【作者】 薛金晶；

【导师】 戴显伟；

【作者基本信息】 中国医科大学 ， 外科学， 2006， 博士

【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IC₅₀。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;G₂+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 cm³,P＜0.01）,抑瘤率增高（31.69% vs.0,P＜0.05）;种植瘤细胞周期中G₂+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。更多还原

【Abstract】 ObjectPancreatic 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 Method1.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 IC₅₀.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.ResultsNCTD 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 cm³,P＜0.01）,cancer inhibition ratio increased（31.69% vs. 0,P＜0.05）; G₂+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.Conclusion1.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.更多还原