【作者】 李海艳；

【导师】 凌昌全；

【作者基本信息】 第二军医大学 ， 中西医结合临床， 2011， 硕士

【摘要】 研究背景肝癌是世界第五大癌症,全球每年50万～100万新发病例和近100万死亡病例,至今还未有特别有效的治疗方法,寻找更好治疗方法成为肝癌治疗面临的重大问题。中医药对肿瘤的治疗有着悠远的历史,在肝癌预防、治疗以及防止复发转移等方面有着独特的优势。然而,由于许多草药的化学成分和其确切的作用机制仍然不明确,阻碍了其进一步研究和被广泛认可。目前,中药有效成分的分离以及药理作用研究是成为研究中药治疗肿瘤的一个重要途径。中药猫人参为对萼猕猴桃的根,是江浙地区临床常用的抗肿瘤药,具有抗肿瘤及抗炎作用,被运用于肝癌、肺癌和骨髓瘤的治疗中。对猫人参有效成分的研究发现该植物中含有一系列的化合物,其中一种三萜类化合物为科罗索酸。科罗索酸作为防治肥胖症和Ⅱ型糖尿病新药,已进入美国FDA的Ⅲ期临床药效学评价。有报道指出科罗索酸对一些人类肿瘤细胞株具有某些细胞毒作用,也有研究发现科罗索酸能够抑制肿瘤细胞存活率、诱导肿瘤细胞凋亡。本课题在前期相关工作的基础上,进一步观察科罗索酸对肝癌体内、体外生长的抑制作用,并初步探讨其作用机制。研究目的1体外观察科罗索酸作用于肝癌细胞株SMMC-7721后细胞的生长状况,观察细胞形态学方面发生的变化。2明确科罗索酸对SMMC-7721的凋亡诱导作用。3了解线粒体膜电位变化及线粒体对细胞色素C的释放情况。4明确科罗索酸对凋亡蛋白Bax和抗凋亡蛋白Bcl-2的表达的影响。5观察科罗索酸对裸鼠肝癌皮下移植瘤的生长抑制作用,了解科罗索酸对SMMC-7721细胞体内生长的影响。研究方法第一部分科罗索酸对肝癌细胞SMMC-7721体外实验作用观察1细胞培养,科罗索酸处理SMMC-7721细胞后,在不同时间点用相差显微镜观察细胞形态学的改变。2通过四氮唑盐还原法(MTT法)检测不同浓度科罗索酸对SMMC-7721细胞增殖的影响,初步确定科罗索酸发挥增殖抑制效果的浓度范围。3采用AnnexinV/PI凋亡试剂盒通过FCM分析检测科罗索酸作用SMMC-7721细胞后的细胞凋亡情况,明确科罗索酸对肝癌细胞SMMC-7721的凋亡诱导作用。4采用Mitocapture试剂盒,通过观察线粒体染色情况,明确科罗索酸处理SMMC-7721细胞后细胞线粒体膜电位的改变。5基于对细胞凋亡诱导的确定性判断,通过制备胞质和线粒体中的蛋白,采用Western blot法检测SMMC-7721细胞线粒体释放细胞色素C的情况。6运用Western blot法获得凋亡蛋白Bax和抗凋亡蛋白Bcl-2的表达,了解科罗索酸作用SMMC-7721后Bax/Bcl-2的比率变化情况。第二部分科罗索酸对肝癌生长抑制作用的体内实验研究构造裸鼠皮下移植瘤模型,随机分组给药10次后,剥取瘤块称重,计算科罗索酸对SMMC-7721细胞的体内肿瘤生长抑制率。研究结果第一部分科罗索酸对肝癌细胞SMMC-7721体外实验作用观察1细胞形态变化相差显微镜下观察发现CRA能够影响细胞形态和贴壁情况。35μM的CRA可以使细胞发生显著的形态学变化,6小时后细胞开始收缩,12小时后培养液中出现了漂浮细胞,18小时后大多数细胞已经失去了原来扁平、多边形的形态。2 MTT结果科罗索酸对SMMC-7721细胞具有增殖抑制作用,25μg/ml科罗索酸作用48小时抑制率为31.64%。3流式细胞仪检测细胞凋亡结果35μM科罗索酸作用24h、36h后,SMMC-7721细胞凋亡比例分别为60.5%、70.6%。4线粒体膜电位改变与对照组相比,科罗索酸作用18小时后,细胞线粒体膜电位明显耗散。5蛋白印迹法分析结果随着药物作用时间的延长,科罗索酸可以使细胞色素C从线粒体内释放入胞质中。6科罗索酸处理SMMC-7721细胞后,随着时间的推移Bax的表达增加,而抗凋亡蛋白Bcl-2的表达水平保持不变,表明科罗索酸可以通过上调Bax/Bcl-2的比率诱导细胞凋亡。第二部分科罗索酸对肝癌生长抑制作用的体内实验研究科罗索酸抑制肝癌体内生长的作用高剂量用药组的抑瘤率为58.7%,表明浓度为50mg/kg的科罗索酸可在体内抑制肿瘤生长。研究结论1科罗索酸能够抑制SMMC-7721细胞增殖,并产生凋亡特征性改变。2科罗索酸能够使SMMC-7721细胞线粒体膜电位耗散,细胞色素C从线粒体内释放入胞质中。3科罗索酸可以通过上调Bax/Bcl-2的比率诱导SMMC-7721细胞凋亡。4浓度为50mg/kg的科罗索酸能够抑制SMMC-7721细胞裸鼠移植瘤增长。更多还原

【Abstract】 IntroductionHuman hepatocellular carcinoma (HCC), with 500,000-1,000,000 new cases and causing approximately 1,000,000 deaths annually, is the fifith most common malignancy in the world. Since there has not yet been a particularly effective treatment, seeking for better treatment has become the major problem facing us. Traditional Chinese Medicine (TCM) with distant history in cancer treatment has its unique advantages in liver cancer prevention, treatment and prevention of recurrence and metastasis. However, the chemical compositions of many herbals and their exact mechanisms remain unclear, which has hindered their further study and being widely recognized. Currently, the separation of effective components of Chinese medicine and researches on the pharmacological effects is becoming an important way to study how Chinese herbal medicine is treating cancer.Traditional Chinese Medicine―mao ren shen‖, the root of Actinidia valvata Dunn, is commonly used in clinical cancer treatment in Jiangsu and Zhejiang provinces. With anti-tumor and anti-inflammatory effects, it was applied to treatment of liver cancer, lung cancer and myeloma. After study on the components of―mao ren shen‖, corosolic acid (CRA), one kind of triterpenoids, was found as one of its effective components. As medicine for prevention and treatment of obesity and typeⅡdiabetes, CRA has entered the PhaseⅢclinical efficacy evaluation of U.S. FDA. Researches show that CRA has cytotoxicity effect on many human tumor cell lines. It was also found to inhibit tumor cell viability and induce tumor cell apoptosis. On the base of previous related work, this subject is to further study the growth inhibitory effect of CRA on liver cancer both in vivo and in vitro, and preliminarily discussed part of its probably relevant mechanisms.Objectives1. To study the effects of CRA on the proliferation of SMMC-7721 cells in vitro, and observe the correspondent morphological variations.2. To explore the apoptosis effect of CRA on SMMC-7721 cells.3. To evaluate the mitochondrial membrane potential changes and the expression of cytochrome C both in mitochondria and cytoplasm.4. To explore the influence of CRA on the expression of apoptosis induce protein Bax and the anti-apoptosis protein Bcl-2.5. To observe the inhibitory action of CRA on nude mouse liver subcutaneous transplanted tumor in vivo.MethodsPART ONE: Experiments of CRA on SMMC-7721 in vitro1. To observe the morphological changes of SMMC-7721 cells after being infected with CRA.2. MTT was applied to detect the proliferation inhibitory effect of CRA on SMMC-7721 at different concentrations, primarily identify the effective concentration range.3. AnnexinV/PI Apoptosis Kit and FCM were applied to detect the apoptosis inducing effect of CRA on SMMC-7721.4. Mitocapture Kit was used to detect the potential changes of mitochondrial membrane in SMMC-7721 cells after being treated wit CRA.5. Western blotting was used to analyze the expression of cytochrome C both in cytoplasm and in mitochondria.6. To detect the changes of Bax/Bcl-2 ratio in SMMC-7721 cells after being treated with CRA through applying Western blotting to observe the expression of Bax and Bcl-2.PART TWO: Researches on the growth inhibitory effect of CRA on liver cancerEstablishing nude mouse liver subcutaneous transplanted tumor model,random grouping,and regular administration.Weighing mice in each group before and after the experiment,measuring cubic capacity and weighing the weight of each tumor.ResultsPART ONE: Experiments of CRA on SMMC-7721 in vitro1. Changes in cell morphology: CRA can influence cell morphology and adherent situation being observed under phase contrast microscope. At 35μM CRA could induce an obvious cell morphological change, after 6 hours some cells start to contract, after 12 hours floating cells appeared in the culture medium, after 18 hours most cells have lost the original flat, polygonal shape.2. MTT results: CRA performs a proliferation inhibitory effect on SMMC-7721, the inhibitory rate was 31.64% after being treated 48 hours with 25μg/ml CRA.3. FCM results: the apoptosis rates of SMMC-7721 after being treated with 35μM CRA for 24 hours and 36 hours are 60.5% and 70.6% separately.4. Changes in mitochondrial membrane potential: compared with the control group, the mitochondrial membrane potential obviously dissipated after being treated with CRA for 18 hours.5. Western blot analysis: cytochrome C was gradually released form mitochondrial into cytosol with treatment of CRA.6. With treatment of CRA on SMMC-7721, the expression of Bax proteins gradually increased, while Bcl-2 remain unchanged, which indicates that CRA could upgrade the Bax/Bcl-2 ratio to induce cell apoptosis.PART TWO: Researches on the growth inhibitory effect of CRA on liver cancerThe tumor inhibitory rate of the high dose group is 58.7%,which indicate at the concentration of 50mg/kg CRA could inhibit the growth of liver tumor. Conclusions1. CRA could inhibit the proliferation of SMMC-7721 cells and produce characteristic changes of apoptosis.2. CRA could cause the mitochondrial membrane potential dissipated and cytochrome C released from mitochondrial into cytosol in SMMC-7721 cells.3. CRA could upgrade the Bax/Bcl-2 ratio to induce SMMC-7721 cell apoptosis.4. CRA could inhibit the growth of nude mouse liver subcutaneous transplanted tumor at the concentration of 50mg/kg.更多还原