【作者】 陈永杰；

【导师】 史仁杰；

【作者基本信息】 南京中医药大学 ， 中医外科学， 2012， 博士

【摘要】 研究目的：滕梨根是临床常用的治疗大肠肿瘤的中草药,其抗肿瘤作用已经为药理学及分子学研究证实,但针对目前对大肠癌抗癌抑癌的作用机理、治疗靶点等缺乏研究。为了明确滕梨根提取物对大肠癌细胞凋亡过程中凋捌亡相关丛因Bcl-2、Bax、Caspase-3的影响及增殖抑制作用的效应,据此了解藤梨根提取物对大肠癌细胞的抗癌抑瘤的部分作用机制与量效关系,开展本研究。研究方法：一、体外实验：采用醇提取工艺提取藤梨根抗癌有效活性成分(简称EERAC),分别配制成10μg/ml、40μg/ml、160Dg/ml、320μg/ml的制剂,将96孔LoVo培养板分成4个组,分别滴加上述不同浓度的EERAC。然后：(1)采用倒置显微镜观察不同浓度EERAC对培养孔内的LoVo细胞形态结构变化的影响,并采用AO／EB和Hoechst33258二种荧光染色观察细胞核内染色质改变、核固缩、细胞膜皱缩,凋亡小体等；(2)采用MTT法检测不同浓度EERAC对大肠癌LoVo细胞的抑制作用；(3)采用琼脂糖凝胶电泳法观察不同浓度EERAC在体外实验组中对LoVo细胞的DNAladder的形成情况；(4)采用流式细胞仪测定不同浓度EERAC对LoVo细胞周期的影响；(5)采用免疫化学法(IHC)测定不同浓度EERAC对LoVo细胞也凋亡相关基因Bcl-2,Bax,Caspase-3的蛋白表达变化；、体内实验：建立大肠癌细胞HT-29荷瘤裸鼠模型,观察：(1)不同剂量(5、10、20mg/kg) EERAC荷瘤裸鼠体重、种植瘤生长抑制作用、对血常规、肝功能和肾功能、免疫器官的影响；(2)并采用LDH释放法测定不同剂量EERAC对裸鼠NK细胞的活性；(3)采用IHC法测定不同剂量EERAC在HT-29荷瘤祼鼠体内凋亡相关基因Bcl-2,Bax,Caspase-3的蛋白表达。研究结果:?、体外实验(1)与空白组比较,在不同浓度的EERAC组,具LoVo细胞密度减低,增殖变慢；细胞逐渐变大、变圆细胞间接触变松,胞浆中颗粒增多,空泡化,脱壁,细胞周围碎片增多,荧光染色观察可见细胞凋亡的特征学形态改变出现。(2)MTT法测定显示,EERAC对LoVo细胞的最佳作用时间为72h,最大抑制率为79.48%；(3)琼脂糖凝胶电泳法检测显示随着实验药物浓度的增高,DNA断裂明显增多,DNA ladder在320μ∥mL组表达最强。(4)滴加EERAC处理24h后,LoVo细胞的Go／G1期细胞比例增高,S期和G2／M期的细胞减少,凋亡率为6.8%、13.1%、18.2%和26.3%,与空白组比较,差异有统计学意义(P<0.05)。(5) EERAC作用LoVo细胞24后,Bcl-2表达明显减弱,Bax、Caspase-3表达水平明显增高,Bcl-2／Bax比值下降,其效应与浓度相关。二、体内实验：(1)不同剂量的EERAC对HT-29移植瘤均有明显的抑制作用,各剂量组的抑瘤率为9.12%,20.13%,37.81%,与药物浓度呈正比；不同剂量的EERAC对荷瘤裸鼠的WBC、脾脏指数等免疫指标均有所增加,对肝肾功能无明显影响。(2)不同剂量的EERAC均可使NK细胞活性度增加。(3)不同剂量的EERAC作用后,LoVo细胞的Bcl-2表达减弱,Bax、Caspase-3表达水平增高,Bcl-2／Bax比值下降,其作用也是呈浓度相关性。研究结论：1、EERAC对大肠癌LoVo细胞具有明显抑制增殖作用,浓度越高、作用时间越长其抑制增殖作用越明显；EERAC能阴滞癌细胞的在有丝分裂周期的S期增殖,能阻断癌细胞DNA物质的合成,高浓度时阻断作用更明显；2、体内外实验研究均证实, EERAC对大肠癌细胞或种植瘤具有抑制增殖炳和诱导凋亡的作用,且其作用强度与浓度或剂量呈正相关。其作用机制在于降低Bcl-2的表达,增强Bax、Caspase-3表达水平,下调Bcl-2／Bax比值。3、EERAC对机体的体重、血常规、肝肾功能无明显影响,同时对机体的免疫功能有一定的增加作用。EERAC其抑癌作用虽差于5-Fu,但其对作用的HT-29荷瘤裸鼠的WBC、脾脏指数等体内免疫指标有提高作用；表明EERAC的抑瘤的同时,有助于提高机体免疫能力,且无5-Fu的明显毒副作用。4、在体内实验中,EERAC对大肠癌细胞的抑制作用与剂量有一定的相关性,高剂量组的疗效较好,且无明显毒副作用。按相关换算法则计算,临床治疗大肠癌时,可以适当提高藤梨根在单张处方中的用量以提高抗癌疗效,初步建议较理想的抗癌剂量为30—35g。更多还原

【Abstract】 Objective:Actinidiae is commonly used in clinical treatment of colorectal cancer of Chinese herbal medicine, its anti-tumor effect of pharmacology and molecular studies confirmed a lack of research, but for the colorectal cancer tumor suppressor mechanism, and therapeutic targets. In order to clarify Actinidiae extracts (Ethanol Extract from Radix of Actinidia Chinensis, EERAC)on apoptosis-related genes in colon cancer cells during apoptosis Bcl-2and Bax in the impact of caspase-3and proliferation inhibition effect, whereby understand Actinidiae extract some of the mechanisms of colorectal cancer anti-cancer dose-effect relationship, to carry out this study.Methods:1. In vitro experiment:the patent alcohol extraction process to extract Actinidiae anticancer effective active ingredient (EERAC), respectively dubbed10μg/ml,40μg/ml,160μg/ml,and320μg/ml concentration, and LoVo were cultured in vitro,the different concentrations of EERAC, and the following experiment (1) using the inverted microscope to observe the different EERAC on LoVo cell morphology changes, and chromatin changes within the two kinds of fluorescence staining nuclei by AO/EB and Hoechst33258nuclear condensation, membrane shrinkage, apoptotic bodies;(2) using different concentrations of MTT assay EERAC inhibition of LoVo cells;(3) using agarose gel electrophoresis observation EERAC in various in vitro experimental groupin the formation DNAladder LoVo cells;(4) using flow cytometry of determination EERAC on LoVo cell cycle;(5) immunochemistry (IHC) determination of the related genes Bel-different concentrations EERAC on apoptosis of LoVo cells2, Bax, Caspase-3expression levels;2In vivo experiments:the establishment of colorectal cancer cells HT-29tumor-bearing mouse model, observed:(1) different doses (5mg/kg,10mg/kg,20mg/kg) EERAC weight of tumor-bearing mice implanted tumor growth inhibitory effect on the blood, liver and renal function, immune organs;(2) and LDH release method deeterminate EERAC tumor-bearing mouse NK cell activity;(3) using the HT-IHC method determination of EERAC processing29tumor-bearing mice, apoptosis related genes Bcl-2, of Bax and Caspase-3expression. Results:Vitro experiment:(1) Compared with the control group, in different concentrations of Actinidiae extract group, LoVo cell density to reduce the proliferation slows down; cells gradually become larger, rounded, cells indirect thixotropic loose, increased particles in the cytoplasm, empty bubble, off the wall, surrounded by cell debris increased, the fluorescence staining characteristics of visible apoptosis school morphological changes appear.(2) Determination by MTT method, the extract Actinidiae time of LoVo cells for72h, and maximal inhibition rate of79.48%;(3) By agarose gel electrophoresis detection of DNA fragmentation increased significantly with the experimental drug concentration increased, the DNA ladder is the strongest expression in320μg/mL group.(4) Dropping EERAC24h increased the proportion of cells in G1/G1phase of LoVo cells, the S and G2/M phase cells reduced the apoptotic rate was6.8%,13.1%,18.2%and26.3%, and the blank group, the difference was statistically significant (P <0.05).(5)After the EERAC role in LoVo cells after24Bcl-2expression was significantly weakened, Bax and Caspase-3expression levels were significantly higher, of Bcl-2/Bax ratio decreased, the effects of concentration.In vivo experiments:(1) EERAC transplanted HT-29tumors significantly inhibited tumor inhibition rate of each dose group was9.12%,20.13%,37.81%, a positive correlation with drug concentration; different doses of EERAC tumor-bearing mice the WBC, spleen index, immune parameters were increased, no significant effect on the liver and kidney function.(2) Different doses of EERAC can NK. cell activity increased.(3) the role of EERAC LoVo cells Bel-2expression was decreased, increased levels of Bax. caspase-3expression of Bcl-2/Bax ratio decreased, the role of a concentration.Conclusion:1.Actinidiae extract inhibit the proliferation of LoVo cells, the inhibition concentration (dose) and time; its mechanism of action and promote apoptosis, mainly LoVo cells, Bcl-2expression by inhibitingenhanced Bax and Caspase-3expression levels, reduction in the Bcl-2/Bax ratio, and its role with the concentration.2.EERAC on colorectal cancer cells HT-29tumor-bearing mice with inhibition of tumor growth and induction of cancer cell apoptosis, mainly through the inhibition of tumor-bearing mice, Bcl-2expression, enhanced Bax and Caspase-3expression level, the Bcl-2/Bax the ratio down, and its role with the concentration, suggesting that its mechanism of action and promote apoptosis. Actinidiae extract can enhance the immune function of tumor-bearing mice, no significant liver and kidney toxicity.3.EERAC had no significant effect on weight of body, blood, liver and kidney function, and can increase the body’s immune function. Inhibitory effect of EERAC was worse than5-FU, but it can enhance WBC, spleen index and immune in HT-29nude. EERAC can inhibit tumor and help the body to improve the immunity without obvious side effects of5-Fu.4. In vivo, the inhibition of colorectal cancer, EERAC dose certain the efficacy of high dose group, and no apparent side effects. Related conversion rules, the clinical treatment of colorectal cancer can be an appropriate increase in the amount of Actinidiae in a single prescription to improve the efficacy of cancer, preliminary proposals for the ideal anti-cancer dosage is30-35g.更多还原