Selective Thrombosis in Orthotopic Tumor Model of Hepatocellular Carcinoma in Mice Induced by Targeted Coagulation Protein tTF-EG3287

肝癌是最常见的恶性肿瘤之一，具有发病率高、复发率高、预后差的特点，经肝动脉化疗栓塞（transarterialchemoembolization，TACE）是目前公认的肝癌非手术治疗的首选方法。目前临床上常用的栓塞材料各有优缺点，在临床应用中如何对症使用尚无统一标准，而且TACE治疗能否提高肝癌病人的生存率，仍然存在争议，TACE治疗存在种种缺陷如栓塞不全、缺乏靶向性、毒副作用大、栓塞剂稳定性与可控性差和伴随血管内皮生长因子升高等。因此，发展新型高效的、具有靶向性、可控性强、无毒的肿瘤血管栓塞剂依然是目前肝癌临床治疗亟待解决的重大问题。 针对以上问题，我们计划采用一种基于靶向性凝血蛋白的选择...Hepatocellular carcinoma, one of the most commonly occurring cancer, is responsible for significant mortality worldwide, with its increasing morbidity, high recurrence and poor prognosis. Transarterial chemoembolization (TACE) is recommended as the current standard care for patients with intermediate-stage hepatocellular carcinoma. However, considering the advantages and disadvantages of conventio...学位：医学硕士院系专业：医学院_肿瘤学学号：2452012115324

Effects of Anti-NRP1 Monoclonal Antibody on Cell Proliferation,Migration and Apoptosis of Melanoma B16F10 Cells

目的研究靶向神经菌毛素1(nEurOPIlIn1,nrP1)单克隆抗体(nrP1 MAb)对黑色素瘤细胞b16f10增殖、迁移和凋亡的影响,并探讨其作用机制。方法 SdS-PAgE检测神经菌毛素1单克隆抗体的纯度;蛋白质印迹法、免疫荧光和免疫组化检测神经菌毛素1单克隆抗体的特异性和b16f10细胞及其移植瘤上神经菌毛素1的表达;四甲基偶氮唑蓝法、划痕实验、AnnEXIn V-fITC/PI双标记流式细胞术等方法检测不同浓度神经菌毛素1单克隆抗体对黑色素瘤细胞增殖、迁移和凋亡的影响;蛋白质印迹法检测神经菌毛素1单克隆抗体作用后细胞内相关信号蛋白的表达。结果纯化的神经菌毛素1单克隆抗体具有高纯度和特异性,同时b16f10细胞及其移植瘤上神经菌毛素1高表达。神经菌毛素1单克隆抗体能有效抑制b16f10细胞的增殖和迁移,抑制作用呈时间和浓度依赖性(P<0.05)。神经菌毛素1单克隆抗体能显著诱导b16f10细胞凋亡,并呈剂量依赖关系(P<0.05)。抗体作用后b16f10细胞中磷酸化的AkT和Erk1/2表达下调,bCl-2和CASPASE 3蛋白表达下调,bAX蛋白的表达上调。结论纯化的神经菌毛素1单克隆抗体能有效地抑制b16f10增殖,迁移并明显诱导凋亡,其可能通过抑制膜上神经菌毛素1与相关细胞因子结合而减少其介导的PI3k/AkT和MEk/Erk1/2信号转导途径的活化来抑制细胞增殖和迁移,并通过bCl-2/bAX/CASPASE3途径诱导细胞凋亡,从而发挥其抗肿瘤作用。OBJECTIVE To investigate the effect of anti NRP1 monoclonal antibody( NRP1 mAb) on melanoma cell B16F10 proliferation,migration and apoptosis,and explore its mechanism.METHODS Western blot,immunofluorescent and immunocytochemistry staining were applied to determine the expression of NRP1 on B16F10 cells and its xenotransplanted tumors by NRP1 mAb.The proliferation,migration and apoptosis of melanoma cells were detected by MTT assay,the scratch test and Annexin V-FITC /PI double labeled flow cytometry after administration with different concentrations of NRP1 mAb.The expression level of some signal molecules was measured by Western blot.RESULTS The purified NRP1 mAb has high purity and specificity.B16F10 cells and its xenotransplanted tumors highly expressed NRP1.NRP1 mAb inhibit the proliferation and migration in a time-and dose-dependent manner( P < 0.05).Moreover NRP1 mAb induce apoptosis of B16F10 in a dose-dependent manner( P < 0.05).The results of western blot revealed down-regulated expression of p-Akt,p-ERK1 /2,Bcl-2 and caspase-3 and up-regulated Bax expression in B16F10 cells treated by NRP1 mAb.CONCLUSION The purified NRP1 mAb can effectively inhibit the proliferation and migration of B16F10 and induce apoptosis.Meanwhile,the changed expression of the proteins suggests that NRP1 mAb may inhibit cell proliferation and migration by ERK1 /2 and PI3 K /Akt pathway and induce cell apoptosis by Bcl-2 /Bax /caspase-3 pathway,consequencely playing anti-tumor role.国家自然科学基金资助项目(30973485;81172970); 福建省属公益类科研专项经费(2011R1039-1;2013J0138

Establishment and Characterization of a Targeting Tumor Vesseles System for Inducing Thrombosis

目的建立一种新型的靶向高效诱发肿瘤血管血栓栓塞体系磁流体-神经纤维网蛋白1抗体A6-链霉亲和素:生物素-短截型组织因子(Mf-A6-SA:b-TTf)。方法化学交联技术制备神经纤维网蛋白1抗体A6-链霉亲和素、磁流体-神经纤维网蛋白1抗体A6-链霉亲和素和生物素-短截型组织因子交联物,凝血因子X活化实验鉴定复合体系活化凝血因子X的活力,荧光显微镜技术和普鲁士蓝染色法同时观察施加外界磁场后复合体系的靶向作用,凝血实验直接观察复合体系引入链霉亲和素:生物素的生物放大效应,体内生物分布实验观察复合体系的安全性。结果成功制备磁流体-神经纤维网蛋白1抗体A6-链霉亲和素及生物素-短截型组织因子,磁流体-神经纤维网蛋白1抗体A6-链霉亲和素:生物素-短截型组织因子体系保留有高效激活凝血因子X的活性,与靶点的结合具有靶向性及高效富集性,体内实验证实能安全有效诱发肿瘤血管栓塞。结论成功制备的具有靶向诱发肿瘤血管血栓栓塞体系磁流体-神经纤维网蛋白1抗体A6-链霉亲和素:生物素-短截型组织因子为进一步探索肿瘤血管的靶向治疗奠定基础。OBJECTIVE To establish a novel targeting tumor vessels system MF-A6-SA:B-tTF for efficiently inducing thrombosis.METHODS Chemical cross-linking technique was used to prepare a cross-linking agent of the A6-Streptavidin( A6-SA),MFA6-SA and Biotein-tTF(B-tTF).FX coagulation assay was used to test MF-A6-SA:B-tTF system's FX activity.Fluorescence microscopy and prussian blue staining were used to simultaneously observe the targeting activity of MF-A6-SA:B-tTF with an external magnetic field.Hemagglutination was directly used to study the system' s biological amplification by SA /B.Biodistribution experiment was used to observe the toxicity of MF-A6-SA:B-tTF.RESULTS MF-A6-SA and B-tTF were successfully prepared.MF-A6-SA:B-tTF system could activate FX,inducing the blood coagulating cascade powerfully.MF-A6-SA:B-tTF could be accumulated to the desired target area with targeting and induce thrombosis in tumor blood vessels in vitro and in vivo.CONCLUSION The double targeting tumor vessels system MF-A6-SA:B-tTF maybe provide a basis for developing the tumor blood vessels targeting therapy.国家自然科学基金资助项目(30973485;81172970); 国家级大学生创新性实验项目(201210384134

基于图像边缘特征的零件分类与定位算法

本论文提出了一种基于图像边缘特征的零件分类与定位算法。算法首先对图像腐蚀膨胀等预处理,然后通过边缘检测得零件的完整边缘轮廓,然后对零件边缘轮廓图像使用方向梯度直方图(HOG)构造训练数据,并采用支持向量机(SVM)在训练数据上训练分类识别模型,最后进行检测。根据检测结果获得图像中零件的种类和位置信息,然后使用棋盘格标定的方法来确定深度。标定时,取标定板上的两个点来计算深度,根据已知的深度,获取实际抓取点的两个图像坐标系的x,y值,计算世界坐标系坐标。此系统能解决零件分类、定位问题,能广泛运用于生产线引导工业机器人进行零件抓取

基于图像边缘特征的零件分类与定位算法

本论文提出了一种基于图像边缘特征的零件分类与定位算法。算法首先对图像腐蚀膨胀等预处理,然后通过边缘检测得零件的完整边缘轮廓,然后对零件边缘轮廓图像使用方向梯度直方图(HOG)构造训练数据,并采用支持向量机(SVM)在训练数据上训练分类识别模型,最后进行检测。根据检测结果获得图像中零件的种类和位置信息,然后使用棋盘格标定的方法来确定深度。标定时,取标定板上的两个点来计算深度,根据已知的深度,获取实际抓取点的两个图像坐标系的x,y值,计算世界坐标系坐标。此系统能解决零件分类、定位问题,能广泛运用于生产线引导工业机器人进行零件抓取

Exacerbated apoptotic effects of anti-human NRP-1 monoclonal antibody in colon cancer cell line LOVOin vitro

目的:研究抗人神经菌毛素1(nEurOPIlIn1,nrP-1)单克隆抗体对结肠癌lOVO细胞的促凋亡作用。方法:小鼠腹水法制备抗nrP-1单克隆抗体(nrP-1 MAb)并用rPrOTEIn A亲和柱纯化,间接ElISA检测抗体的滴度水平。细胞免疫荧光检测nrP-1蛋白在结肠癌细胞株lOVO上的表达,MTT法检测nrP-1 MAb对lOVO的生长抑制作用,dAPI荧光显微镜法观察细胞凋亡形态,AnnEXIn V-fITC/PI流式细胞仪法检测nrP-1 MAb诱导lOVO细胞凋亡,WESTErn blOTTIng检测凋亡相关蛋白CASPASE-3、ClEAVEd CASPASE-3蛋白的表达。结果:SdS-PAgE和间接ElISA检测纯化的nrP-1 MAb纯度为95%、效价为2.56x10-5;细胞免疫荧光染色显示nrP-1定位于lOVO细胞膜;MTT法检测和AnnEXIn V-fITC/PI流式细胞仪法检测结果显示,nrP-1 MAb对lOVO细胞有生长抑制作用和促凋亡作用;dAPI染色·荧光显微镜观察发现,nrP-1MAb可以诱导lOVO细胞核浓缩与碎裂;WESTErn blOTTIng检测到在nrP-1 MAb作用下,lOVO细胞裂解液ClEAVEd CASPASE-3条带信号增强、CASPASE-3条带信号减弱。结论:纯化的nrP1 MAb能抑制lOVO细胞的生长并诱导其凋亡。OBJECTIVE To investigate the exacerbated apoptotic effect of anti-human nerve pilifactor 1(neuropilin-1,NRP-1)monoclonal antibody(NRP-1 MAb)on colon cancer LOVO cells in vitro.METHODS NRP-1 MAb was prepared from ascites and purified by rProtein A affinity column and the titers were measured by indirect ELISA.The NRP-1 expression in colon cancer cell LOVO was detected by immunofluorescence;growth inhibition of LOVO cells treated with different concentration NRP-1 MAb was analyzed by MTT assay;DAPI staining was used to observe the morphology of apoptotic cells with Fluorescence microscopy;apoptosis rate of LOVO cells was determined by flow cytometry assay with Annexin V-FITC/PI;Western blotting was used to detect Caspase-3 protein expression.RESULTS The results of SDS-PAGE and indirect ELISA showed the purity of NRP-1 MAb was 95%and the titer was 2.56×10-5.Immunofluorescence staining indicated that the NRP-1 was localized on the membrane of LOVO cells.MTT assay and Annexin V-FITC/PI flow cytometry assay indicated that NRP-1MAb inhibited LOVO cells growth and induced LOVO cells apoptosis.Degradation and fragmentation of nuclear in LOVO cells treated with NRP-1 MAb were observed under a fluorescence microscope by DAPI staining;Western blotting detected that cleaved Caspase-3 band signal enhanced,while Caspase-3 band signal decreased after incubated LOVO cells with the NRP-1MAb.CONCLUSION NRP-1 MAb can inhibit proliferation and increase apoptosis of the LOVO cells.国家自然科学基金(编号:30973485;81172970); 福建省属公益类科研专项经费(编号:2011R1039-1