛋白表达

图2,低浓度至高浓度ZCO作用HeLa细胞24,48及72 h,hsp-70逐渐降低,其作用方式呈时间-剂量依赖性。

表1 6组细胞抑制情况比较
Tab.1 Comparison of the inhibition rate among six groups of cells x¯±s,n=6
组别与时间 A 抑制率/%
正常对照组
24 h 0.93±0.02 -
48 h 0.95±0.02 -
72 h 0.98±0.03 -
5 mg·L-1ZCO组
24 h 0.90±0.12 7±2
48 h 0.87±0.01 9±1
72 h 0.88±0.04 8±1
10 mg·L-1ZCO组
24 h 0.88±0.01 9±2
48 h 0.85±0.00 11±0
72 h 0.85±0.02 21±1
20 mg·L-1ZCO组
24 h 0.82±0.02 13±1*1
48 h 0.75±0.01 20±1*1
72 h 0.74±0.02 21±1*1
40 mg·L-1ZCO组
24 h 0.75±0.01 18±1*1
48 h 0.68±0.01 31±2*1
72 h 0.70±0.01 29±1
80 mg·L-1ZCO组
24 h 0.68±0.02 22±1
48 h 0.50±0.00 55±1
72 h 0.53±0.02 56±1

Compared with normal control group , *1P<0.05

与正常对照组比较, *1P<0.05

表1 6组细胞抑制情况比较

Tab.1 Comparison of the inhibition rate among six groups of cells x¯±s,n=6

图1 4组HeLa细胞形态 (A0/EB染色,×200)
A.正常对照组;B. 5 mg·L-1 ZCO组;C. 20 mg·L-1 ZCO组;D. 80 mg·L-1 ZCO组

Fig.1 Morphology of four groups of HeLa cells (A0/EB staining,×200)
A. normol control group;B. 5 mg·L-1 ZCO group;C. 20 mg·L-1 ZCO group;D. 80 mg·L-1 ZCO group

表2 4组HeLa细胞 48 h caspase-3酶活性比较
Tab.2 Comparison of caspase-3 activity among four groups of HeLa cells at 48 h x¯±s,n=6
组别 A 酶活性
正常对照组 0.81±0.01 -
5 mg·L-1 ZCO组 0.72±0.00 1.05±0.12*1
20 mg·L-1 ZCO组 0.15±0.01 1.91±0.03*1
80 mg·L-1 ZCO组 0.07±0.00 3.31±0.01*1

Compared with normal control group , *1P<0.05

与正常对照组比较,*1P<0.05

表2 4组HeLa细胞 48 h caspase-3酶活性比较

Tab.2 Comparison of caspase-3 activity among four groups of HeLa cells at 48 h x¯±s,n=6

图2 ZCO对HeLa细胞hsp-70蛋白表达的影响

Fig.2 Effect of ZCO on the protein expression of hsp-70 in HeLa cells

2.5 细胞凋亡

ZCO作用HeLa细胞48 h后,细胞出现不同程度的凋亡,由低浓度至高浓度,凋亡逐渐增加,尤其以早期凋亡较为明显。见图3。

图3 4组HeLa细胞凋亡情况
A.正常对照组;B. 5 mg·L-1 ZCO组;C. 20 mg·L-1 ZCO组;D. 80 mg·L-1 ZCO组

Fig.3 Apoptosis of four groups of HeLa cells
A. normol control group;B. 5 mg·L-1 ZCO group;C. 20 mg·L-1 ZCO group;D. 80 mg·L-1 ZCO group

2.6 细胞周期变化

ZCO作用HeLa细胞48 h,随药物浓度增加,G2期细胞逐渐增加,细胞阻滞在G2/M期。

3 讨论

珊瑚姜能药食两用,有广泛的药理活性,且毒性小[13-14]。ZCO具有抗细菌及抗真菌等作用,但很少有抗肿瘤方面的研究报道。本研究显示,ZCO对HeLa细胞的生长具有抑制作用, 随着药物浓度增加, 抑制作用增强,其作用方式呈现时间-剂量依赖性,且当药物作用48 h,抑制作用较为明显。吖啶橙透过正常细胞膜,使细胞核呈绿色或黄绿色均匀荧光,其使凋亡细胞染上致密浓染的黄绿色荧光或黄绿色碎片颗粒;而坏死细胞黄色荧光减弱甚至消失[15-17]。本实验中,ZCO由低浓度至高浓度,黄绿色荧光逐渐减少,而红色荧光逐渐增多,细胞变成圆形或椭圆形,见数量不等的漂浮细胞及细胞碎片。流式细胞仪检测凋亡结果亦证明,随药物浓度增加,凋亡程度愈加明显。

Caspase-3最主要的底物是多聚(ADP-核糖)聚合酶[poly(ADP-ribose) polymerase,PARP],该酶与DNA修复、基因完整性监护有关[18-19]。在细胞凋亡启动时,PARP在Asp216-Gly217之间被Caspase-3剪切成两个片段,使PARP中与DNA结合的两个锌指结构与羧基端的催化区域分离,不能发挥正常功能[20]。本实验结果显示,随着药物浓度增加,caspase-3 酶活性逐渐增强,呈时间-剂量依耐性,从而证实了caspase-3 参与ZCO诱导细胞凋亡过程。

正常情况下hsp-70位于细胞质内,当细胞遭受应激作用时,hsp-70迅速移入细胞核内并包围核仁,细胞质内只有少量存在;而应激消除后细胞处于恢复阶段时,细胞核内hsp-70又返回细胞质,在细胞质内呈低水平表达,再次应激又重新返回细胞核[21],其在许多肿瘤中呈高表达,如宫颈癌组织的hsp-70阳性率高于正常对照组织,且低分化宫颈癌组织的阳性率高于高分化宫颈癌。本实验中,随药物浓度增加及作用时间延长,hsp-70表达水平逐渐降低,证实ZCO通过影响hsp-70蛋白表达而抑制细胞增殖。

当细胞周期紊乱时,细胞出现恶性增殖,导致肿瘤的发生[22]。本实验显示,随着ZCO浓度增加,G2期细胞增多,细胞周期阻滞在G2/M期,阻断了细胞的有丝分裂,从而对HeLa细胞增殖起到抑制作用。

总之, ZCO可抑制HeLa细胞增殖,其机制是通过激活凋亡途径关键酶,使凋亡相关hsp-70蛋白表达下调,细胞周期阻滞在G2/M期,最终阻止肿瘤细胞的增殖与分化。

The authors have declared that no competing interests exist.

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[5] LUO S Q, PENG Q C, YANG X O, et al.Volatiles and inhibitory phytopathogens fungi activities of essential oil extracted from Zingiber corallinum Hance[J]. Guangdong Agricultural Sci ,2013,40(17):84-86.
To investigate antimicrobial activities of essential oil of Zingiber corallinum Hance against phytopathogenic fungi, the essential oil hydrodistillated was analyzed by gas chromatography- mass spectrometry(GC-MS). Using microbial growth inhibition assays in vitro, the oil was tested for fungitoxic effects against 4 phytopathogenic fungi, which were Rhizoctonia solani K眉hn, Fusarium gram inearum, Fusarium nivale, Pyricularia oryzae. Eighteen different components representing 91.87% of the compounds in the oil were identified. Sabinene(54.07%), 1-Terpinen-4-ol(23.74%), Triquinacene,1,4-bis(methoxy)-(6.47%) and 纬-Terpinen(4.60%) were found to be predominant components. The oil exhibited considerable antifungal activity against tested fungi, median effectively lethal concentration(EC50) of the oil against 4 phytopathogenic fungi were 3.64, 11.20, 11.33, 14.19 mg/L, respectively, and it was most significantly toxic against Rhizoctonia solani K眉hn. The research results provided theoretical basis for the development of environment friendly pesticide and comprehensive application of Zingiber corallinum Hance.
DOI:10.1177/0333102412467514      URL    
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[6] DE-SHUN Y U, FEI-FEI Y E, YANG X Q. Antioxidant activity of essential oil from Zingiber corallinum Hance[J]. Food Sci ,2011,32(17):164-167.
A natural plant essential oil was extracted from Zingiber corallinum Hance as a common herb for Miao ethnic minority in Guizhou by supercritical carbon dioxide extraction.The antioxidant activity of the essential oil was evaluated by comparing changes in the peroxide value(POV) of concentrated fish oil during storage at 60-65 鈩 in the respective presence of the essential oil,vitamin E(VE) and butylated hydroxytoluene(BHT).Forty-four compounds were identified in the essential oil by GC-MS,including 13 monoterpenoids(25.12%),16 oxygen-containing monoterpenoids(26.43%),9 sesquiterpenes(8.82%) and 6 oxygen-containing sesquiterpenes,benzene derivatives or others(39.63%).The essential oil as a natural antioxidant was superior to VE and BHT.The fish oil can be protected effectively by adding 0.3% essential oil.
DOI:10.1111/j.1759-6831.2010.00113.x      URL    
[本文引用:1]
[7] 袁琦,曹煜,瘳芬,.珊瑚姜油消毒液的消毒杀菌研究[J].贵阳医学院学报,2011,36(1):34-37.
目的:研究珊瑚姜油达到消毒杀菌剂的条件.方法:观察中和剂对消 毒剂的中和作用以达到作为消毒剂的基本条件.采用细菌定量杀灭试验,测定珊瑚姜油消毒液对微生物的杀灭效果;采用悬液定量杀菌试验对消毒液的稳定性进行测 试.结果:在(20±1)℃的条件下,用含2%甘氨酸、0.3%卵磷脂、3%吐温-80的0.03 mol/L PBS作中和剂,可有效中和金黄色葡萄球菌和白色念珠菌表面残留消毒液对受试菌的作用;珊瑚姜油消毒液对金黄色葡萄球菌、大肠杆菌、铜绿假单胞菌、白色念 珠菌都有很好杀灭效果,且具有很好的稳定性.结论:珊瑚姜油消毒液的消毒作用强,稳定性好.
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[8] YANG C, ZHOU L L, WANG H Y, et al.The inhibitory effect of Zingiber corallinum Hance essential oil on drug-resistant bacteria and evaluation of its acute toxicity[J]. Med Sci Monitor, 2011,17(5):139-146.
BACKGROUND: The excessive and irregular use of antibiotics could result in the generation and diffusion of drug-resistant bacteria. The aim of this study was to investigate the inhibitory effect of Zingiber corallinum Hance essential oil (ZCHO) on drug-resistant bacteria, especially on drug-resistant Acinetobacter baumannii. MATERIAL/METHODS: Susceptibility testing was used to evaluate the effect of ZCHO on growth inhibition of drug-resistant bacteria by paper disk method. Mice orally administered with ZCHO were used to observe acute toxicity and to determine median lethal dose (LD6368) of ZCHO. Broth dilution method was used to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ZCHO on drug-resistant Acinetobacter baumannii. RESULTS: ZCHO exhibited an obvious inhibitory effect not only on gram-negative drug-resistant bacteria including Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Acinetobacter baumannii, but also on gram-positive drug-resistant bacteria including Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus. The ZCHO containing 79% terpinen-4-ol revealed better bacteriostatic effect than ZCHO with 34% terpinen-4-ol. The LD6368 of ZCHO was 1790.427 mg/kg. The MIC and MBC of ZCHO on drug-resistant Acinetobacter baumannii were 1457.81 mg/L. CONCLUSIONS: ZCHO has obvious bacteriostasis and bactericidal effects, especially against drug-resistant Acinetobacter baumannii. Therefore, ZCHO is a promising natural bioactive component with antibacterial effect and satisfactory safety due to its low toxicity.
DOI:10.12659/MSM.881760      PMID:21525802      URL    
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[9] LOOVEREN M V, GOOSSENS H, GROUP T A S. Antimicrobial resistance of Acinetobacter spp. in Europe[J]. Clin Microbiology Inf ,2004,10(8):684-704.
Bacteria of the genus Acinetobacter are ubiquitous in nature. These organisms were invariably susceptible to many antibiotics in the 1970s. Since that time, acinetobacters have emerged as multiresistant opportunistic nosocomial pathogens. The taxonomy of the genus Acinetobacter underwent extensive revision in the mid-1980s, and at least 32 named and unnamed species have now been described. Of these, Acinetobacter baumannii and the closely related unnamed genomic species 3 and 13 sensu Tjernberg and Ursing (13TU) are the most relevant clinically. Multiresistant strains of these species causing bacteraemia, pneumonia, meningitis, urinary tract infections and surgical wound infections have been isolated from hospitalised patients worldwide. This review provides an overview of the antimicrobial susceptibilities of Acinetobacter spp. in Europe, as well as the main mechanisms of antimicrobial resistance, and summarises the remaining treatment options for multiresistant Acinetobacter infections.
DOI:10.1111/j.1469-0691.2004.01035.x      PMID:15301671      URL    
[本文引用:1]
[10] 涂云华,康颖倩,周英,. 姜黄挥发油对THP-1细胞增殖及凋亡的影响[J]. 山东大学学报(医学版),2015,53(5):46-51.
目的研究姜黄挥发油(TVO)对急性单核细胞白血病THP-1细胞株增殖及凋亡的影响,并探讨其作用机制。方法不同浓度(5~80mg/L)TV0体外作用于THP-1细胞。应用噻唑蓝(MTT)法检测细胞增殖抑制率;经吉姆萨染色,倒置显微镜观察细胞形态变化;DNA片段化测细胞凋亡;流式细胞仪检测细胞凋亡及周期;比色法测Caspase-3酶活性。结果TVO具有抑制THP-1细胞株增殖的作用,并表现出时间一剂量依赖性。同时,TVO能诱导THP-1细胞凋亡,呈现剂量依赖性,镜下见典型细胞凋亡形态,Caspase-3酶活性随药物浓度增加而增强。经TVO作用后的细胞周期被阻滞在G1期,G2/M期及s期细胞数减少。结论TVO对THP-1细胞株具有增殖抑制及促凋亡的作用,其机制可能是使细胞周期阻滞在G1期,且激活了细胞凋亡途径中关键酶Caspase-3活性,从而抑制肿瘤细胞的增殖与分化。
[本文引用:2]
[11] HWANG T S, HAN H S, CHOI H K, et al.Differential, stage-dependent expression of Hsp70, Hsp110 and Bcl-2 in colorectal cancer[J]. J Gastroent Hepatology,2003,18(6):690-700.
The presence of hypoxic cells in solid tumors has been suggested to contribute to the malignant progression of various tumors. Recently, we reported an activation of heat shock transcription factor (HSF) and expression of heat shock proteins (Hsp) in murine tumor cells by hypoxia.To search for a possible role of Hsp in the malignant progression of human tumors, we analyzed the expression profiles of Hsp family proteins in weakly and highly metastatic human colorectal cancer (CRC) cell lines. We also analyzed the expression profiles of Bcl-2 family proteins because the altered expression of these proteins has been demonstrated in various solid tumors.In the present paper we showed among various Hsp and Bcl-2 family proteins that the expression of Hsp70 and Hsp110 was elevated in highly metastatic CX-1 and HT-29 cells, while the expression of Bcl-2 was elevated in weakly metastatic MIP-101 and Clone A cells. Subsequent immunohistochemical analysis of 81 primary human CRC tissues demonstrated that the expression of Hsp70 and Hsp110 was highly correlated with the advanced clinical stages and positive lymph node involvement. The expression of Bcl-2, in contrast, was correlated to the early clinical stage and negative lymphovascular invasion.Taken together, our study demonstrated for the first time a differential, stage-dependent expression of Hsp70, Hsp110 and Bcl-2 in CRC. We suggest that the molecular mechanisms underlying the differential expression of Hsp and Bcl-2 family members deserves a more rigorous future study, the results of which might offer novel modes of rationale and strategy to predict and manipulate the malignant progression of colorectal cancers.
DOI:10.1046/j.1440-1746.2003.03011.x      PMID:12753152      URL    
[本文引用:1]
[12] 吴江群,聂兴举,秦泽莲. 氧化苦参碱对病理性瘢痕成纤维细胞增殖凋亡的影响[J].中国微创外科杂志,2011,11(3):259-263.
目的 观察氧化苦参碱(oxymatrine,OM)对人病理性瘢痕成纤维细胞增殖、凋亡、细胞周期及细胞外信号调节激酶1(ERK1)的影响,并且与瘢痕治疗 常规药物氢化可的松(hydrocortisone,HC)的作用进行比较. 方法 原代的瘢痕疙瘩、增生性瘢痕和正常皮肤的成纤维细胞(KFb、HFb、NFb)采用组织块培养法培养,将氧化苦参碱(2×10-6 mol/L)、氢化可的松(5.5×10-9 mol/L)分别作用于KFb、HFb、NFb,用四甲基偶氮唑蓝微量酶反应比色法(MTT)检测细胞增殖活力,流式细胞仪检测细胞凋亡、细胞周期、 ERK1的变化.结果 OM能明显抑制KFb、HFb和NFb的增殖活性(抑制率分别为12%,14%,10%),促进KFb的凋亡(增加72%).HC明显抑制KFb、 HFb、NFb的增殖(抑制率分别为21%,21%,15%),并促进其凋亡(分别增加184%、121%和148%).上述改变均有统计学意义.OM、 HC对三种细胞的细胞周期及ERK1表达无显著作用. 结论 类似氢化可的松,氧化苦参碱通过抑制瘢痕疙瘩、增生性瘢痕、正常皮肤成纤维细胞的增殖和促进瘢痕疙瘩成纤维细胞的凋亡作用而可能应用于瘢痕疙瘩和增生性瘢 痕的治疗.
[本文引用:1]
[13] 张润宇,余德顺. 民间传统药用植物珊瑚姜的研究与开发进展[J]. 四川中医,2004,22(10):26-28.
珊瑚姜是一种极具潜在药用价值的民间传统药用植物,近20年来受到人们的广泛关注.本文综述了相关文献,对已有珊瑚姜的研究和开发成果作了系统介绍,展望了其进一步深入开发前景.
[本文引用:1]
[14] 彭霞,赵应红. 傣药珊瑚姜的生药学研究[J]. 中华现代临床医学杂志,2004,2(12B):1172-1174.
目的 研究傣药珊瑚姜的生药鉴定特征。 方法 植物形态分类及中药鉴定学常规方法技术。 结果 珊瑚姜为姜科姜属植物Zingiber corallinum Hance的根茎,其植物形态、药材性状、显微特征、理化鉴别、薄层色谱及紫外吸收光谱有其特有的鉴别特征。 结论 为珊瑚姜药材的鉴别、开发利用及质量标准的制订提供了依据。
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[本文引用:1]
[15] 连海,金宁一,李霄,.新城疫病毒HN基因诱导人肺癌细胞SPC-A1凋亡的作用机制[J].中国生物化学与分子生物学报,2006,22(3):222-227.
为了探索新城疫病毒血凝素神经氨酸酶(HN)基因对人肺癌细胞SPC- A1的作用及机制,将含有HN基因的重组质粒pVHN经脂质体介导转染人肺癌细胞SPC-A1,通过MTT方法检测细胞活力;采用吖啶橙/溴化乙锭(AO /EB)染色分析肿瘤细胞凋亡;罗丹明123和DCFA染色测定线粒体跨膜电位(△ψm)和活性氧水平变化;流式细胞仪分析MHC-Ⅰ分子表达;底物染色 反应检测caspase-3活性.结果重组质粒pVHN转染人肺癌细胞SPC-A1 48 h后,细胞活力明显降低;AO/EB染色可见明显的细胞凋亡形态学变化;与空质粒对照相比,线粒体△ψm下降(P<0.01),活性氧水平升高 (P<0.05);细胞表面MHC-I分子表达上调(P>0.05);caspase-3活性增强(P<0.01).以上结果提示,新城疫病毒HN基因能 够上调SPC-A1细胞表面MHC-Ⅰ分子表达,并通过上调ROS水平,下调线粒体△ψm,进而激活caspase-3,最终诱导人肺癌细胞凋亡.
DOI:10.3969/j.issn.1007-7626.2006.03.008      Magsci     URL    
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[16] CAO X, WANG A H, JIAO R Z, et al.Surfactin induces apoptosis and G2/M arrest in human breast cancer MCF-7 cells through cell cycle factor regulation[J]. Cell Biochem Bioph ,2009,55(3):163-171.
Surfactin, purified from Bacillus subtilis natto TK-1, inhibited proliferation of human breast cancer MCF-7 cells in a dose- and time-dependent manner, with IC 50 at 24, 48, and 72聽h of 82.6, 27.3, and 14.8聽M, respectively. Surfactin-induced cell death was considered to be apoptotic by observing the typical apoptotic morphological change by acridine orange/ethidium bromide staining and Transferase-mediated dUTP Nick End-labeling assay. [Ca 2+ ]i measurement revealed that surfactin induced a sustained increase in concentration of intracellular [Ca 2+ ]i. Flow cytometric analysis also demonstrated that surfactin caused time-dependent apoptosis of MCF-7 cells through cell arrest at G 2 /M phase. Western blot revealed that surfactin induced accumulation of the tumor suppressor p53 and cyclin kinase inhibitor p21 waf1/cip1 , and inhibited the activity of the G 2 -specific kinase, cyclin B1/p34 cdc2 . Based on our findings, surfactin inhibited proliferation in MCF-7 cells by inducing apoptosis and the elevation of [Ca 2+ ]i may play an important role in the apoptosis. The mechanism which surfactin caused G 2 /M arrest seems to be through cell cycle factor regulation.
DOI:10.1007/s12013-009-9065-4      PMID:19669740      URL    
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[17] DEACIUC I V, FORTUNATO F, D'SOUZA N B, et al. Modulation of caspase-3 activity and Fas ligand mRNA expression in rat liver cells in vivo by alcohol and lipopolysaccharide[J]. Alcoh Clin Exp Res ,1999,23(2):349-356.
The purpose of this study was to determine if exacerbation of apoptosis precedes liver injury during chronic exposure of rats to alcohol. After 7 weeks of feeding an alcohol- or dextrin-containing liquid diet, the animals were treated with gram-negative bacterial lipopolysaccharide (1 mg x kg(-1) body weight, intravenously) or sterile saline and sacrificed 3 hr after the treatment. Alanine:2-oxoglutarate aminotransferase (ALT) and lactate:NAD oxidoreductase [lactate dehydrogenase (LDH)] were measured in plasma. The caudate lobe of the liver was resected for histology, while the rest of the organ was perfused with collagenase to isolate hepatocytes, Kupffer cells (KCs), and sinusoidal endothelial cells (SECs) by centrifugal elutriation. Hepatocyte mitochondria were isolated by differential centrifugation of the cell homogenate. Reduced and oxidized glutathione (GSH and GSSG) in isolated hepatocytes and hepatocyte mitochondria, and malondialdehyde in hepatocytes were assayed. Caspase-3 activity and Fas ligand mRNA expression were determined in hepatocytes, KCs, and SECs. Plasma ALT and LDH activity, liver histology, GSH, GSSG and their ratio, and malondialdehyde content were not affected by alcohol treatment Caspase-3 activity was significantly increased in alcohol-treated rats in all three cell types, with the lowest response observed in hepatocytes and the highest in KCs. Fas ligand mRNA expression, which had the highest level in SECs, followed by KCs and hepatocytes, was not affected by alcohol administration. Lipopolysaccharide had the following effects: an increase in ALT in both pair- and alcohol-fed rats, and LDH only in alcohol-fed rats, a decrease in GSH + GSSG levels in both mitochondria and hepatocytes, an elevation of malondialdehyde content in hepatocytes, a raise in caspase-3 activity in all groups and cell types, and an augmentation of Fas ligand expression in hepatocytes and KCs, but not in SECs. These data suggest that, during chronic alcohol consumption, an exacerbated apoptosis precedes alcohol-induced liver injury.
DOI:10.1111/j.1530-0277.1999.tb04121.x      PMID:10069567      URL    
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[18] LU H F, SUE C C, YU C S, et al.Diallyl disulfide (DADS) induced apoptosis undergo caspase-3 activity in human bladder cancer T24 cells[J]. Food Chem Toxi ,2004,42(10):1543-1552.
Diallyl disulfide (DADS), one of the major components of garlic (Allium sativum), is well known to have chemopreventative activity against human cancer such as colon, lung and skin. But the exact mechanism of the action is still unclear. In this study, we investigated how DADS––induced cell cycle arrest and apoptosis in T24 human bladder cancer cells in vitro. Apoptosis induction, cell viability, cell cycle arrest, caspases-3, -9 activity and gene expression were measured to determine their variation by flow cytometric assay, western blot, and determination of caspase-3 activity, PCR and cDNA microarray. There are significant differences in cell death (decreased viable cells then increased the amounts of apoptosis) of T24 cells that were detected between DADS (5–75 μM) treated and untreated groups. A significant increase was found in apoptosis induction when cells were treated with DADS (50 μM) compared to without DADS treated groups. DADS also promoted caspase-3 activity after exposure for 1, 3, 6, 12, and 24 h, which led to induce apoptosis. DADS also increased the product of intracellular hydrogen peroxide. Furthermore, the DADS-induced apoptosis on T24 cells was blocked by the broad-spectrum caspase inhibitor, z-VAD-fmk and antioxidant (catalase). DADS also increased cyclin E and decreased CDK2 gene expression which may lead to the G2/M arrest of T24 cells.
DOI:10.1016/j.fct.2003.06.001      PMID:15304301      URL    
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[19] IRAJ J A, MOJTABA S, FATEMEH V, et al.Evaluation of insulin and ascorbic acid effects on expression of Bcl-2 family proteins and caspase-3 activity in hippocampus of STZ-induced diabetic rats[J]. Cell Moler Neurob ,2009,29(1):133-140.
We assessed the expression of Bcl-2 family members at both mRNA and protein levels as well as the Caspase-3 activity, in order to investigate the occurrence of apoptosis in hippocampus of STZ-induced diabetic rats. We selected twenty-four Wistar rats; half of them were made diabetic by intraperitoneal injection of a single 60 mg/kg dose of streptozotocin (STZ, IP), while the others received normal saline and served as controls. The expressions of Bcl-2, Bcl-x(L), and Bax mRNA and proteins were measured using RT-PCR and western blotting, respectively. Caspases-3 activity was determined by using the Caspase-3/CPP32 Fluorometric Assay Kit. The result showed that mRNA and protein levels of Bcl-2 and Bcl-x(L) were lower in hippocampus of diabetic group than that of the control group, whereas expressions of Bax in hippocampus of diabetic rats were higher than that of controls at both mRNA and protein levels (P < .01). Hyperglycemia was found to raise 6.9-fold hippocampal caspase-3 activity in diabetic group compared with control group (P < .001). Therefore, the induction of diabetes is associated with increased ratios of Bax/Bcl-2, Bax/Bcl-x(L), and increased caspase-3 activity in hippocampus which shows that apoptosis is favored in hippocampal region.
DOI:10.1155/2008/638467      PMID:2566751      URL    
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[20] MOSSER D D, CARON A W, BOURGET L, et al.Role of human heat shock protein hsp70 in protection against stress-induced apoptosis[J]. Mole Cell Biology,1997,17(9):5317-5327. [本文引用:1]
[21] MOSSER D D, CARON A W, BOURGET L, et al.The chaperone function of hsp70 is required for protection against stress-induced apoptosis[J]. Mole Cell Biology,2000,20(19):7146-7159.
Cellular stress can trigger a process of self-destruction known as . Cells can also respond to stress by adaptive changes that increase their ability to tolerate normally lethal conditions. Expression of the major heat-inducible protein protects cells from heat-induced . has been reported to act in some situations upstream or downstream of , and its protective effects have been said to be either dependent on or independent of its ability to inhibit activation. Purified has been shown to block procaspase processing in vitro but is unable to inhibit the activity of active caspase 3. Since some aspects of function can occur in the absence of its chaperone activity, we examined whether lacking its ATPase domain or the C-terminal EEVD sequence that is essential for was required for the prevention of . We generated stable cell lines with -regulated expression of , , and chaperone-defective mutants lacking the ATPase domain or the C-terminal EEVD sequence or containing AAAA in place of EEVD. Overexpression of or protected cells from heat shock-induced by preventing the processing of procaspases 9 and 3. This required the chaperone function of since mutant proteins did not prevent procaspase processing or provide protection from . activation was inhibited by both and and by each of the domain mutant proteins. The chaperoning activity of is therefore not required for inhibition of activation, and inhibition was not sufficient for the prevention of . Release of from mitochondria was inhibited in cells expressing full-length but not in cells expressing the protein with ATPase deleted. Together with the recently identified ability of to inhibit -mediated procaspase 9 processing in vitro, these data demonstrate that can affect the apoptotic pathway at the levels of both release and initiator and that the chaperone function of is required for these effects.
DOI:10.1128/MCB.20.19.7146-7159.2000      PMID:86268      URL    
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[22] ELDER R T, BENKO Z, ZHAO Y.HIV-1 VPR modulates cell cycle G2/M transition through an alternative cellular mechanism other than the classic mitotic checkpoints.[J]. Front Biosci ,2002,7(2):d349-d357.
Abstract HIV-1 Vpr induces cell cycle G2/M arrest in both human and fission yeast cells, suggesting a highly conserved activity of this viral protein. In this review, we summarize the current understanding of Vpr-induced G2 arrest based on studies from both mammalian cells and the fission yeast (Schizosaccharomyces pombe) model system. Fission yeast has proven to be an excellent model system to investigate cell cycle G2/M control of eukaryotic cells. Similarly, fission yeast has also been instrumental in defining the molecular mechanism underlying the G2 arrest induced by Vpr. We have compared the classic DNA-damage and DNA-replication checkpoint controls of the cell cycle G2/M transition to the G2 arrest conferred by Vpr. Based on the current findings, we hypothesize that Vpr induces cell cycle G2 arrest through an alternative novel cellular pathway(s) rather than through the classic mitotic checkpoint controls. A number of cellular proteins which may be involved in this new cellular pathway(s) have been identified and are discussed.
DOI:10.2741/elder      PMID:11815283      URL    
[本文引用:1]
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关键词(key words)
珊瑚姜油
宫颈
HeLa细胞
细胞周期

oil
Carcinoma
cervical
HeLa cell
Cell cycle

作者
吴春维
涂云华
李明娥
叶振源
薛月萃
曹煜

WU Chunwei
TU Yunhua
LI Minge
YE Zhenyuan
XUE Yuecui
CAO Yu