【作者】 张彦民；

【导师】 贺浪冲；

【作者基本信息】 西安交通大学 ， 药物分析学， 2008， 博士

【摘要】 塔斯品碱是一种阿朴啡类生物碱,是从被称为lion’s tail（或leontik）的小檗科植物Leontice ewersmannii中首次分离获得。塔斯品碱具有多种药理学活性,如抑菌作用、抗炎作用、促进伤口愈合作用、细胞毒作用、抗病毒作用等,但至今未发现塔斯品碱有抗血管生成作用的相关报道。本课题以塔斯品碱为研究对象,应用药理学、组织化学、细胞生物学以及分子生物学等方法,对其抗血管生成作用和相关作用机制进行研究。实验内容主要包括以下几个方面:1.塔斯品碱对小鼠S₁₈₀肉瘤抑制作用建立小鼠S₁₈₀肉瘤模型,塔斯品碱分组给药后计算抑瘤率和脾脏指数。将称重后瘤体进行4%多聚甲醛固定,进行免疫组织化学实验,考察塔斯品碱对小鼠S₁₈₀肉瘤分泌的VEGF、bFGF、Bax、Bcl-2和CD34的影响,计算各组微血管密度。结果表明塔斯品碱可抑制小鼠S₁₈₀肉瘤的生长,与阴性对照组比较,3个剂量组瘤重明显低于对照组,抑瘤率有显著性差异（P<0.05）。根据免疫组化实验结果,塔斯品碱组3个剂量组CD34、VEGF、bFGF、和Bcl-2表达总量显著低于模型对照组,Bax表达总量显著高于模型对照组,说明塔斯品碱能够抑制小鼠S₁₈₀肿瘤分泌的VEGF、bFGF、Bcl-2的表达,上调Bax的表达,降低微血管密度。2.塔斯品碱抑制鸡胚尿囊膜血管生成作用研究建立了鸡胚尿囊膜实验模型和A549人肺腺癌移植瘤模型,考察塔斯品碱对鸡胚尿囊膜（CAM）血管生成,对A549人肺腺癌移植瘤的生长抑制作用和血管生成的影响。结果表明塔斯品碱在0.25-4.0μg /egg剂量范围内可剂量依赖性地抑制CAM血管生成,降低CAM血管数量和血管面积;在该剂量范围内,塔斯品碱能够抑制A549人肺腺癌移植瘤的生长,降低瘤体内微血管密度和VEGF表达。3.塔斯品碱对血管内皮细胞和肿瘤细胞生长抑制作用研究体外培养人脐静脉血管内皮细胞HUVEC,人肺腺癌细胞LTEP-a-2,人非小型肺腺癌细胞A549,人肺腺癌细胞SPC-A-1,人大细胞肺癌NCI-H460,人外阴上皮癌A431细胞,加入不同浓度的塔斯品碱,MTT法观察塔斯品碱对各细胞活力的影响,细胞计数法考察塔斯品碱对HUVEC和A549细胞生长曲线和倍增时间的影响,流式细胞术测定塔斯品碱对HUVEC和A549细胞周期的影响。结果表明:塔斯品碱对各细胞活力均有较好的抑制作用（A431除外）,能够抑制HUVEC和A549细胞增殖并延长其倍增时间。同时,塔斯品碱能够转化HUVEC和A549细胞周期,使细胞停滞在S期。4.塔斯品碱对HUVEC和A549细胞凋亡作用研究体外培养HUVEC和A549细胞,加入不同浓度的塔斯品碱,流式细胞术测定塔斯品碱对HUVEC和A549细胞凋亡的影响,HE染色法和透射电镜观察细胞形态、结构变化,免疫组织化学法观察塔斯品碱对HUVEC和A549细胞Bax和bcl-2蛋白表达的影响。结果表明塔斯品碱在0.06-0.66μg/mL剂量范围内能够诱导HUVEC和A549细胞凋亡,随着药物浓度和作用时间的增加,凋亡率逐渐增加,与对照组相比有显著性差异（p<0.05）。细胞经塔斯品碱作用后依次出现形状不规则,细胞收缩变圆变小,与周围细胞失去联系,胞浆浓缩,细胞核内染色质凝聚,核仁萎缩和凋亡小体等。免疫组化实验表明塔斯品碱能够降低HUVEC和A549细胞Bcl-2表达,升高Bax表达,具有较好的剂量依赖性,但是与对照组比较只有0.54μg/mL和0.66μg/mL具有显著性差异,说明塔斯品碱在低剂量时能抑制细胞增殖,在高剂量时诱导细胞凋亡。5.塔斯品碱抑制血管生成的分子机制研究建立体外细胞划伤模型,观察塔斯品碱对HUVEC和A549迁移的影响。结果表明:不同浓度（0.06-0.44μg/mL）塔斯品碱作用于HUVEC和A549细胞后,可抑制HUVEC和A549细胞的迁移,随着药物浓度的增加,迁移速度呈降低趋势,与对照组相比有显著性差异（p<0.05）。采用酶联免疫吸附实验（ELISA）法和Western blot测定塔斯品碱对HUVEC和A549细胞分泌的VEGF和bFGF的影响。结果表明:不同浓度（0.06-0.44μg/mL）塔斯品碱作用于HUVEC和A549细胞后,可抑制HUVEC和A549细胞分泌VEGF和bFGF,抑制作用具有时间和剂量依赖性。采用反转录聚合酶链式反应（RT-PCR）考察塔斯品碱对A549细胞的VEGF mRNA表达和HUVEC细胞的VEGF、Flt-1和Flk-1/KDR mRNA表达的影响。结果表明:不同浓度（0.06-0.44μg/mL）塔斯品碱作用于HUVEC和A549细胞后,可抑制A549细胞的VEGF mRNA表达以及HUVEC的VEGF和Flk-1/KDR mRNA表达,与对照组相比有显著性差异（p<0.05）,但不能显著性地抑制Flt-1 mRNA的表达。综上所述,本课题对塔斯品碱的抗血管生成作用及其作用机制进行了研究。研究发现:（1）塔斯品碱能够抑制小鼠S180肉瘤的生长和鸡胚尿囊膜A549人肺腺癌移植瘤的生长,降低瘤体微血管密度,抑制S180肉瘤和A549移植瘤的血管生成。（2）塔斯品碱对HUVEC细胞和A549细胞增殖、迁移、倍增时间均有较好的抑制作用,能够转换HUVEC细胞和A549细胞周期,诱导HUVEC细胞和A549细胞凋亡。（3）塔斯品碱抑制血管生成作用机制是通过抑制血管内皮细胞和肿瘤细胞分泌的VEGF和bFGF的蛋白,下调A549细胞VEGF mRNA表达以及HUVEC的VEGF和Flk-1/KDR mRNA表达,从而抑制血管内皮细胞增殖、降低形成血管的能力,最终起到抗血管生成的作用。本研究为将塔斯品碱作为血管生成抑制剂的候选化合物研究开发奠定了基础。更多还原

【Abstract】 Taspine was a kind of aporphines alkaloid and was isolated from Leontice ewersmannii named lion’s tail (or leontik). Taspine had many pharmacological activities, such as bacteriostatic action, anti-inflammatory, anti-ulcer, promoting wound healing, cytotoxic activity and inhibiting viral DNA polymerase. There were not reports published about anti-angiogenesis of taspine. In this study, we investigated taspine’s anti-angiogenesis and the mechanism of action by pharmacology, histochemistry, cell biology and cell biology. The contents were as follows:1. Inhibitory effect of taspine on mouse S180 sarcoma and tumor angiogenesisThe mouse S180 sarcoma model was established. The rate of inhibiting tumor growth and spleen index were calculated after treatment by taspine. The weighed tumors were fixed by 4% paraformaldehyde. The immunohistochemistry was used to investigate the protein expression of VEGF、bFGF、Bax、Bcl-2 and CD34. Effects of Taspine on the tumor angiogenesis and MVD were estimated by immunohistochemistry. The results showed taspine could inhibit mouse S180 sarcoma growth in a good dose-dependent manner. The inhibition rates by taspine on tumor at low, middle and high concentrations were significant compared with the negative control group (P<0.05). The microvessel density and protein expression of the VEGF、bFGF、Bcl-2 and Bax in the tumor were decreased compared with the negative control. The ratio of Bax to Bcl-2 was increased. The study indicated taspine had anti-tumor action, and the mechanism may involve decreasing the expression of the VEGF, bFGF, Bcl-2/Bax and MVD and inducing the vascular endothelial cell apoptosis.2. Effect on CAM angiogenesis action by taspineThe CAM model and CAM tumor transplantation of A549 model were established. The taspine’s effects on CAM angiogenesis, growth inhibition and angiogenesis of the tumor transplantation were demonstrated by the models. The results indicated taspine could inhibit CAM angiogenesis in a dose-dependent manner, and decrease the blood vessel number and area within the 0.25-4.0μg/egg. At the same time, taspine could inhibit growth of tumor transplantation, decrease MVD and VEGF protein expression. 3. Study of growth inhibition on HUVEC and tumor cell by taspineThe HUVEC, LTEP-a-2, A549, SPC-A-1, NCI-H460 and A431 cells were incubated in vitro. The cells were treated with taspine at different concentrations. The cell viability was examined by MTT assay, the HUVEC and A549 cell growth curve and double time were determined by cell counting and the HUVEC and A549 cell cycle was investigated by flow cytometry. The results showed taspine could inhibit the cell viability (except A431 cell), HUVEC and A549 cell proliferation, prolong the double time of cells, and change the cell cycle. The cell cycle was significantly stopped at the S phase.4. Study of inducing apoptosis by taspineThe HUVEC and A549 cells were incubated in vitro. The cells were treated with taspine at different concentrations (0.06-0.66μg/mL). The apoptosis was examined by flow cytometry. The cell morphology and ultramicrostructure were observed by HE dyeing and transmission electron microscope. The Bax and Bcl-2 expression was determined by the immunohistochemical method. The results showed taspine could induce apoptosis of HUVEC and A549 cell. The apoptosis rate was increased with the increased concentration of taspine. There was a significant difference between the treatment group and the control group (p<0.05). After the treatment of taspine, the cells showed irregular appearance, volume reduction, endochylema concentration, nuclear chromatin of maldistribution, nuclear fragmentation and apoptosis bodies with increased concentrations. The immunohistochemistry result indicated taspine decreased Bcl-2 expression while increased Bax expression. Compared with the control, there was a significant difference at the 0.54μg/mL for HUVEC and 0.66μg/mL for A549 cell. This showed taspine could inhibit cell proliferation at low concentrations and induce apoptosis at high concentrations.5. The molecular mechanism of anti-angiogenesisIn order to evaluate the effect on migration of HUVEC and A549 by taspine, the cell scrape model was established. The experimental result showed taspine could inhibit the HUVEC and A549 migration within the concentration range of 0.06-0.44μg/mL. The migration speed decreased significantly (p<0.05) with the increased doses compared with the negative control group.The secreted VEGF and bFGF of HUVEC and A549 were examined by enzyme-linked immunosorbent assay and western blot assay. The experimental data indicated taspine could decrease the protein level secreted by HUVEC and A549 within the concentration range of 0.06-0.44μg/mL.In addition, the VEGF mRNA of A549 cell and VEGF、Flt-1 and Flk-1/KDR mRNA of HUVEC were evaluated by reverse transcriptase polymerase chain reaction. The results showed VEGF mRNA of A549 and the VEGF and Flk-1 mRNA of HUVEC were down-regulated significantly (p<0.05) by taspine compared with the control.In summary, the present study suggests that taspine can inhibit the growth of mouse S180 sarcoma and CAM tumor transplantation of A549, decrease their MVD and inhibit their angiogenesis. Taspine has a good inhibitiory effect on the proliferation, migration and double time of HUVEC and A549 cell. Taspine can change cell cycle and induce apoptosis of HUVEC and A549 cell. The mechanism of angiogenesis action is that taspine inhibits VEGF and bFGF protein expression of HUVEC and A549, and down-regulates VEGF mRNA of A549 cell and VEGF、Flt-1 and Flk-1/KDR mRNA of HUVEC. As a result, taspine inhibits the ability of HUVEC proliferation and migration to vascularize, and thus inhibits the angiogenesis. The results from our study make it possible that taspine acts as a potential angiogenesis inhibitor.更多还原