【作者】 张锐；

【导师】 崔永安；

【作者基本信息】 扬州大学 ， 中西医结合临床， 2009， 硕士

【摘要】 1.目的:近年来,抗肿瘤血管生成已成为肿瘤治疗的基础和临床研究热点,是最有希望的肿瘤导向治疗靶标,与传统的肿瘤治疗相比,抗肿瘤血管生成治疗的最大特点在于它不但具有良好的特异性,而且克服了药物达到肿瘤组织的不足和肿瘤耐药性等问题。我国的中医药治疗与血管生成相关的病症有着悠久的历史,具有不同于西医的特殊性,并在这些病症的治疗上有极大的优势。因此,研究中药抗肿瘤血管生成,在阐明中医基础理论、临床治疗及中药研究中具有十分重要的意义。乌骨藤,又叫通光藤,为萝藦科牛奶菜属植物,主产于云南、贵州,据记载:该药始载于《滇南本草》,性味苦,微甘、凉,入肺胃膀胱经;具有止咳平喘、消炎镇痛、通乳利尿等功效,用于治疗慢性支气管炎。民间用乌骨藤治疗癌症获得良效,引起广大医药工作者的关注。本试验旨在研究并证明传统中药乌骨藤抗肿瘤活性,从血管内皮生长因子、碱性成纤维细胞生长因子、基质金属蛋白酶-2以及抑癌基因p53等角度探讨乌骨藤的抗肿瘤作用机制。2.方法:2.1 MTT法采用四氮唑盐(MTT)还原法测定不同浓度的乌骨藤注射液的体外对人肝癌细胞Bel-7402细胞增殖的影响,并根据吸光度(OD)值作图得到剂量-效应图谱。2.2 PI单染法检测细胞凋亡和周期采用流式细胞术检测不同浓度的乌骨藤注射液对人肝癌细胞Bel-7402凋亡和周期的影响。2.3 ELISA法利用ELISA试剂盒检测不同浓度的乌骨藤注射液对人肝癌细胞Bel-7402不同时间段表达VEGF的影响。2.4免疫细胞化学法采用细胞免疫化学SP染色法检测不同浓度的乌骨藤注射液作用于人肝癌细胞Bel-7402后bFGF和MMP-2蛋白的阳性表达率。2.5流式细胞术检测抑癌基因p53蛋白的表达采用流式细胞术,通过间接荧光标记法测定不同浓度的乌骨藤注射液作用于人肝癌细胞Bel-7402后p53蛋白的表达情况。p53蛋白荧光指数(Fluoresence Index,FI)表示定量的p53蛋白。绿荧光LOG收集的荧光强度峰值数转化成线性刻度,对照管作为阴性表达FI=1。FI计算公式如下:FI=检测管细胞平均荧光强度/对照管细胞平均荧光强度。如FI>1.0为阳性表达,FI≤1.0为阴性表达。3.结果:3.1不同浓度的乌骨藤注射液(10mg/ml、20mg/ml、40mg/ml)对人肝癌细胞Bel-7402均有抑制作用,且呈剂量时间依赖,经统计学分析有差异。3.2人肝癌细胞Bel-7402经不同浓度的乌骨藤注射液(20mg/ml、40mg/ml、80mg/ml)作用后,细胞凋亡率明显上升,且呈剂量依赖,经统计学分析有意义。然而,细胞周期变化不大,未证实细胞被阻滞于特定周期。3.3利用ELISA试剂盒检测不同浓度的乌骨藤注射液(10mg/ml、20mg/ml、40mg/ml)作用于人肝癌细胞Bel-7402后VEGF的表达情况,随着药物浓度的增加,VEGF表达逐渐减少,且与阴性对照组相比均有统计学差异。3.4免疫细胞化学的结果显示,不同浓度的乌骨藤注射液(10mg/ml、20mg/ml、40mg/ml)作用于人肝癌细胞Bel-7402后,与阴性对照组相比,bFGF和MMP-2的表达均有所减少,经统计学分析有差异。3.5流式细胞免疫荧光检测p53蛋白的结果显示,随着药物浓度的增加,荧光强度增加,p53峰总体向右偏移。根据FI计算公式计算出FI值,经不同浓度的乌骨藤注射液作用24h后,p53均阳性表达,且FI值随剂量的增加而增高。4.结论:乌骨藤注射液体外具有较强的抗肿瘤作用,一方面乌骨藤注射液可以下调肿瘤细胞VEGF、bFGF、MMP-2的表达,从而阻断了肿瘤的血管生成和转移侵袭,另一方面,乌骨藤注射液可以激活p53基因的表达诱导肿瘤细胞的凋亡,且p53的激活又可以抑制VEGF的表达,进一步阻断了肿瘤血管的生成。乌骨藤注射液可能是通过诱导肿瘤细胞凋亡和阻断肿瘤血管生成与转移侵袭发挥抗肿瘤作用的。更多还原

【Abstract】 1.Objectives:Recently, the anti-tumor angiogenesis tumor therapy, which is the most promising treatment of tumor targeted therapy, has become a basic and clinical research hot spots. Compared with the traditional treatment of cancer, the most prominent feature of anti-angiogenesis therapy is that it not only is well specific, but also overcomes the deficient of drug resistance and the difficulty of medicine reaching neoplastic tissue .The history of the treatment of the traditional Chinese medicine on angiogenesis is very long in time . With different specificity of Western medicine it shows a great grace. Therefore, research on anti-angiogenesis therapy in the area of the treatment of cancer by traditional Chinese medicine is very significant in the territories of elucidating the basic theory of traditional Chinese medicine, clinical treatment and research on Chinese medicine.Glaucescent Fissistigma Root, also known as Marsdenia tenacissima(Roxb.)Wight et Arn, which is bitter, cool and a little sweet,mainly grows in Yunnan and Guizhou province.According to records,it was first recorded in the "Materia Medica polyanthum" . It can stop coughing and gasping ,eliminate inflammation,relieve pain and has the effect of lactogenesis and diuresis.It was used to treat chronic bronchitis. People obtain fine effect in the process of using Glaucescent Fissistigma Root treating cancers. So it draws attention by many Chinese medicine practitioners. This test is to study and prove that traditional Chinese medicine Glaucescent Fissistigma Root can treat tumor.We investigate the mechanism of anti-tumor from the facts of vascular endothelial growth factor , basic fibroblast growth factor, MMP-2 and p53 .2.Methods:2.1 MTT methodApply MTT deoxidized method to survey the rate of Glaucescent Fissistigma Root inhibiting tumor cell in vitro. Hepatoma carcinoma cell strain Bel-7402 is effected by different density of Glaucescent Fissistigma Root.We get dosage-effectiveness atlas by the OD.2.2 PI single-staining methodApply flow cytometry to detect cell cycle and apoptosis cell population of Bel-7402 cells effected by different concentrations of Glaucescent Fissistigma Root.2.3 ELISA methodApply ELISA Kit to detect the expression of VEGF of Bel-7402 cells in different time effected by different concentrations of Glaucescent Fissistigma Root.2.4 Immunocytochemical methodApply Immunocytochemical method to detect the positive expression rate of b-FGF and MMP-2 protein of Bel-7402 cells effected by different concentrations of Glaucescent Fissistigma Root.2.5 Flow cytometry detect anti-oncogene-p53 proteinumApply flow cytometry to survey p53 proteinum transmutation expressed by Bel-7402 cells, through indirect fluorescent labelling method.P53 proteinum Fluoresence Index(FI) express quantitative p53 proteinum. Green fluoresence LOG collect fluorescence intensity peak amplitude number, which convert linearity scale. It take peak amplitude number of contrast control tube as negative expression FI=1. FI calculated formula as follow: FI= mean fluorecence intensity of detector tube cell / mean fluorecence intensity of detector tube cell contrast control tube. For instance FI>1.0 for masculine expression, FI≤1.0 for negative expression.3.Results:3.1 Four densities of Glaucescent Fissistigma Root(10,20,40mg/ml) have determinate depressant effect on Bel-7402 cells.And the effection is dose-time-dependent. It is significant through statistics analysising discrepancy(P<0.01).3.2 A marked increase in the apoptosis rate of human liver cancer cells Bel-7402 by different concentrations of Glaucescent Fissistigma Root(20,40,80mg/ml) appeared. It is significant through statistics analysising discrepancy.However, there is little change in the cell cycle, which did not confirm that cells were arrested at a specific cycle.3.3 Apply ELISA Kit to detect the expression of VEGF in human liver cancer cell Bel-7402 effected by different concentrations of Glaucescent Fissistigma Root(10,20,40mg/ml). With the increase in drug concentration, VEGF expression gradually reduced.Compared with the negative control group,it is significant.3.4 The result of immunocytochemistry showed that compared with the negative control group,the expression of bFGF and MMP-2 of Bel-7402 cells effected by different concentrations of Glaucescent Fissistigma Root(10,20,40mg/ml) has been reduced.3.5 Utilizing flow cytometry immunofluorescence technic detect p53 gene product, along with the accrescence of medicine density, p53 peak offset rightward overall. According to FI formula calculation, it could calculate FI numerical value. The tumor cells are effected by different concentrations of Glaucescent Fissistigma Root(10,20,40mg/ml) for 24 hours, p53 proteinum in them all show masccline expression,and the FI numerical value increase along with the accrescence of dosage.4.Conclusion: Glaucescent Fissistigma Root has a strong anti-tumor effects in vitro.It can inhibit VEGF, b-FGF, MMP-2 and other substances in the expression of angiogenesis. The mechanism was probably related to the activation of tumor suppressor gene p53 and inhibition of other angiogenic factors, inhibiting tumor cell and endothelial cell proliferation and inducing apoptosis of tumor cells to block tumor angiogenesis.更多还原