翔科生物蛋白芯片技术在医学研究的应用重庆肿瘤.ppt

Scott Luo Ph.D. R & D associate Director or Cell:(China) 217-721-9160 (US),广州瑞博奥生物科技有限公司 广州华南生物芯片研究中心 TelFaxWebsite: Or ,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 Biomedical Research and Translational Medicine,RayBio C Series Array: Mouse Cytokine Antibody Array III,IL-6,IL-6 Knockout mouse,IL-6,Wild type mouse,High-content (高密度) 每张芯片同一样品可同时检测多达数百种指标。 Omics Tool (蛋白质组学的工具) 可用于分析反应的模式。 High Throughput (高通量) 每天完成多至上千样品的测试。 Miniaturization 微型化 节省样品和试剂, 实验空间,仪器设备 High sensitivity, specificity and reproducibility 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,Benefits of protein and antibody arrays 蛋白和抗体芯片的优点,抗体芯片的载体,膜芯片 玻璃芯片,Sandwich Method,Direct Label Method,Competitive Method,Label-free Method,A.,B.,C.,D.,Signal detection in most antibody arrays relies on common labels (biotin, fluors, or enzymes) attached to the antibody or antigen. The sandwich method (A) requires an immobilized capture antibody and a labeled, in-solution detection antibody. This method has the advantages of excellent specificity and sensitivity. The competitive method (B) relies on competitive binding between the analyte and a labeled antigen. In the direct-label method (C), the label is attached to the analyte itself, while the label-free method (D) relies on an inherent property of the analyte (such as mass per unit area or surface plasmon resonance) to produce a signal.,Processing Quantibody Data,Data Extraction (ArrayVision, GenePix),Standard Curve (Raybio Q-Analyzer),Quantitative Data Export,全自动化蛋白质芯片检测仪：整合自动加样，清除，孵化，检测， 结果分析为一体的仪器， 已完成整体设计和样机制作。Automatic protein chip analyzer: integration of sample handling, wash, incubation, detection and data analysis into a single device.,全自动化蛋白质芯片检测仪 (Automatic protein chip analyzer),Alzheimers Disease the search for biomarkers,Alzheimers 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,259 patients,AD: Alzheimers disease OD: Other dementias MCI: Mild cognitive impairment NDC: Control (non-demented),Does Alzheimers have a distinct cytokine signature detectable in plasma?,Training set,43 AD, 40 NDC,18 cytokines associated with Alzheimers,Ray S, et al. Classification and prediction of clinical Alzheimers diagnosis based on plasma signaling proteins (2007) Nature Med.,Statistical analysis,RayBio Cytokine Array 1000,Search for Alzheimers Biomarkers,DISCOVERY,Ray S, et al. Classification and prediction of clinical Alzheimers diagnosis based on plasma signaling proteins Nature Med.,Training set 43 AD, 40 NDC,18个细胞因子 与 Alzheimers相关,Blinded test set 42 AD, 39 NDC, 11 OD,Alz vs. 其他老年痴呆症 准确度 89%,Blinded test set 47 MCI,预测 Alz 准确度 81%,人细胞因子抗体芯片1000 (AAH-CYT-1000),2012年7月最新Nature,肿瘤微环境通过分泌 HGF引起对RAF抑制剂的先天抵抗,癌细胞不死之谜,Nature. 2012 Jul 26;487(7408):500-4.,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（AAH-CYT-G4000(274个因子) （AAH-BLG-1 (507个因子),肿瘤细胞 (45)和stromal cell line or primary cells共培养 (23),HGF responsible for rescue.,鉴定HGF的作用,Nature. 2012 Jul 26;487(7408):500-4.,PLX4720,AAH-CYT-G4000(274个因子) AAH-BLG-1 (507个因子),Nature. 2012 Jul 26;487(7408):500-4.,Stromal cell line Rescue score LL 86 * 0.35 Wi-38 * 0.33 PC60163A1* 0.31 Hs888Lu * 0.24 CCD-13Lu * 0.23 PC87985B1 * 0.21 CCD-8Lu 0.07 HS-5 0.06 CCD-1068Sk 0.06 HDF 0.05 HUVEC-CS 0.05 PC87399A1 0.05 PC87322A1 0.05 CCD-1090Sk 0.04 CCD-1117Sk 0.04 CCD-1065Sk 0.04 HMF 0.03 CCD-1069Sk 0.0,Correlations between proteins secreted by stromal cells and stromal average melanoma rescue scores,鉴定HGF的作用,其他RTK配体是否也会与抗药性相关,In vitro 鉴定,WB 鉴定 HGF/MET通路机制,In vivo 鉴定,BRAFi PLX4720,肿瘤微环境通过分泌 HGF引起先天抵抗 RAF抑制剂,（AAH-CYT-G4000(274个因子) （AAH-BLG-1 (507个因子),Without our technology, such breakthrough discovery will not be possible.,2010年8月:,皮质类固醇抑制婴幼儿血管瘤源性干细胞内的VEGF-A分泌,uPAR, IL-6, MCP1, and MMP1 are down-regulated in HemSC3 treated with dexamethasone,人血管生成抗体玻璃芯片-1: AAH-ANG-G1000； （检测43种不同的细胞因子）,uPAR, IL-6, MCP1, and MMP1 are down-regulated in HemSC3 treated with dexamethasone,人血管生成抗体玻璃芯片-1: AAH-ANG-G1000；,Identification of key factors in tumorigenesis,YAP (yes-associated protein) A transcriptional regulator with key roles in both malignancy and in normal organ proliferation Promotes epithelial-to-mesenchymal transition Highly upregulated in cancers,Preliminary Data,MCF10A: a mammary epithelial cell line (non-transformed) (3D culture growth),MCF10A cells,YAP transfection OR recombinant EGF,ProliferationSurvival,YAP-expressing cells also induce proliferation in non-YAP-expressing cells in co-culture.,What are the key transcriptional targets involved in YAP-mediated cell proliferation?,Empty vector,YAPS127A,Amphiregulin (AR),(+) EGF,(-) EGF,RayBio Human Growth Factor Array I (AAH-GF-1),IGFBP6,MCSF-R,PDGF-AA,Supernates from transfected MCF10A 3D culture system,Amphiregulin is the key transcriptional target of YAP,Since Amphiregulin is a ligand for the ErbB receptors, RayBio EGFR Phosphorylation Antibody Arrays were utilized.,Empty Vector,(+) EGF,(-) EGF,YAP activation increases the phosphorylation of classic EGFR target residues,RayBio EGFR Phosphorylation Array (AAH-PER-1),Transfected MCF10A 3D cultureed cell lysates,YAPS127A,EGFR Tyr845,ErbB4 Tyr1284,EGFR Tyr1148,Conclusion,Zhang J, et al. YAP-dependent induction of amphiregulin identifies a non-cell-autonomous component of the Hippo pathway. Nat Cell Biol 2009; 11(12): 1444-1451.,Amphiregulin,EGFR family,YAP,Cell proliferation,Amphiregulin,Reversible tyrosine phosphorylation is governed by the balanced action of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Dysregulation of PTP activity results in aberrant tyrosine phosphorylation, which is frequently implicated in the progression of various diseases including diabetes, rheumatoid arthritis, and cancer . The first PRL associated with cancer metastasis was PRL-3 (PTP4A3), which was found to be consistently expressed at high levels in all 18 human colorectal cancer (CRC) liver metastases examined, but at lower levels in the corresponding primary tumors and normal epithelium.,Hypothesis: PRL-3 plays a key role in cancer metastasis?,Al-aidaroos et al. J Clin Invest. 2013;123(8):34593471. doi:10.1172/JCI66824.,PRL-3 overexpression activates the EGFR in A431 cells,PRL-3induced EGFR activation is partially dependent on SRC activity and may not involve changes in autocrine stimulation.,PRL-3 induces EGFR hyperphosphorylation via PTP1B downregulation,Proposed model of PRL-3induced oncogenic addiction to hyperactivated EGFR,Figure 1 Wnt heterogeneity in primary tumours and in CSC culture.,Figure 5 Myofibroblasts support stem-cell properties and regulate Wnt signalling.,肾移植监测,同时检测人体血和尿中20种蛋白标记物,新药毒性的监测,人体血和尿,监测治疗性药物,及时调整用药,早期发现肾脏损伤性疾病,Kidney transplantation Drug treatment (cisplatin) Kidney disease,Different stage,AKI arrays,Early diagnosis Predictive markers,AKI,Normal,肿瘤标志物检测抗体芯片-定量检测十六种常见肿瘤血清标志物 (Cancer detection antibody arrays: simultaneous detection of 16 common cancer biomarkers),采用肿瘤标志物检测抗体芯片同时检判断一次同时检测十六种常见肿瘤血清标志物，省时，省钱，有利于在广大基层开展长期监测十六种常见肿瘤血清标志物的动态变化，有利于检测出早期肿瘤。,一滴血,One drop of blood,16 common cancer biomarkers can be measured simultaneously using cancer biomarker antibody arrays. It is cost effective and convenient. The dynamic change of those cancer biomarkers can be monitored longitudinally. It will benefit for cancer screening and early detection,上千篇New England Journal of Medicine 、Nature、Science, Nature Medicine、Cell、Lancet、PNAS等高水平论文均使用了其所开发的技术和产品 (thousand of papers using our technologies and products have been published in the literatures)；,抗体芯片的国际刊物引用数,中国区2013年应用Raybiotech产品发表的SCI论文 200多篇,RayBiotech芯片产品和技术的客户,Examples of Diseases have been applied using RayBiotech Technologies and products,Wide range of samples Conditioned media Plasma Serum Urine Amniotic fluid Sputum Cerebrospinal fluid Synovial fluid Breath condensates Bronchoalveolar lavage Gingival crevicular fluid Laser capture-microdissected tissue Saliva Breast interstitial fluid Liver cyst fluids Tear Cell lysates Tissue lysates Other,根据客户需求 灵活多变,产品定制 多种多样,RayBiotech公司抗体抗原库的资源 客户拥有的原材料 市场上可购买到,定制服务,RayBiotech Custom Array,Raybiotech 亚特兰大总部,广州瑞博奥,谢谢！,联 络 我 们,广州瑞博奥生物科技有限公司 电 话: 020 - 3205 1923, 3229 0761 （免费: 4