流式细胞术在信号转导通路研究中的作用

17
GMCSF
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FCM 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。
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Patient
Inhibitor
Unstim 2.5’ 5’ 8’
p-erk
Unstim 2.5’ 5’ 8’
p-erk
Side Scatter
AML-M2
75% blasts CD34+,CD117+,CD13+ CD33+,MPO+,CD7+,Flt3+
Cytogenetics
Normal
Mutational Status
UO126 X
p-erk
PI3
LY X X
akt
mTor
Rap X
p-S6
Prol/apop Trans reg
Apoptosis
Proliferation
Proliferation
Apoptosis
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p-stat5
FCM signal transduction analysis in AML
Early
Intermediate
Mature
Granulocyte Maturation
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FCM 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
FCM signal transduction analysis in AML Normal bone marrow
p-S6
p-erk
Response
Response
Response
GMC1 SF
S2CF
F3lt3
IL4 -3
p-stat5
GMCSF SCF
Flt3
IL-3
GMCSF SCF
Flt3
CD34+CD117+
Inhibitor
CD34+CD117+
Inhibitor
CD34-CD117+
CD34-CD117+
Inhibitor
Inhibitor
4 min
4 min
4 min
4 min
6 min
6 min
6 min
6 min
8 min
p-erk
8 min
p-akt
8 min
p-erk
Flt3 (ITD) neg Flt3 (D835) neg NPM1 pos
SCF
neg
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FCM signal transduction analysis in AML AML Patient #5
– 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。
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FCM 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+
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Signaling:
Western Blot
Untreated Treated
Signaling Cytometry (SC)
250 150
pSTAT5 100 75 50
• 均一的溶解产物 • 得到定性结果 •实验时间较长
Treated Green Untreated Blue
Number of cells Number of cells
CD45 pAKT Alexa 647
FCM signal transduction analysis in AML AML Patient #1
Flt3 stimulation, CD34+ population
Normal
Inhibitor
IL-3
Lymphs Mono Mature granulocytes Intermediate granuloctyes Immature granuloctyes CD34+CD117+ CD34-CD117+
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FCM signal transduction analysis in AML Normal bone marrow
8 min
p-akt
Company Confidential
FCM signal transduction analysis in AML Normal bone marrow
p-erk response, GM-CSF stimulation
Magnitude of response
CD34+/ CD117+
UO126 X
p-erk
PI3
LY X X
akt
mTor
Rap X
p-S6
Prol/apop Trans reg
Apoptosis
Proliferation
Proliferation
Apoptosis
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p-stat5
FCM signal transduction analysis in AML Normal bone marrow
CD45 CD117
CD15
Side Scatter
CD34
CD15
CD15
Side Scatter
Side Scatter
Side Scatter
CD13
CD13
CD33
CD33
CD33
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CD13
Number of cells Number of cells
FCM signal transduction analysis in AML Normal bone marrow
Cell 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
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信号转导的应用
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
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FCM Signal Transduction Responses: Normal Bone Marrow
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FCM signal transduction analysis in AML Normal bone marrow
Lymphs
9
Blasts
10
Monos
21
9
8
13
Number of cells
11
65
54
9
8
13
10
Baidu Nhomakorabea
13
65
Phosphorylated S6
Immature grans
9
Unstimulated
9
Rapamycin (Rap)
12
Stem cell factor (SCF)
10
SCF + Rap
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Signal transduction in AML:
• 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
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Signal transduction in AML
IL-3/GM-CSF Jak2* Receptor
SCF/Flt-3
Receptor
PI3
shp2
p-stats
akt
MAPK
mTor
Rap X
p-S6
p-mek
UO126 X
p-erk
p38
MAPK
p-mek
• 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
SSigignnaalilninggCCyyttoommeettrryyffoorraannaalylyssisisooffccoommpplelexxssaammppleless
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Single cell (Flow Cytometry) vs Bulk assay (Western Blot)
Signaling Cytometry
Signaling Simplified
赵静 贝克曼库尔特公司
What is Cell Signaling?
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•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
•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
Number of cells
Blasts
13 71
21 117
Monos
18
83
Immature grans
21
Mature grans
11
Unstimulated
45
23
IL-3
24
26
14
Stem cell factor (SCF)
142
229
100
GMCSF
Phosphorylated stat5
Western Blot
% Pos Cells
Relative Protein Expression
1%
50%
100
50
Flow Cytometry
100% 1
Relative Fluorescence
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Signaling Cytometry: Use of one or multiple signal transduction markers in conjunction with cell surface markers to dissect complex Biological processes in heterogeneous populations
– Raf kinase inhibitors – Mek inhibitors • Rapamycin • PI3 kinase inhibitors
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Signal 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?
• 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 Confidential
Company Confidential
Signal transduction in AML
IL-3/GM-CSF Jak2* Receptor
SCF/Flt-3
Receptor
PI3
shp2
p-stats
akt
MAPK
mTor
Rap X
p-S6
p-mek
UO126 X
p-erk
p38
MAPK
p-mek