去甲斑蝥素影响胰腺癌细胞生长的实验研究
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摘要
目的
胰腺癌是临床上常见的恶性肿瘤,发病率高,预后差。目前尚无完全可靠有效的治疗方法。已知肿瘤是一种细胞增殖与死亡障碍性疾病,肿瘤细胞凋亡的调控机制失常在恶性肿瘤的演进过程中起重要作用。肿瘤防治的关键手段之一就是抑制肿瘤细胞的增殖和促进肿瘤细胞的凋亡。本研究探讨传统中药的提取物去甲斑蝥素(norcantharidin,NCTD)对人胰腺癌AsPC-1细胞增殖、细胞周期、细胞生长的相关凋亡基因Bcl-2、Caspase-3的影响变化情况,寻求其作用效果及机制。
材料和方法
1.人胰腺癌AsPC-1细胞购自第四军医大学。去甲斑蝥素(norcan-tharidin,NCTD)由沈阳药科大学赵明宏博士馈赠。流式细胞仪;美国Bec-ton Dickinson公司产的荧光激活分选仪(FACS)420型。免疫组化鼠抗人Bcl-2抗体、鼠抗人Caspase-3抗体购于武汉博士德生物工程有限公司。图像分析仪器型号;MetaMorph/DP10/BX41;生产厂;UIC/OLYMPUS,US/JP。图象分析软件;1D Kodar成像分析系统。
2.培养人胰腺癌AsPC-1细胞株并传代。
3.体外细胞杀伤实验;①以NCTD浓度为横轴、吸光值为纵轴,绘制药物浓度曲线,求50%抑制率对应的药物浓度即为半数抑制浓度IC_(50)。②NCTD对AsPC-1细胞的抑制率为;抑制率=(1-实验组A值/对照组A值)×100%。找出NCTD抑制作用最明显的时间。
4.细胞形态学观察;①一般形态学观察。②超微结构观察。
5.流式细胞术;检测细胞周期、细胞凋亡率。
6.Western blot;分别收集对照组(n=6)和实验组(n=6)胰腺癌细胞进行Western blot实验。以β-actin为内对照。照片用1D Kodar图像分析软件进行光密度积分值半定量分析,以相应蛋白条带的平均光密度值来表示Capase-3的表达程度。
7.胰腺癌裸鼠模型建立,裸鼠种植瘤生长抑制实验;荷瘤鼠随机分成对照组(n=8)、NCTD组(n=8)、5-FU组(n=8)、NCTD+5-FU联合用药组(n=8)4组,各组用药前裸鼠体重和瘤体大小无差异。从种植AsPC-1细胞后第2周始,分别自腹腔注射生理盐水、5-FU(24mg/kg,)、NCTD(28mg/kg)、5-FU(24mg/kg)+NCTD(28mg/kg),每周2次,共6周。
8.荷瘤裸鼠标本免疫组化处理;荷瘤鼠随机分成对照组(n=8)、NCTD组(n=8)。用药前2组裸鼠体重和瘤体大小无差异。从种植AsPC-1细胞后第2周始,分别自腹腔注射生理盐水、NCTD(28mg/kg,每周2次,共6周。待第7周以颈椎脱位法处死裸鼠,切取种植瘤标本固定、切片。Bcl-2免疫组化处理,封片后在光镜下观察。根据细胞浆中出现棕黄染物质的光密度判读结果,随机框取相同放大倍数与面积的细胞区域,采用MetaMor-ph/DP10/BX41型图象分析系统测定其相对光密度。
结果
1.NCTD浓度为10μg/ml时,对AsPC-1的生长有抑制作用,且随药物浓度升高其抑制作用增强,呈剂量-效应关系;NCTD作用6h后对AsPC-1生长具有抑制作用,48 h时抑制作用最明显,呈时间-效应关系。经NCTD作用后,流式细胞仪显示;G_2+M期的细胞明显增多,S期细胞明显减少,细胞凋亡率上升;光镜检查显示;AsPC-1可出现细胞固缩,胞膜突出,核碎裂等现象;电镜显示;细胞微绒毛卷缩、高尔基体、线粒体等细胞器衰退,并可见典型的凋亡小体。
2.20μg/ml的NCTD液作用于体外培养的人胰腺癌AsPC-1细胞48h,Western blot分析显示实验组Caspase-3的表达显著增加(P＜0.05);裸鼠移植瘤切片经免疫组化分析,实验组Bcl-2的表达显著减少(P＜0.01)。
3.NCTD可使裸鼠种植瘤明显缩小(2.22±0.18 vs.3.25±0.18 cm~3,P＜0.01),抑瘤率增高(31.69% vs.0,P＜0.05);种植瘤细胞周期中G_2+M期细胞百分率明显增多(51.24±1.92 vs.23.31±1.81,P＜0.01),凋亡率上升(0.23±0.05 vs.4.21±0.52,P＜0.01);与5-FU合用时上述作用更明显。NCTD作用后的种植瘤细胞经光镜检查见胞核固缩,核碎裂等现象;电镜显示典型的凋亡细胞及凋亡小体。
结论
1.NCTD能抑制人胰腺癌AsPC-1细胞的生长,其作用呈剂量、时间-效应关系。其机制可能与NCTD诱导AsPC-1细胞凋亡,抑制细胞增殖,干扰细胞生长周期,抑制DNA合成并影响代谢有关。
2.NCTD可以诱导人胰腺癌AsPC-1细胞发生凋亡,该凋亡过程的作用机制可能与调控癌变基因Bcl-2、Capase-3的表达有关。
3.NCTD可明显抑制裸鼠胰腺癌种植瘤的增殖和生长,若与5-FU合用呈协同效应,可加强其抗癌作用。
Object
Pancreatic cancer is a common malignant tumor that we meet everyday in clinical practice.It has a high morbidity and mortality,very poor prognosis. There is no any reliable curing therapy for pancreatic cancer.It is known that cancer is an incontrollable proliferation and death disorder disease,the mecha-nism of controlling cancer cell apoptosis disorder plays a role in developing of a cancer.The key point of prevention of a cancer is suppresses cancer cell prolif-eration and accelerates the apoptosis.We investigated the efficacy and mecha-nism of the extraction of a traditional medicine norcantharidin(NCTD)on hu-man pancreatic cancer cell proliferation,cell circle,related apoptotic gene ex-pression of Bcl-2,Caspase-3.
Material and Method
1.Human pancreatic cancer cell line AsPC-1 was bought from The Fourth Military Medical University.Norcantharidin(NCTD)was donated by Doctor Zhao Minghong from Shenyang Pharmaceutical University.Flow Cytometry (FACS 420)was bought from Becton Dickinson Company.Immunohistochemis-try mouse antihuman Bcl-2 antibody,mouse antihuman Caspase-3 antibody was bought from Boside Biotechnology Company.Photo analysis system is Meta-Morphrph/DP10/BX41(UIC/Olympus,US/JP).
2.Culture human pancreatic cancer cell line AsPC-1 and proliferation, passage.
3.In Vitro Cytotoxic Test;Fulfill a NCTD drug concentration versus cell suppression curve by using NCTD concentration and extinction value,get 50% inhibition ratio IC_(50).Calculate the inhibition ratio by the formula below;
Inhibition ration=(1-actual value of experimental group A value/con-trol group A value)(100%,find out the maximum work time and duration4. Cell morphologic study;To investigate the general morphologic change and ul-tramicrostructure changes.
5.Flow Cytometric Study;To investigate cell circle and apoptotic ratio of the cancer cells.
6.Western Blot study;Collected cancer cells from the control group(N= 6)and experimental group(N=6)to test by using western blot.Useβ-actin as a control medicine.Photodensity value was analyzed semiquantitatively by Flours Mutilmager Analyzer(BioRad),the average photodensity of the protein strip stood for the expression value of the Casepase-3.
7.In Vivo;Setting up the node mouse model of growth inhibition on the cancer cells.Tumor growing nude mice randomly divided into control group(N =8),NCTD group(N=8),5-FU group(N=8),NCTD+5-FU Group (N=8),respectively.Body weight and cancer size showed no statistic differ-ence pre-experiment.From the second week of AsPC-1 implantation,normal saline or 5-FU(24mg/kg),or NCTD(28mg/kg)or 5-FU(24mg/kg)plus NCTD(28mg/kg)was administrated to the mice,respectively,twice a week,6 weeks in total.
8.Immunohistochemistry study;Tumor growing nude mice randomly divid-ed into control group(N=8)and NCTD group(N=8).Mice body weight and tumor size showed no statistic difference.Normal saline or NCTD(28mg.kg) was administrated respectively to mouse after two weeks of AsPc-1 cancer cells implantation intra-peritoneally,twice a week,6 weeks in total.Mouse was eu-thanasia at 7 weeks post cancer cells implantation.Cancer was taken out for fur-ther study.Specimen was stained immunohistochemically by Bcl- 2 antibody. Optical microscopic analysis was sequentially done.The positive result was de-fined by the degree of buffy stained matter in Cytoplasm.Randomly selected 100 cancer cells under 5 microscopic areas,MetaMorph/DP10/BX41 system ana-lyzed the relative optical density to determine the stained degree of the stained area.
Results
NCTD inhibited the cancer cell growth when cells were administrated at 10 (g/ml concentration,and the effect enhanced when the concentration adminis-trated higher at a dose dependent manner.It showed a suppression effect on the ASPC-1 cancer cells growth after administration of NTCD 6 hours,the effect reached maximum after NCTD administration 48 hours at a time dependent man-ner.Flow cytometric analysis showed G2 stage cancer cells group enlarged after NCTD administration,while S stage cancer cells decreased,apoptotic rate of the cancer cells increased.It showed in optical microscopic view as ASPC-1 canc-er cell nuclear pyknosis,vela cell membrane,nuclear chip phenomenon,apop-totic body was shown.Electron microscope analysis showed cellular crisp mi-crovilli,Retrogression cellular organelle of Golgi's body and mitochondria,apop-totic body was shown.
Caspase-3 expression was enhanced in experimental group after adminis-tration of 20(g/ml NCTD 48 hours by western blot analysis(P＜0.05); While Bcl-2 expression decreased by immunohistochemical assay at same group(P＜0.01).
NCTD administration suppressed transferred tumor's growth(2.22±0.18 vs.3.25±0.18 cm~3,P＜0.01),cancer inhibition ratio increased(31.69% vs. 0,P＜0.05); G_2+M stage cell increased in implanted tumor cell circle(51.24±1.92 vs.23.31±1.81,P＜0.01); apoptotic rate increased(0.23±0.05 vs.4.21±0.52,P＜0.01); All the effects above showed stronger when mice were coadministrated with 5-Fu.Optical microscopic analysis showed nuclear pycnosis,karyorrhexis phenomenon in implanted tumor cells; Electronic micro-scopic analysis showed classic apoptotic cells and apoptotic body in cancer cells.
Conclusion
1.NCTD suppressed human pancreatic cancer cell line AsPC-1 growth, the effect was showed at a dose and time dependent manner.The possible mech-anism assumed as NCTD induced AsPC-1 cell apoptosis,suppressed human pancreatic cancer cell proliferation,interfered cell circle,inhibited DNA syn-thesis and affected metabolism of cancer cells.
2.NCTD could induce human pancreatic cancer cell line AsPC-1 apopto-sis,the mechanism of action probably related with the expression of regulatory gene Bcl-2 and Caspase-3.
3.NCTD inhibited nude mice implanted tumor proliferation and growth,It has synergistic effect with 5-Fu when they were given together,and could en-hance anticancer effect when both drugs were given.
胰腺癌是临床上常见的恶性肿瘤,发病率高,预后差。目前尚无完全可靠有效的治疗方法。已知肿瘤是一种细胞增殖与死亡障碍性疾病,肿瘤细胞凋亡的调控机制失常在恶性肿瘤的演进过程中起重要作用。肿瘤防治的关键手段之一就是抑制肿瘤细胞的增殖和促进肿瘤细胞的凋亡。本研究探讨传统中药的提取物去甲斑蝥素(norcantharidin,NCTD)对人胰腺癌AsPC-1细胞增殖、细胞周期、细胞生长的相关凋亡基因Bcl-2、Caspase-3的影响变化情况,寻求其作用效果及机制。
材料和方法
1.人胰腺癌AsPC-1细胞购自第四军医大学。去甲斑蝥素(norcan-tharidin,NCTD)由沈阳药科大学赵明宏博士馈赠。流式细胞仪;美国Bec-ton Dickinson公司产的荧光激活分选仪(FACS)420型。免疫组化鼠抗人Bcl-2抗体、鼠抗人Caspase-3抗体购于武汉博士德生物工程有限公司。图像分析仪器型号;MetaMorph/DP10/BX41;生产厂;UIC/OLYMPUS,US/JP。图象分析软件;1D Kodar成像分析系统。
2.培养人胰腺癌AsPC-1细胞株并传代。
3.体外细胞杀伤实验;①以NCTD浓度为横轴、吸光值为纵轴,绘制药物浓度曲线,求50%抑制率对应的药物浓度即为半数抑制浓度IC_(50)。②NCTD对AsPC-1细胞的抑制率为;抑制率=(1-实验组A值/对照组A值)×100%。找出NCTD抑制作用最明显的时间。
4.细胞形态学观察;①一般形态学观察。②超微结构观察。
5.流式细胞术;检测细胞周期、细胞凋亡率。
6.Western blot;分别收集对照组(n=6)和实验组(n=6)胰腺癌细胞进行Western blot实验。以β-actin为内对照。照片用1D Kodar图像分析软件进行光密度积分值半定量分析,以相应蛋白条带的平均光密度值来表示Capase-3的表达程度。
7.胰腺癌裸鼠模型建立,裸鼠种植瘤生长抑制实验;荷瘤鼠随机分成对照组(n=8)、NCTD组(n=8)、5-FU组(n=8)、NCTD+5-FU联合用药组(n=8)4组,各组用药前裸鼠体重和瘤体大小无差异。从种植AsPC-1细胞后第2周始,分别自腹腔注射生理盐水、5-FU(24mg/kg,)、NCTD(28mg/kg)、5-FU(24mg/kg)+NCTD(28mg/kg),每周2次,共6周。
8.荷瘤裸鼠标本免疫组化处理;荷瘤鼠随机分成对照组(n=8)、NCTD组(n=8)。用药前2组裸鼠体重和瘤体大小无差异。从种植AsPC-1细胞后第2周始,分别自腹腔注射生理盐水、NCTD(28mg/kg,每周2次,共6周。待第7周以颈椎脱位法处死裸鼠,切取种植瘤标本固定、切片。Bcl-2免疫组化处理,封片后在光镜下观察。根据细胞浆中出现棕黄染物质的光密度判读结果,随机框取相同放大倍数与面积的细胞区域,采用MetaMor-ph/DP10/BX41型图象分析系统测定其相对光密度。
结果
1.NCTD浓度为10μg/ml时,对AsPC-1的生长有抑制作用,且随药物浓度升高其抑制作用增强,呈剂量-效应关系;NCTD作用6h后对AsPC-1生长具有抑制作用,48 h时抑制作用最明显,呈时间-效应关系。经NCTD作用后,流式细胞仪显示;G_2+M期的细胞明显增多,S期细胞明显减少,细胞凋亡率上升;光镜检查显示;AsPC-1可出现细胞固缩,胞膜突出,核碎裂等现象;电镜显示;细胞微绒毛卷缩、高尔基体、线粒体等细胞器衰退,并可见典型的凋亡小体。
2.20μg/ml的NCTD液作用于体外培养的人胰腺癌AsPC-1细胞48h,Western blot分析显示实验组Caspase-3的表达显著增加(P＜0.05);裸鼠移植瘤切片经免疫组化分析,实验组Bcl-2的表达显著减少(P＜0.01)。
3.NCTD可使裸鼠种植瘤明显缩小(2.22±0.18 vs.3.25±0.18 cm~3,P＜0.01),抑瘤率增高(31.69% vs.0,P＜0.05);种植瘤细胞周期中G_2+M期细胞百分率明显增多(51.24±1.92 vs.23.31±1.81,P＜0.01),凋亡率上升(0.23±0.05 vs.4.21±0.52,P＜0.01);与5-FU合用时上述作用更明显。NCTD作用后的种植瘤细胞经光镜检查见胞核固缩,核碎裂等现象;电镜显示典型的凋亡细胞及凋亡小体。
结论
1.NCTD能抑制人胰腺癌AsPC-1细胞的生长,其作用呈剂量、时间-效应关系。其机制可能与NCTD诱导AsPC-1细胞凋亡,抑制细胞增殖,干扰细胞生长周期,抑制DNA合成并影响代谢有关。
2.NCTD可以诱导人胰腺癌AsPC-1细胞发生凋亡,该凋亡过程的作用机制可能与调控癌变基因Bcl-2、Capase-3的表达有关。
3.NCTD可明显抑制裸鼠胰腺癌种植瘤的增殖和生长,若与5-FU合用呈协同效应,可加强其抗癌作用。
Object
Pancreatic cancer is a common malignant tumor that we meet everyday in clinical practice.It has a high morbidity and mortality,very poor prognosis. There is no any reliable curing therapy for pancreatic cancer.It is known that cancer is an incontrollable proliferation and death disorder disease,the mecha-nism of controlling cancer cell apoptosis disorder plays a role in developing of a cancer.The key point of prevention of a cancer is suppresses cancer cell prolif-eration and accelerates the apoptosis.We investigated the efficacy and mecha-nism of the extraction of a traditional medicine norcantharidin(NCTD)on hu-man pancreatic cancer cell proliferation,cell circle,related apoptotic gene ex-pression of Bcl-2,Caspase-3.
Material and Method
1.Human pancreatic cancer cell line AsPC-1 was bought from The Fourth Military Medical University.Norcantharidin(NCTD)was donated by Doctor Zhao Minghong from Shenyang Pharmaceutical University.Flow Cytometry (FACS 420)was bought from Becton Dickinson Company.Immunohistochemis-try mouse antihuman Bcl-2 antibody,mouse antihuman Caspase-3 antibody was bought from Boside Biotechnology Company.Photo analysis system is Meta-Morphrph/DP10/BX41(UIC/Olympus,US/JP).
2.Culture human pancreatic cancer cell line AsPC-1 and proliferation, passage.
3.In Vitro Cytotoxic Test;Fulfill a NCTD drug concentration versus cell suppression curve by using NCTD concentration and extinction value,get 50% inhibition ratio IC_(50).Calculate the inhibition ratio by the formula below;
Inhibition ration=(1-actual value of experimental group A value/con-trol group A value)(100%,find out the maximum work time and duration4. Cell morphologic study;To investigate the general morphologic change and ul-tramicrostructure changes.
5.Flow Cytometric Study;To investigate cell circle and apoptotic ratio of the cancer cells.
6.Western Blot study;Collected cancer cells from the control group(N= 6)and experimental group(N=6)to test by using western blot.Useβ-actin as a control medicine.Photodensity value was analyzed semiquantitatively by Flours Mutilmager Analyzer(BioRad),the average photodensity of the protein strip stood for the expression value of the Casepase-3.
7.In Vivo;Setting up the node mouse model of growth inhibition on the cancer cells.Tumor growing nude mice randomly divided into control group(N =8),NCTD group(N=8),5-FU group(N=8),NCTD+5-FU Group (N=8),respectively.Body weight and cancer size showed no statistic differ-ence pre-experiment.From the second week of AsPC-1 implantation,normal saline or 5-FU(24mg/kg),or NCTD(28mg/kg)or 5-FU(24mg/kg)plus NCTD(28mg/kg)was administrated to the mice,respectively,twice a week,6 weeks in total.
8.Immunohistochemistry study;Tumor growing nude mice randomly divid-ed into control group(N=8)and NCTD group(N=8).Mice body weight and tumor size showed no statistic difference.Normal saline or NCTD(28mg.kg) was administrated respectively to mouse after two weeks of AsPc-1 cancer cells implantation intra-peritoneally,twice a week,6 weeks in total.Mouse was eu-thanasia at 7 weeks post cancer cells implantation.Cancer was taken out for fur-ther study.Specimen was stained immunohistochemically by Bcl- 2 antibody. Optical microscopic analysis was sequentially done.The positive result was de-fined by the degree of buffy stained matter in Cytoplasm.Randomly selected 100 cancer cells under 5 microscopic areas,MetaMorph/DP10/BX41 system ana-lyzed the relative optical density to determine the stained degree of the stained area.
Results
NCTD inhibited the cancer cell growth when cells were administrated at 10 (g/ml concentration,and the effect enhanced when the concentration adminis-trated higher at a dose dependent manner.It showed a suppression effect on the ASPC-1 cancer cells growth after administration of NTCD 6 hours,the effect reached maximum after NCTD administration 48 hours at a time dependent man-ner.Flow cytometric analysis showed G2 stage cancer cells group enlarged after NCTD administration,while S stage cancer cells decreased,apoptotic rate of the cancer cells increased.It showed in optical microscopic view as ASPC-1 canc-er cell nuclear pyknosis,vela cell membrane,nuclear chip phenomenon,apop-totic body was shown.Electron microscope analysis showed cellular crisp mi-crovilli,Retrogression cellular organelle of Golgi's body and mitochondria,apop-totic body was shown.
Caspase-3 expression was enhanced in experimental group after adminis-tration of 20(g/ml NCTD 48 hours by western blot analysis(P＜0.05); While Bcl-2 expression decreased by immunohistochemical assay at same group(P＜0.01).
NCTD administration suppressed transferred tumor's growth(2.22±0.18 vs.3.25±0.18 cm~3,P＜0.01),cancer inhibition ratio increased(31.69% vs. 0,P＜0.05); G_2+M stage cell increased in implanted tumor cell circle(51.24±1.92 vs.23.31±1.81,P＜0.01); apoptotic rate increased(0.23±0.05 vs.4.21±0.52,P＜0.01); All the effects above showed stronger when mice were coadministrated with 5-Fu.Optical microscopic analysis showed nuclear pycnosis,karyorrhexis phenomenon in implanted tumor cells; Electronic micro-scopic analysis showed classic apoptotic cells and apoptotic body in cancer cells.
Conclusion
1.NCTD suppressed human pancreatic cancer cell line AsPC-1 growth, the effect was showed at a dose and time dependent manner.The possible mech-anism assumed as NCTD induced AsPC-1 cell apoptosis,suppressed human pancreatic cancer cell proliferation,interfered cell circle,inhibited DNA syn-thesis and affected metabolism of cancer cells.
2.NCTD could induce human pancreatic cancer cell line AsPC-1 apopto-sis,the mechanism of action probably related with the expression of regulatory gene Bcl-2 and Caspase-3.
3.NCTD inhibited nude mice implanted tumor proliferation and growth,It has synergistic effect with 5-Fu when they were given together,and could en-hance anticancer effect when both drugs were given.
引文
1姚燕丹,林少芒.去甲斑蝥素的研究进展及其乳腺癌的关系 中国医药导刊,2005,7(1);58-598
2Chen YN,Chen JC,Yin SC,et al.Effector mechanisms of norcantharidin - Induced mitotic arrest and apoptosis in human hepatoma cells.Int J Cancer,2002,100(2);158-65.
3Chen YN,Cheng CC,Chen JC,et al.Norcantharidin - induced apoptosis is via the extra cellular signal regulated kinase and c - Jun - NH2 - terminal kinase signaling pathways in human hepatoma HepG2 cells.Br J Pharmacol,2003; 140(3);461-470
4Li JL,Cai YC,Liu XH,et al.Norcantharidin inhibits DNA replication and induces apoptosis with the cleavage of initiation protein Cdc6 in HL - 60 cells.,2006; 17(3);307-14
5范祖,赵泽明,陈春球,等.去甲斑蝥素对荷瘤裸鼠胆囊癌移植瘤增殖和凋亡的体内干预实验.中华普通外科杂志,2005;20(7);438-441.
6孙震晓,赵天德,魏玉林,等.斑蝥素及去甲斑蝥素诱导人红白血病细胞凋亡的细胞学研究.解剖学报,2000,31(1);56-60
7赵聚山,陈璇.去甲斑蝥素对体外两种肿瘤细胞生长的影响.辽宁中医杂志,2004,31(2);157-158
8刘晓兰,陈家旭,刘燕,等.去甲斑蝥素诱导HL60细胞凋亡的研究.北京中医药大学学报,2000,23(4);35-37
9唐龙英,王昭梅,郑钦岳,等.去甲斑蝥素对卵巢癌3AO和AO细胞的体外抑制作用.第二军医大学学报,1999,20(2);97-99
10Modonnell TJ,Beham A,Sarkiss M,et al.Impotance of the Bcl - 2 family in cell death regulation,Exprientia,1996,52(10-11);1008-1112
11Yin C,Kundoscon C M,Kanemeyer S J,et al Bax suppresses of tumor genesis and stimulates apoptosis in vivo.Nature,1997,385(6617);737-640
12郑俊猛,王得坤,张柱林,等.Bel-2硫代反义寡核苷酸提高肺癌细胞对Fas介导细胞毒作用的敏感性.中华肿瘤杂志,2002,24;549.
13Manne U,Weiss HL,Grizzle WE.BCL-2 expressions associated with im- proved prognosis in patients with sistal colorectal adenocarcimomas.Int J Cancer,2000,89(5);423-30
14滕军放,冯建玉,卢宏,等.局灶性脑缺血再灌注后大鼠脑皮质Caspase-3,Bcl-2的表达.中华医学杂志,85(42);3012-3018
15Chakraborty C,Saha G,Sarkar B,et al.Caspase - 3 induced apoptosis in transgenic zebrafish.Biotechnol Lett.2006; 28(3);189-96.
1姚燕丹,林少芒.去甲斑蝥素的研究进展及其乳腺癌的关系.中国医药导刊,2005,7(1);58-598
2张泰松,程树仓,臧恒昌,等.去甲斑蝥素诱导细胞凋亡的研究进展.海峡药学,2005,17(3);125-127
3孙震晓,赵天德,魏玉林,等.去甲斑蝥素诱导人肝癌BEL-7402细胞凋亡的研究.解剖学报,1999,30(1);65-68
4赵聚山,陈璇.去甲斑蝥素对体外两种肿瘤细胞生长的影响.辽宁中医杂志,2004,31(2);157-158
5刘晓兰,陈家旭,刘燕,等.去甲斑蝥素诱导HL60细胞凋亡的研究.北京中医药大学学报,2000,23(4);35-37
6唐龙英,王昭梅,郑钦岳,等.去甲斑蝥素对卵巢癌3AO和AO细胞的体外抑制作用.第二军医大学学报,1999,20(2);97-99
7范跃祖,傅锦业,赵泽明,等.去甲斑蝥素对原发性胆囊癌GBE-SD细胞系增殖和凋亡的影响.上海医学,2003,26(增刊);1-4
8范跃祖,傅锦业,赵泽明,等.去甲斑蝥素对人原发性胆囊癌GBC-SD细胞系增殖及相关基因的干预效应.中华实验外科杂志,2004,21(5);554-556
9范跃祖,赵泽明,陈春球,等.去甲斑蝥素对人原发性荷瘤裸鼠胆囊癌GBC-SD移植瘤增殖和凋亡的体内干预实验.中华普通外科杂志,2005,20(7);438-441
10安巍巍,龚显峰,王敏伟,等.去甲斑蝥素诱导人黑色素瘤A375-S2细胞凋亡.中国医院药学杂志,2005,25(6);501-504
11张卫东,赵惠儒,阎影,等.斑蝥素诱导人肺癌A549细胞凋亡及其分子机制的研究.中华肿瘤杂志,2005,27(6);330-333
12赵泽明,范跃祖,傅锦业,等.去甲斑蝥素对荷瘤裸鼠胆囊癌移植瘤nm23、基质金属蛋白酶-2和基质金属蛋白酶组织抑制因子-2的影响..中华普通实验外科杂志,2005,22(3);307-311
13安巍巍,薛莲,王敏伟,等.去甲斑蝥素诱导小鼠肺纤维瘤L929细胞凋亡.中国癌症杂志,2005,15(1);22-25
14Huang S,Pettaway CA,Uehara H et al.Blockade of NF - kappaB activity in human prostate cancer cells is associated with suppression of angiogenesis,invasion,and metastasis[J].Oncogene,2001,20(31);4188-97
15Lin A,Karin M.NF - kappaB in cancer.a marked target[J].S emin Cancer Biol,2003,13(2);107-114
16何太平,莫丽儿,梁念慈.斑蝥素诱导高转移卵巢癌细胞HO-8910PM细胞凋亡的研究.中国药科大学学报,2005,36(2);164-167
17孙震晓,李家实.去甲基斑蝥素抗肿瘤研究热点.西北药学杂志,1998,13(5);227-228
18郑兰英,史春雷,贺延新.去甲斑蝥素治疗肝癌的研究.河北医学,2005,11(9);820-822
19周文丽,考军,范清玲.去甲斑蝥素片辅助治疗胃肠道癌肝转移30例临床观察.山东医药,2005,45(20);32-33
20郝素华.埃迪注射液联合化疗治疗晚期恶性肿瘤的疗效观察.肿瘤研究与临床,2003,15(3);202-203
21挛祖鹏,李晓东,马敏.去甲斑蝥素注射液联合氟尿嘧啶治疗晚期肝癌的临床研究.河北医药,2005,27(7);542-544
22吴光兴,杨志华,陈恩碧.化疗联合复方斑蝥素注射液治疗恶性淋巴瘤临床观察.中国医师杂志,2005,7(10);1425-1428
23王怀璋,王程虎,王瑞香.化疗联合埃迪注射液治疗晚期非小细胞肺癌疗效观察.中国治疗临床与康复,2004,11(4);358-359
24史健,魏速菊,单保恩.斑蝥酸钠注射液治疗癌性胸腔积液的临床观察.中国中西医结合杂志,2005,25(5);451-453
25郭艳霞,叶志雄.复方斑蝥汤治疗晚期鼻咽癌疗效分析.现代医院,2005,5(9);80-81
26陈双彪,陈祖红,苏腾良,等.水蛭斑蝥汤治疗良性前列腺增生症30例疗效观察.新中医,2006,38(1);44-45
27王新华.尤斯洛(斑蝥素)治疗尖锐湿疣30例报道.中国民康医学杂志.2004,16(11);680-681
2Chen YN,Chen JC,Yin SC,et al.Effector mechanisms of norcantharidin - Induced mitotic arrest and apoptosis in human hepatoma cells.Int J Cancer,2002,100(2);158-65.
3Chen YN,Cheng CC,Chen JC,et al.Norcantharidin - induced apoptosis is via the extra cellular signal regulated kinase and c - Jun - NH2 - terminal kinase signaling pathways in human hepatoma HepG2 cells.Br J Pharmacol,2003; 140(3);461-470
4Li JL,Cai YC,Liu XH,et al.Norcantharidin inhibits DNA replication and induces apoptosis with the cleavage of initiation protein Cdc6 in HL - 60 cells.,2006; 17(3);307-14
5范祖,赵泽明,陈春球,等.去甲斑蝥素对荷瘤裸鼠胆囊癌移植瘤增殖和凋亡的体内干预实验.中华普通外科杂志,2005;20(7);438-441.
6孙震晓,赵天德,魏玉林,等.斑蝥素及去甲斑蝥素诱导人红白血病细胞凋亡的细胞学研究.解剖学报,2000,31(1);56-60
7赵聚山,陈璇.去甲斑蝥素对体外两种肿瘤细胞生长的影响.辽宁中医杂志,2004,31(2);157-158
8刘晓兰,陈家旭,刘燕,等.去甲斑蝥素诱导HL60细胞凋亡的研究.北京中医药大学学报,2000,23(4);35-37
9唐龙英,王昭梅,郑钦岳,等.去甲斑蝥素对卵巢癌3AO和AO细胞的体外抑制作用.第二军医大学学报,1999,20(2);97-99
10Modonnell TJ,Beham A,Sarkiss M,et al.Impotance of the Bcl - 2 family in cell death regulation,Exprientia,1996,52(10-11);1008-1112
11Yin C,Kundoscon C M,Kanemeyer S J,et al Bax suppresses of tumor genesis and stimulates apoptosis in vivo.Nature,1997,385(6617);737-640
12郑俊猛,王得坤,张柱林,等.Bel-2硫代反义寡核苷酸提高肺癌细胞对Fas介导细胞毒作用的敏感性.中华肿瘤杂志,2002,24;549.
13Manne U,Weiss HL,Grizzle WE.BCL-2 expressions associated with im- proved prognosis in patients with sistal colorectal adenocarcimomas.Int J Cancer,2000,89(5);423-30
14滕军放,冯建玉,卢宏,等.局灶性脑缺血再灌注后大鼠脑皮质Caspase-3,Bcl-2的表达.中华医学杂志,85(42);3012-3018
15Chakraborty C,Saha G,Sarkar B,et al.Caspase - 3 induced apoptosis in transgenic zebrafish.Biotechnol Lett.2006; 28(3);189-96.
1姚燕丹,林少芒.去甲斑蝥素的研究进展及其乳腺癌的关系.中国医药导刊,2005,7(1);58-598
2张泰松,程树仓,臧恒昌,等.去甲斑蝥素诱导细胞凋亡的研究进展.海峡药学,2005,17(3);125-127
3孙震晓,赵天德,魏玉林,等.去甲斑蝥素诱导人肝癌BEL-7402细胞凋亡的研究.解剖学报,1999,30(1);65-68
4赵聚山,陈璇.去甲斑蝥素对体外两种肿瘤细胞生长的影响.辽宁中医杂志,2004,31(2);157-158
5刘晓兰,陈家旭,刘燕,等.去甲斑蝥素诱导HL60细胞凋亡的研究.北京中医药大学学报,2000,23(4);35-37
6唐龙英,王昭梅,郑钦岳,等.去甲斑蝥素对卵巢癌3AO和AO细胞的体外抑制作用.第二军医大学学报,1999,20(2);97-99
7范跃祖,傅锦业,赵泽明,等.去甲斑蝥素对原发性胆囊癌GBE-SD细胞系增殖和凋亡的影响.上海医学,2003,26(增刊);1-4
8范跃祖,傅锦业,赵泽明,等.去甲斑蝥素对人原发性胆囊癌GBC-SD细胞系增殖及相关基因的干预效应.中华实验外科杂志,2004,21(5);554-556
9范跃祖,赵泽明,陈春球,等.去甲斑蝥素对人原发性荷瘤裸鼠胆囊癌GBC-SD移植瘤增殖和凋亡的体内干预实验.中华普通外科杂志,2005,20(7);438-441
10安巍巍,龚显峰,王敏伟,等.去甲斑蝥素诱导人黑色素瘤A375-S2细胞凋亡.中国医院药学杂志,2005,25(6);501-504
11张卫东,赵惠儒,阎影,等.斑蝥素诱导人肺癌A549细胞凋亡及其分子机制的研究.中华肿瘤杂志,2005,27(6);330-333
12赵泽明,范跃祖,傅锦业,等.去甲斑蝥素对荷瘤裸鼠胆囊癌移植瘤nm23、基质金属蛋白酶-2和基质金属蛋白酶组织抑制因子-2的影响..中华普通实验外科杂志,2005,22(3);307-311
13安巍巍,薛莲,王敏伟,等.去甲斑蝥素诱导小鼠肺纤维瘤L929细胞凋亡.中国癌症杂志,2005,15(1);22-25
14Huang S,Pettaway CA,Uehara H et al.Blockade of NF - kappaB activity in human prostate cancer cells is associated with suppression of angiogenesis,invasion,and metastasis[J].Oncogene,2001,20(31);4188-97
15Lin A,Karin M.NF - kappaB in cancer.a marked target[J].S emin Cancer Biol,2003,13(2);107-114
16何太平,莫丽儿,梁念慈.斑蝥素诱导高转移卵巢癌细胞HO-8910PM细胞凋亡的研究.中国药科大学学报,2005,36(2);164-167
17孙震晓,李家实.去甲基斑蝥素抗肿瘤研究热点.西北药学杂志,1998,13(5);227-228
18郑兰英,史春雷,贺延新.去甲斑蝥素治疗肝癌的研究.河北医学,2005,11(9);820-822
19周文丽,考军,范清玲.去甲斑蝥素片辅助治疗胃肠道癌肝转移30例临床观察.山东医药,2005,45(20);32-33
20郝素华.埃迪注射液联合化疗治疗晚期恶性肿瘤的疗效观察.肿瘤研究与临床,2003,15(3);202-203
21挛祖鹏,李晓东,马敏.去甲斑蝥素注射液联合氟尿嘧啶治疗晚期肝癌的临床研究.河北医药,2005,27(7);542-544
22吴光兴,杨志华,陈恩碧.化疗联合复方斑蝥素注射液治疗恶性淋巴瘤临床观察.中国医师杂志,2005,7(10);1425-1428
23王怀璋,王程虎,王瑞香.化疗联合埃迪注射液治疗晚期非小细胞肺癌疗效观察.中国治疗临床与康复,2004,11(4);358-359
24史健,魏速菊,单保恩.斑蝥酸钠注射液治疗癌性胸腔积液的临床观察.中国中西医结合杂志,2005,25(5);451-453
25郭艳霞,叶志雄.复方斑蝥汤治疗晚期鼻咽癌疗效分析.现代医院,2005,5(9);80-81
26陈双彪,陈祖红,苏腾良,等.水蛭斑蝥汤治疗良性前列腺增生症30例疗效观察.新中医,2006,38(1);44-45
27王新华.尤斯洛(斑蝥素)治疗尖锐湿疣30例报道.中国民康医学杂志.2004,16(11);680-681