Company ConfidentialSignaling CytometrySignaling Simplified赵静 贝克曼库尔特公司Company ConfidentialWhat is Cell Signaling?控制细胞行为和相关细胞活动的通讯系统控制细胞行为和相关细胞活动的通讯系统 能够对微环境的变化正确地作出反应能够对微环境的变化正确地作出反应 是发育，组织修复和免疫的基础是发育，组织修复和免疫的基础 信号处理的错误可导致各种疾病信号处理的错误可导致各种疾病Company ConfidentialSignaling:Signaling Cytometry for analysis of complex samplesSignaling Cytometry for analysis of complex samplesWestern Blot2501501007550UntreatedTreatedpSTAT5 均一的溶解产物 得到定性结果实验时间较长 不同的完整细胞对不同亚群进行分析 得到定量结果 快速有效Treated Green Untreated BlueSignaling Cytometry (SC)Company ConfidentialSingle cell (Flow Cytometry) vs Bulk assay (Western Blot)Western BlotFlow Cytometry% Pos Cells1%50%100%Relative Protein Expression100501Relative FluorescenceCompany ConfidentialSignaling CytometrySignaling Cytometry: Use of one or multiple signal transduction markers in conjunction with cell surface markers to dissect complex Biological processes in heterogeneous populationsCompany ConfidentialCell Signaling Pathways?肿瘤信号通路?自身性免疫性疾病?细胞调控?应激反应?药物动力学监测Company Confidential在AML 诊断和监测的应用 在CLL 诊断和监测的应用 在细胞周期调控中的应用信号转导的应用Company Confidential 靶向治疗的发展 在AML的具有特征性突变信号转导蛋白 Flt3 (1/3 of AML) Ras (10-15% of AML) SCF( 5-10% of AML) PDGF, FGF, JAK2, SHP2 转录因子，胞浆、细胞核转运子等 直接抑制突变的信号分子 Flt3 receptor inhibitors Ras (farmesyl transferease inhibitors) SCF (Gleevec)抑制下游效应分子 MAPK pathway inhibitors Raf kinase inhibitors Mek inhibitors Rapamycin PI3 kinase inhibitorsSignal transduction in AML:Company ConfidentialSignal transduction in AML对于AML中异常的信号转导，FCM 可提供敏感和特异的检测比目前标准的方法提供更特异的疾病治疗监测的方法。 直接判定目前靶向治疗或联合治疗的疗效。 预测预后?Company ConfidentialSignal transduction in AMLReceptorJak2*p-statsUO126 XProliferationp-mekp-erkMAPKshp2Prol/apop Trans regRap Xp-S6PI3mToraktApoptosisp-stat5UO126 XRap XProliferationReceptorp-S6PI3mToraktApoptosisp-mekp-erkMAPKp38IL-3/GM-CSFSCF/Flt-3LY X XCompany ConfidentialFCM signal transduction analysis in AML 建立正常的信号表达/表型文库 建立在正常骨髓中与免疫表型相关的信号转导反应 在AML中评价异常信号转导的特征，变异性以及是否容易 检测。Company ConfidentialFCM signal transduction analysis in AML正常和AML骨髓中的信号反应 未刺激/刺激样本 SCF, Flt-3, GMCSF, GCSF, MCSF, IL-3 Rapamycin, UO126, LY294002 Formaldehyde fix at 37oC for 10 at 2, 5, 10, 20 minutes Detergent/methanol permeabilization Chow et al, Cytometry 67A:4-17, 2005 Jacobberger et al, Cytometry 54A:75-88, 2003 流式检测 p-erk, p-S6, p-akt, p-stat5 CD45, CD64, CD11b, CD15, CD34, CD117, CD13, CD33, CD16, CD14 评价 lymphs, monos, mature granulocytes, immature granulocytes, CD34+CD117+, CD34-CD117+Company ConfidentialFCM Signal Transduction Responses: Normal Bone MarrowCompany Confidential131811217183234521241426117142100229FCM signal transduction analysis in AML Normal bone marrowUnstimulatedIL-3Stem cell factor (SCF)GMCSFPhosphorylated stat5Number of cellsBlastsMonosMature gransImmature gransCompany ConfidentialSignal transduction in AMLReceptorJak2*p-statsUO126 XProliferationp-mekp-erkMAPKshp2Prol/apop Trans regRap Xp-S6PI3mToraktApoptosisp-stat5UO126 XRap XProliferationReceptorp-S6PI3mToraktApoptosisp-mekp-erkMAPKp38IL-3/GM-CSFSCF/Flt-3LY X XCompany Confidential91021998139115412981310101365176565FCM signal transduction analysis in AML Normal bone marrowPhosphorylated S6Number of cellsLymphsBlastsMonosImmature gransUnstimulatedRapamycin (Rap)Stem cell factor (SCF)SCF + RapGMCSFCompany ConfidentialFCM Signal Transduction Analysis in AML: Normal Bone Marrow部分总结目前可以用免疫表型（10个抗原） 定义15群细胞，对每群分析6种 刺激剂刺激后的6个磷酸化信号 分子。Company ConfidentialFCM signal transduction analysis in AML Normal bone marrow1234ResponseResponseResponseGMCSFGMCSFGMCSFSCFSCFSCFFlt3Flt3Flt3IL-3IL-3IL-3LymphsMono Mature granulocytes Intermediate granuloctyes Immature granuloctyes CD34+CD117+ CD34-CD117+p-S6p-stat5p-erkCompany ConfidentialFCM signal transduction analysis in AML Normal bone marrowSide ScatterSide ScatterSide ScatterCD13CD15CD33CD33CD33CD15CD15CD13CD13Side ScatterCD34CD117CD45Company ConfidentialFCM signal transduction analysis in AML Normal bone marrowp-aktp-aktp-erkp-erkNumber of cellsCD34+CD117+CD34-CD117+CD34-CD117+CD34+CD117+Number of cellsInhibitorInhibitorInhibitorInhibitor4 min4 min4 min4 min6 min6 min6 min6 min8 min8 min8 min8 minCompany ConfidentialFCM signal transduction analysis in AML Normal bone marrowMagnitude of responseGranulocyte MaturationCD34+/ CD117+EarlyIntermediateMaturep-erk response, GM-CSF stimulationCompany ConfidentialFCM signal transduction analysis in AML Normal bone marrow得到与免疫表型相关的信号反应的详细图谱 反应变化的幅度和动力学变化 SCF, Flt-3, GMCSF, GCSF, MCSF, IL-3骨髓中不同的细胞亚群检测信号转导反应 不同的细胞亚群信号转导反应明显不同 在同一亚群中用不同的刺激反应明显不同 敏感 与细胞系/WB具有高度相关性。但还能够提供 . 独特的细胞因子/生长因子下的骨髓环境的数据。 全新的与成熟相关的动力学变化/ 变化幅度的数据Company ConfidentialFCM signal transduction analysis in AML AML Patient #1Flt3 stimulation, CD34+ populationp-erkNumber of cellsNormalInhibitorUnstim2.558p-erkNumber of cellsPatientInhibitorUnstim2.558CD45Side ScatterAML-M2 75% blasts CD34+,CD117+,CD13+ CD33+,MPO+,CD7+,Flt3+ Cytogenetics Normal Mutational Status Flt3 (ITD) neg Flt3 (D835) neg N