【作者】 王春阳；

【导师】 韩瑞发；

【作者基本信息】 天津医科大学 ， 外科学， 2010， 博士

【摘要】 第一部分化学遗传学筛选抑制斑马鱼血管生成小分子化合物目的：筛选具有抑制斑马鱼血管生成功能的小分子化合物。方法：1.Tg(flk1:EGFP)斑马鱼胚胎搜集。采用转基因斑马鱼品系Tg(flk1:EGFP),其内皮细胞表达flk1启动子控制的增强型绿色荧光蛋白(enhanced green fluorescent protein, EGFP),因而完整的脉管系统可在荧光显微镜下直接观察。斑马鱼胚胎由Tg(flkl:EGFP)纯合子或杂合子与野生型配对自然交配获得。每次交配5-6对,平均每对可获得100-150个胚胎。胚胎在28℃培养基中孵育。2.药物筛选。用于筛选的化合物库为MicroSource公司Spectrum Collection化合物库。斑马鱼胚胎置于96孔板内,在发育到受精后12-14小时阶段胚胎外膜被1.5%蛋白酶消化去除。小分子化合物溶解在0.5%DMSO中,然后分别以0.1μM、1μM和5μM的浓度添加在胚胎培养基中。作用24、48、72小时后观察胚胎大体形态、胚胎活力、血管发育、心率和循环情况。相同体积的0.5%DMSO和5gM血管生成抑制剂PD173074分别作为阴性和阳性对照。结果：共筛选出7种具有抑制斑马鱼血管生成功能药物,选出率为0.35%。它们分别是异鱼藤酮、dihydromunduletone、马兜铃酸、美伐他汀、辛伐他汀、洛伐他汀和罗伐他汀。药物的最适浓度范围在0.3μM(辛伐他汀)至10μM(马兜铃酸和罗伐他汀)之间。这7种化合物根据生物活性可归为三组：鱼藤酮类(异鱼藤酮、dihydromunduletone)、马兜铃酸和他汀类药物(美伐他汀、辛伐他汀、洛伐他汀和罗伐他汀)。进一步发现,罗伐他汀不影响代表血管发生的背主动脉和已存在血管的生长发育,提示罗伐他汀的血管生成抑制作用是特异性的而非细胞毒性。第二部分罗伐他汀抗血管生成作用机制的体外实验研究目的：探讨罗伐他汀抑制血管生成的作用机制。方法：1.内皮细胞增殖、血管小管形成和迁移实验。分别应用Promega CellTiter96(?)试剂盒、内皮细胞血管小管形成实验和Cultrex(?)细胞迁移检测评价罗伐他汀对人脐静脉内皮细胞(human umbilical vein endothelial cell, HUVEC)增殖、血管小管形成和迁移能力的影响。2.内皮细胞周期和凋亡检测。采用流式细胞术检测罗伐他汀对细胞周期和凋亡的影响。细胞周期检测采用PI染色,应用FlowJo软件进行参数分析；而凋亡检测则进行PI和Annexin V双染色,利用Cell Quest软件分析数据。3.内皮细胞血管生成调控基因表达检测。应用RT2Profiler基因芯片技术,观察罗伐他汀引起的HUVEC血管生成通路基因谱的差异表达。结果：罗伐他汀以浓度依赖性方式显著抑制HUVEC体外增殖(IC50,5.87μM)。同时,分别用5μM、10μM、罗伐他汀处理的内皮细胞形成的管化分支平均长度分别是对照组的91%(P=0.01)、76%(P<0.05)、7%(P<0.01)。另外,内皮细胞迁移实验发现,与对照组相比,罗伐他汀处理组HUVEC平均迁移率分别下降了79%(5μM, P<0.01),84%(10μM, P<0.01)、87%(20μM, P<0.01).流失细胞术研究发现,HUVEC在5μM罗伐他汀作用48小时后,与对照组相比G1期细胞平均数量显著上升12.5%(61.9vs49.4%,P<0.05),而S期细胞却下降了19.9%(5.4vs25.1%,P<0.05),说明罗伐他汀可诱导内皮细胞G1期阻滞。我们还发现AnnexinV和PI双阳性细胞数量显著上升了8.5%(6.1vs14.6%,P<0.05),说明药物可能诱导内皮细胞晚期凋亡增加。进一步PCR芯片检测结果显示,在HUVEC内88个人血管生成通路调控基因中,43个基因表达水平在罗伐他汀的作用下发生显著变化(大于2倍),其中包括21个下调基因和22个上调基因。在下调基因中绝大多数为促血管生成基因、如成纤维生长因子-1、肝细胞生长因子-1、血管内皮生长因子C、转换生长因子B1：与之相反,上调基因多为抑制血管生成基因如组织金属蛋白酶抑制剂-1、稳定素1等。第三部分罗伐他汀治疗前列腺癌的实验研究目的：观察罗伐他汀对人前列腺癌的治疗效果方法：1.细胞增殖检测。应用Promega CellTiter96(?)试剂盒检测罗伐他汀对人前列腺癌细胞系PPC-1细胞增殖的影响。2.免疫缺陷鼠人前列腺癌PPC-1细胞移植瘤生长抑制实验。建立人前列腺癌PPC-1细胞免疫缺陷鼠(NOD/SCID)移植瘤模型,动物随机分为对照组和罗伐他汀治疗组,每组6只。罗伐他汀以每天40mg/kg腹腔注射15天,每天记录动物一般状态、体重和肿瘤体积。3.微血管密度检测。切取肿瘤组织,免疫组化CD31染色计数肿瘤微血管密度。4.药物毒性研究。分别取心、肝、结肠、肺、肾组织HE染色显微镜下观察。结果：罗伐他汀浓度依赖性地抑制PPC-1细胞增殖(IC50.14μM)。在治疗的第15天,给药组肿瘤平均体积较对照组明显减小(802.87±306.04mm3vs1801.26±395.7mm3, P<0.01),相应地,肿瘤重量在两组中也差异显著(0.86±0.39g vs1.39±0.88g, P<0.05)。此外,给药组肿瘤切片内CD31阳性血管数量较对照组显著减少(13.7±3.5/HPF vs2.1±1.4/HPF,P<0.01)。最后,动物体重(16.2±1.86g vs18016.3±1.24g,P>0.05)和主要脏器组织病理形态也无明显差异。结论：1斑马鱼化学遗传学小分子化合物筛选是研发血管生成抑制剂以及抗癌药物的良好方法；2罗伐他汀通过抑制内皮细胞功能和改变血管生成基因表达谱抑制血管生成；3罗伐他汀利用降低肿瘤微血管密度和抗增殖双靶点作用机制安全有效地抑制小鼠体内前列腺癌的生长,从而有望成为前列腺癌治疗新药。更多还原

【Abstract】 Part I. Chemical Genetic Screen to Identify Small Molecules Inhibiting Zebrafish AngiogenesisObjective:To identify small molecules compounds with the activity to inhibit zebrafish angiogenesis.Methods:1. Tg(flk1:EGFP) zebrafish husbandry. A transgenic zebrafish line, Tg(flkl:EGFP), with the endothelial cell-specific flk1promoter directing enhanced green fluorescent protein (EGFP) expression was used. The entire vascular network of the fish is marked by EGFP and could be visualized under fluorescence microscopy. For each mating, one Tg(flk1:EGFP) fish of either homozygous or heterozygous genetic makeup was paired with one wild type fish and5-6such pairs were set up. On average, each pair generated100-150embryos, and embryos were incubated in Holtfreter’s solution at28℃.2. Drug screen. The Library of Micro Source Spectrum Collection containing2000compounds was used for this screen. The dechorionated embryos were placed in Holtfreter’s solution within96-well plates with3-5embryos/well at the12-14hpf stage of development. Next, the compounds, diluted in0.5%dimethyl sulfoxide (DMSO), were added to each well at an initial concentration of0.1μM,1μM, and5μM for72h. After24,48and72h of exposure to the compound, the embryos were visually inspected for viability, gross morphological defects, heart rate and circulation. The0.5%DMSO with equal volume and the known angiogenic inhibitor, PD173074, were used as negative and positive control, respectively.Results:A total of seven hits (hit rate0.35%,7/2000) were identified according to the definition aforementioned with the optimal concentrations varying from0.3μM (simvastatin) to10μM (aristolochic acid and rosuvastatin). Interestingly, these lead compounds can be classified into three groups based on their bioactivities:rotenoids (isorotenone, dihydromunduletone), aristolochic acid and statins (simvastatin, mevastatin, lovastatin and rosuvastatin). Furthermore, we found the anti-angiogenic activity of rosuvastain was specific, rather than cytotoxic. Part II. Experimental Study on Anti-angiogenic Mechanisms of Rosuvastatin in vitro.Objective:To explore the anti-angiogenic mechanisms of rosuvastain in vitro..Methods:1. HUVEC Cell proliferation, tube formation and migration assays. The effects of rosuvastain on HUVEC proliferation, tube formation and migration were determined by the Promega CellTiter96(?) non-radioactive cell proliferation assay, the Endothelial Tube Formation Assay kit and the Cultrex(?) Cell Migration Assay, respectively.2. Cell cycle and apoptosis assays. For the cell cycle assay, the cells were stained with propidium iodide (PI) and analyzed using FlowJo software to determine the cell cycle parameters. For the apoptosis assay, cells were double-stained with annexin V and PI and analyzed by FACScan flow cytometer using Cell Quest software.3. Human angiogenesis PCR array. The effect of rosuvastatin on human angiogenesis pathway gene profiling in HUVEC was analyzed with human angiogenesis RT2Profiler(?) PCR array.Results:Rosuvastatin significantly suppressed HUVEC proliferation (IC50,5.87μM) in a dose-dependent manner. Moreover, after treatment with5u.M of rosuvastatin for48h, the average number of HUVECs arrested in the G1phase was significantly increased by12.5%(61.9vs49.4%, P<0.05) and the number of cells in the S-phase was decreased by19.7%(5.4vs25.1%, P<0.05) compared to the control, indicating rosuvastatin arrested HUVEC cell-cycle progression at the G1phrase. Meanwhile, the average number of the double positive annexin V+/PI+cells was increased by8.5%in rosuvastatin-treated HUVECs relative to the control (6.1vs14.6%, P<0.05), indicating that5μM of rosuvastatin could induce apoptosis in HUVECs in vitro. Subsequently, the expression levels in43of the88examined genes in various angiogenesis pathways were significantly changed (>2-fold) in the rosuvastatin-treated HUVECs, including21down-and22up-regulated genes. Of those, most of the proangiogenic genes, such as FGF-1(fibroblast growth factor1), HGF (hepatocyte growth factor), VEGF (vascular endothelial growth factor), and TGF β1(transforming growth factor, beta1) were decreased, whereas numerous anti-angiogenic factors, such as TIMP-1(tissue inhibitor of metalloproteinase1), and STAB1(Stabilin1) were elevated.Part Ⅲ. Experimental Study on the Efficacy of Rosuvastatin in Treatment of Prostate Cancer.Objective:To observe the therapeutic efficacy of rosuvastatin in human prostate cancer.Methods:1. Cell proliferation assay. The effect of rosuvastain on human prostate cancer cell line PPC-1proliferation was determined by the Promega CellTiter96(?) non-radioactive cell proliferation assay.2. Xenograft mouse model. A human prostate cancer cell line PPC-1xenograft mouse model was established in immunodeficient NOD/SCID mice. When tumors were established and of measurable size, mice were randomly assigned to the rosuvastatin treatment group (n=6) or the control group (n=6). Rosuvastatin (40mg/kg) was administered daily by intraperitoneal injection (i.p.). Tumor volume and animal weight were recorded daily.3. Microvessel density assay. Immunohistochemistry method was used to determine MVD in tumor tissue sections stained with anti-mouse CD31.4. Drug toxicity. The heart, liver, colon, lung and kidney tissues of xenograft mice were fixed for HE staining to assess rosuvastatin toxicity.Results:Rosuvastatin effectively inhibited human prostatic cancer cell PPC-1growth in vitro (IC5014μM). At the day15, the average tumor size of the rosuvastatin-treated group was markedly decreased relative to that of the control group (802.87±306.04mm3vs1801.26±395.7mm3, P<0.01). Accordingly, the average tumor weight of the rosuvastatin-treated group was also significantly reduced compared to that of the control group (0.86±0.39g vs1.39±0.88g, P<0.05). Also the reduction in vessel number within tumors of rosuvastatin-treated mice was statistically significant in comparison to that of the control group at day15(13.7±3.5/HPF vs2.1±1.4/HPF, P<0.01), suggesting tumor angiogenesis was markedly inhibited by rosuvastatin. In addition, no significant difference was detected in body weight and histopathologic morphology in important organs tissues between the rosuvastatin-treated group and the control group. Conclusions:Zebrafish chemical genetic screen is an excellent method for antiangiogenic and anticancer drugs discovery; the antiangiogenic mechanisms of rosuvastatin could be associated with the inhibition of endothelial cell functions and the alteration of human angiogenesis pathway gene profiling; our study may offer the preclinical data for the novel therapeutic potential of rosuvastatin in prostate carcinoma by targeting angiogenesis and cell proliferation.更多还原