蛋白抗体芯片在医学研究中的应用

1、罗树红：罗树红：美国伊利诺伊州大学高级研究员，美国伊利诺伊州大学高级研究员，2010年任南方医科大学特聘教授，博士生导师。年任南方医科大学特聘教授，博士生导师。2012年年4月任美国月任美国Raybiotech研发部主任，研究员，科学家。从事病原体与宿主相互作用分子机制及致病机理研究，具有广泛的分子生物学，免疫学以研发部主任，研究员，科学家。从事病原体与宿主相互作用分子机制及致病机理研究，具有广泛的分子生物学，免疫学以及蛋白组学领域的理论知识和实验技术。现为多家国外杂志评审，更多实验问题以及论文撰写发表联系：及蛋白组学领域的理论知识和实验技术。现为多家国外杂志评审，更多实验问题以及论文撰写发表

2、联系：Scott Luo Ph.D. R & D associate Director E-mail: Cell:(China) Raybiotech山东省总代理山东省总代理济南翔科生物技术有限公司济南翔科生物技术有限公司TelQ:541785194Cell-mail:Website:O蛋白质与抗体芯片在生物医学研究和转化医学中的应用蛋白质与抗体芯片在生物医学研究和转化医学中的应用Arrays of Discovery: The application of Protein Antibody Arrays in

3、Biomedical Research and Translational Medicine抗体芯片技术和产抗体芯片技术和产品的疾病研究应用品的疾病研究应用广泛的液体样品广泛的液体样品 Conditioned mediaPlasmaSerumUrineAmniotic fluidSputum Cerebrospinal fluid Synovial fluid Breath condensatesBronchoalveolar lavageGingival crevicular fluidLaser capture-microdissected tissueSalivaBreast inter

4、stitial fluidLiver cyst fluidsTearCell lysatesTissue lysates艾滋病艾滋病 过敏过敏 阿尔茨海默氏症阿尔茨海默氏症 哮喘哮喘 动脉粥样硬化动脉粥样硬化 自闭症自闭症 自身免疫性糖尿病自身免疫性糖尿病 癌症癌症 结肠炎结肠炎 克罗恩病克罗恩病 青光眼青光眼 移植排斥反应移植排斥反应高血压高血压 感染感染 缺血再灌注缺血再灌注 肥大细胞增多症肥大细胞增多症 多发性硬化症多发性硬化症 慢性阻塞性肺病慢性阻塞性肺病 银屑病银屑病 类风湿关节炎类风湿关节炎 败血症败血症 系统性红斑狼疮系统性红斑狼疮 IL-6IL-6 Knockout miceI

5、L - 6基因敲除小鼠基因敲除小鼠 IL-6Wild type mice野生小鼠野生小鼠Mouse Cytokine Antibody Array III 样本图片：小鼠细胞因子抗体芯片样本图片：小鼠细胞因子抗体芯片IIIIL-6 IL-6 敲除前后其它多种指标的表达情况也发生了明显变化，说明各细胞因子敲除前后其它多种指标的表达情况也发生了明显变化，说明各细胞因子之间是以相互作用的网状形式在机体内发挥作用之间是以相互作用的网状形式在机体内发挥作用 ，而单纯做，而单纯做ELISA ELISA 和和Western blotWestern blot就不会达到这样的效果就不会达到这样的效果IL-6 I

6、L-6 敲除前后其它多敲除前后其它多种指标的表达情况种指标的表达情况lHigh-content (高密度高密度)每张芯片同一样品可同时检测多达数百种指标。每张芯片同一样品可同时检测多达数百种指标。lOmics Tool (蛋白质组学的工具蛋白质组学的工具)可用于分析反应的模式。可用于分析反应的模式。lHigh Throughput (高高通通量量)每天完成多至上千样品的测试。每天完成多至上千样品的测试。lMiniaturization 微型化微型化节省节省样品和试剂样品和试剂, 实验空间实验空间,仪器设备仪器设备lHigh sensitivity, specificity and reprod

7、ucibility高灵敏，高特异性，重复性高灵敏，高特异性，重复性蛋白芯片是通过芯片矩阵平台同时检测多种蛋白质表达及功能的技术蛋白芯片是通过芯片矩阵平台同时检测多种蛋白质表达及功能的技术蛋白芯片和抗体芯片的技术原理蛋白芯片和抗体芯片的技术原理Biomarker-生物标志物生物标志物identifynewbiomarker发现新生物标志物发现新生物标志物earlydiagnosis早期诊断早期诊断multiplediagnosis多重诊断多重诊断Drugdiscoveryanddevelopment-新药研发新药研发identifydrugtarget发现药物靶标发现药物靶标identifyle

8、adcompound发现有前景新药发现有前景新药investigatedrugaction研究药物作用研究药物作用determinepotentialsideeffect确定潜在副作用确定潜在副作用Biomedicalresearch-生物医学科研生物医学科研Multiplexabundanceprofile-蛋白表达谱蛋白表达谱Multiplexfunctionprofile-蛋白功能谱：蛋白质间相互作用等蛋白功能谱：蛋白质间相互作用等蛋白芯片和抗体芯片的应用蛋白芯片和抗体芯片的应用Alzheimers Disease the search for biomarkers Alzheimers

9、 is a progressive, fatal neurodegenerative brain disorder the most common form of dementia Diagnosis is made by a process of elimination using a combination of imaging, cognitive and neuropsychological exams No chemistry-based diagnostic currently exists应用一：标志物筛选应用一：标志物筛选259 patientsAD: Alzheimers d

10、iseaseOD: Other dementiasMCI: Mild cognitive impairmentNDC: Control (non-demented)Does Alzheimers have a distinct cytokine signature detectable in plasma?应用一：标志物筛选应用一：标志物筛选Training set43 AD, 40 NDC18 cytokines associatedwith Alzheimers Ray S, et al. Classification and prediction of clinical Alzheime

11、rs diagnosis based on plasma signaling proteins (2007) Nature Med. Statistical analysisRayBio Cytokine Array 1000Search for Alzheimers BiomarkersDISCOVERYBlinded test set42 AD, 39 NDC, 11 ODPAM algorithm: 18 cytokinesOverall accuracy 89%VALIDATIONRayBio Cytokine Array 1000Classification of Alz vs. o

12、ther dementiasBlinded test set47 MCIOverall accuracy 81%PAM algorithm: 18 cytokinesRayBio Cytokine Array 1000Prediction of Alz应用一：标志物筛选应用一：标志物筛选Ray S, et al. Classification and prediction of clinical Alzheimers diagnosis based on plasma signaling proteins Nature Med. Training set43 AD, 40 NDC18个细胞因子

13、 与 Alzheimers相关Blinded test set42 AD, 39 NDC, 11 ODAlz vs. 其他老年痴呆症准确度准确度 89%Blinded test set47 MCI预测 Alz准确度准确度 81%人细胞因子抗体芯片人细胞因子抗体芯片1000 (AAH-CYT-1000)应用一：标志物筛选应用一：标志物筛选应用二：作用机制研究应用二：作用机制研究In a seminal study (Cell. 2013 Jan 17;152(1-2):51-67) by Anura Rambukkana and colleagues at the Medical Researc

14、h Council Center for Regenerative Medicine in London and the Rockefeller Institute in New York, the RayBio Mouse Chemokine/Cytokine Antibody Array C series 1000 was utilized to test for the presence of biologically active proteins in media conditioned by adult Schwann cell reprogrammed progenitor/st

15、em-like cells. The application of this array allowed for the identification of immunomodulatory factors secreted by these cells that promote bacteria-laden macrophage survival and migration, thus exacerbating the effect of leprosy bacterium infection. Please click here for a link to the publication.

16、应用二：作用机制研究应用二：作用机制研究Toshihiro Masaki , Jinrong Qu , Justyna Cholewa-Waclaw , Karen Burr , Ryan Raaum , Anura RambukkanaReprogramming Adult Schwann Cells to Stem Cell-like Cells by Leprosy Bacilli Promotes Dissemination of InfectionCell Volume 152, Issues 1?2 2013 51 - 67The proposed model: Schwann c

17、ells in the adult PNS infected with ML undergo a reprogramming process that coverts Schwann cells to pSLC that promote bacterial dissemination.应用二：作用机制研究应用二：作用机制研究Toshihiro Masaki , Jinrong Qu , Justyna Cholewa-Waclaw , Karen Burr , Ryan Raaum , Anura RambukkanaReprogramming Adult Schwann Cells to S

18、tem Cell-like Cells by Leprosy Bacilli Promotes Dissemination of InfectionCell Volume 152, Issues 1?2 2013 51 - 67Reprogrammed pSLC secrete Immune/growth factors that promote macrophage survival and migrationThe RayBio Mouse Chemokine/Cytokine Antibody Array C series 1000 (96)Axl, bFGF, BLC, CD30 Li

19、gand, CD30, CD40, CRG-2, CTACK, CXCL16, DPPIV/CD26, Dtk, Eotaxin-1, Eotaxin-2, E-Selectin, Fas Ligand, Fc gamma RIIB, Flt-3 Ligand, Fractalkine, G-CSF, GITR, GM-CSF, HGF R, ICAM-1, IFN-gamma, IGFBP-2, IGFBP-3, IGFBP-5, IGFBP-6, IGF-1, IGF-2, IL-1 beta, IL-10, IL-12 p40/p70, IL-12 p70, IL-13, IL-15,

20、IL-17, IL-17B R, IL-1 alpha, IL-2, IL-3, IL-3 Rb, IL-4, IL-5, IL-6, IL-7, IL-9, I-TAC, KC/CXCL1, Leptin/OB, Leptin R, LIX, L-Selectin, Lungkine, Lymphotactin, MCP-1, MCP-5, M-CSF, MDC, MIG, MIP-1 alpha, MIP-1 gamma, MIP-2, MIP-3 beta, MIP-3 alpha, MMP-2, MMP-3, Osteopontin, Osteoporotegerin, PF-4, P

21、ro-MMP-9, P-Selectin, RANTES, Resistin, SCF, SDF-1 alpha, Shh-N, sTNFRI, sTNFRII, TARC, TCA-3, TECK, Thymus CK-1, TIMP-1, TIMP-2, TNF alpha, Thrombopoietin, TRANCE, TROY, TSLP, VCAM-1, VEGF-A, VEGFR1, VEGFR2, VEGFR3, VEGF-D应用二：作用机制研究应用二：作用机制研究Toshihiro Masaki , Jinrong Qu , Justyna Cholewa-Waclaw ,

22、Karen Burr , Ryan Raaum , Anura RambukkanaReprogramming Adult Schwann Cells to Stem Cell-like Cells by Leprosy Bacilli Promotes Dissemination of InfectionCell Volume 152, Issues 1?2 2013 51 - 67Reprogrammed pSLC secrete Immune/growth factors that promote macrophage survival and migrationThe RayBio M

23、ouse Chemokine/Cytokine Antibody Array C series 1000 (96)Axl, bFGF, BLC, CD30 Ligand, CD30, CD40, CRG-2, CTACK, CXCL16, DPPIV/CD26, Dtk, Eotaxin-1, Eotaxin-2, E-Selectin, Fas Ligand, Fc gamma RIIB, Flt-3 Ligand, Fractalkine, G-CSF, GITR, GM-CSF, HGF R, ICAM-1, IFN-gamma, IGFBP-2, IGFBP-3, IGFBP-5, I

24、GFBP-6, IGF-1, IGF-2, IL-1 beta, IL-10, IL-12 p40/p70, IL-12 p70, IL-13, IL-15, IL-17, IL-17B R, IL-1 alpha, IL-2, IL-3, IL-3 Rb, IL-4, IL-5, IL-6, IL-7, IL-9, I-TAC, KC/CXCL1, Leptin/OB, Leptin R, LIX, L-Selectin, Lungkine, Lymphotactin, MCP-1, MCP-5, M-CSF, MDC, MIG, MIP-1 alpha, MIP-1 gamma, MIP-

25、2, MIP-3 beta, MIP-3 alpha, MMP-2, MMP-3, Osteopontin, Osteoporotegerin, PF-4, Pro-MMP-9, P-Selectin, RANTES, Resistin, SCF, SDF-1 alpha, Shh-N, sTNFRI, sTNFRII, TARC, TCA-3, TECK, Thymus CK-1, TIMP-1, TIMP-2, TNF alpha, Thrombopoietin, TRANCE, TROY, TSLP, VCAM-1, VEGF-A, VEGFR1, VEGFR2, VEGFR3, VEG

26、F-D应用二：作用机制研究应用二：作用机制研究2012年年7月最新月最新Nature肿瘤微环境通过分泌肿瘤微环境通过分泌 HGF引起对引起对RAF抑制剂的先天抵抗抑制剂的先天抵抗 癌细胞不死之谜癌细胞不死之谜Nature. 2012 Jul 26;487(7408):500-4.应用三：通路机制研究应用三：通路机制研究实验思路实验思路 1 提出假设-肿瘤微环境与肿瘤的抗药性有关 2 设计实验 筛选上清因子（筛选上清因子（AAH-CYT-G4000(274个因子个因子) （AAH-BLG-1 (507个因子个因子)肿瘤细胞肿瘤细胞 (45)和和stromal cell line or prima

27、ry cells共培养共培养 (23)HGF responsible for rescue.鉴定HGF的作用Nature. 2012 Jul 26;487(7408):500-4.PLX4720应用三：通路机制研究应用三：通路机制研究AAH-CYT-G4000(274个因子个因子)AAH-BLG-1 (507个因子个因子)Nature. 2012 Jul 26;487(7408):500-4.Stromal cell line Rescue scoreLL 86 * 0.35Wi-38 * 0.33PC60163A1* 0.31Hs888Lu * 0.24CCD-13Lu * 0.23PC87985B1 * 0.21CCD-8Lu 0.07HS-5 0.06CCD-1068Sk 0.06HDF 0.05HUVEC-CS 0.05PC87399A1 0.05PC87322A1 0.05CCD-1090Sk 0.04CCD-1117Sk 0.04CCD-1065Sk 0.04HMF 0.03CCD-1069Sk 0.0Correlations between proteins secreted by stromal cells and stromal