Studies of a Monoclonal Antibody Targeting Neuropilin-1 on Growth Inhibition of Skin Cancer Cells and Its Mechanisms

研究背景和目的 在日照丰富的国家，皮肤癌的患病率极高，其病因复杂，发病机制各异，而传统的放疗、化疗手段存在抗肿瘤效果差、副作用大等一系列缺点。分子靶向治疗却有着良好的肿瘤靶向性和较低的毒副作用，为癌症治疗提供了新的治疗手段。近期研究显示神经鞭毛素蛋白-1（Neuropilin-1，NRP-1)是癌症治疗的新靶标，NRP-1是一个新型的多功能受体，是Ⅰ型跨膜糖蛋白。NRP-1可作为轴突导向因子3(semaphorin3，Sema3）、血管内皮细胞因子（vascularendothelialgrowthfactor，VEGF）、肝细胞生长因子（hepatocytegrowthfactor，HGF...Background and Purpose In the abundant sunshine state, the morbidity rate of skin cancer is extremely high, its etiology is complex and pathogenesis is different. But traditional radiotherapy and chemotherapy are used in a restrict range for the disadvantages of low effects and large side effects. Molecular targeted drugs with high tumor-targeting ability and low side effects provide a new strat...学位：理学硕士院系专业：医学院_药理学学号：2452011115336

靶向Neuropilin-1的抗肿瘤研究进展

Neuropilin-1(NRP-1)又称神经鞭毛素蛋白,是一个大小为130～140 kD的非酪氨酸激酶跨膜蛋白,参与脊椎动物胚胎的神经和心血管系统发育。它作为一个多功能受体调节VEGF(血管内皮生长因子),PDGF(血小板衍生生长因子),bFGF(成纤维生长因子)等细胞因子的信号通路,与肿瘤血管新生和肿瘤转移密切相关。NRP-1被认为是一个新的肿瘤治疗靶点,近年来针对NRP-1为靶点的药物研究有不少新进展。本文主要介绍NRP-1的生物学特征,对肿瘤作用以及相关药物研究

靶向Neuropilin-1的抗肿瘤研究进展

Neuropilin-1(NRP-1)又称神经鞭毛素蛋白,是一个大小为130～140 kD的非酪氨酸激酶跨膜蛋白,参与脊椎动物胚胎的神经和心血管系统发育。它作为一个多功能受体调节VEGF(血管内皮生长因子),PDGF(血小板衍生生长因子),bFGF(成纤维生长因子)等细胞因子的信号通路,与肿瘤血管新生和肿瘤转移密切相关。NRP-1被认为是一个新的肿瘤治疗靶点,近年来针对NRP-1为靶点的药物研究有不少新进展。本文主要介绍NRP-1的生物学特征,对肿瘤作用以及相关药物研究

抗人NRP1单克隆抗体的活性鉴定及初步应用

目的鉴定抗人神经菌毛素1(neuropilin1,NRP1)单克隆抗体的生物活性并利用该抗体检测常见肿瘤细胞株NRP1蛋白的表达情况。方法腹水法制备4株抗NRP1单克隆抗体并用rProtein A亲和柱纯化抗体,间接ELISA检测4株抗体的滴度水平,Western blot分析4株抗体结合NRP1蛋白的特异性,流式细胞术分析4株抗体结合NRP1蛋白的亲和力,共聚焦免疫荧光实验进一步验证抗体结合NRP1蛋白的能力;细胞免疫组织化学染色检测14株肿瘤细胞表达NRP1蛋白的情况。结果 4株抗体均能特异结合NRP1蛋白,其中A6结合NRP1能力最强,能结合在表达NRP1蛋白的细胞膜表面。细胞免疫组织化学染色结果显示NRP1在HepG2、C6、HEK293、BEL-7402、MDA-MB-453呈高水平表达,在U87、MGC803、MCF7、MDA-MB-231中表达水平相对较弱,而在U251、BGC823、H6、HT-29的表达介于两者之间,在SMMC-7721细胞中未检测到表达。结论 4株抗NRP1单克隆抗体均能特异结合NRP1蛋白,多种肿瘤细胞株均不同程度表达NRP1蛋白,为进一步探讨NRP1蛋白与肿瘤发生发展的关系奠定了基础

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

HTRA1基因杂合突变相关遗传性脑小血管病1例并文献复习 Heterozygous HTRA1-related Hereditary Cerebral Small Vessel Disease: A Case Report and Literature Review

本文对1例60岁男性轻度认知障碍、头晕患者的临床症状、影像学和基因检测结果进行分析。该患者既往有高血压、高脂血症、高尿酸血症病史，以及长期吸烟、饮酒史，其母亲曾患痴呆，头颅MRI示多发腔隙性脑梗死、脑白质变性和多发微出血病灶，基因检测报告示HTRA1基因c.1174T>C杂合突变，以上结果符合HTRA1基因杂合突变相关遗传性脑小血管病的诊断。 Abstract: Clinical symptoms, imaging findings, and genetic testing results of a 60-year-old male patient with mild cognitive impairment and dizziness were analyzed. The patient had a history of hypertension, hyperlipidemia, hyperuricemia, and long-term smoking and alcohol consumption. His mother had dementia. Cranial magnetic resonance images revealed multiple lacunar infarctions, white matter degeneration, and multiple microbleeding lesions. Genetic testing report showed a heterozygous mutation in the HTRA1 gene (c.1174T > C). All of above results are consistent with the diagnosis of heterozygous HTRA1-related hereditary cerebral small vessel disease

中国裸子植物的物种多样性格局及其影响因子

物种多样性的大尺度空间格局是宏观生态学和生物地理学研究的核心问题之一。本文利用中国裸子植物分布数据,结合气候、地形等环境信息,分析了中国裸子植物物种多样性的大尺度格局及其影响因素,比较了不同类群之间物种多样性格局和主导因子的差异,并探讨了裸子植物在植物区系中所占比重的地理格局。结果表明,中国裸子植物的物种多样性总体上呈现南高北低的趋势,物种多样性在横断山区最高。在裸子植物的三个主要类群中,松柏亚纲的物种多样性格局与整体相似,买麻藤亚纲的多样性高值区则出现在中国西北部的干旱地区,苏铁亚纲的分布区较为狭窄,主要集中在南方地区。线性回归分析结果表明,空间异质性和降水因子对中国裸子植物多样性格局的解释率最高,末次冰期以来的气温变化、海拔高差和能量因子次之。这表明中国裸子植物物种多样性的格局受到了多种因素的影响,其中空间异质性和降水因子影响最大。进一步分析发现,物种多样性格局的主导因子在不同类群之间具有显著差异,这可能反映了这些类群的进化历史以及生理适应的差异。裸子植物与被子植物的比例具有明显的空间格局:在东部、南部气候环境优越的地区,裸子植物与被子植物的比例低于0.06;而在西部、北部等气候环境比较恶劣的地区,裸子植物的比例则显著上升。回归分析表明,能量和水分因子显著影响了裸子植物与被子植物的比例。随着能量的降低和降水的减少,裸子植物与被子植物的比例会显著升高,这可能是由于被子植物在温暖湿润地区具有较强竞争优势,但裸子植物对极端环境具有更好的适应

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