通过自我更新因子标记肿瘤干细胞

daviddow

Wnt activity defines colon cancer stem cells and is regulated by the microenvironment. Nat Cell Biol. 2010 May;12(5):468-76. Epub 2010 Apr 25.( G. ~  y0 x/ j, e8 b
Most tumor cells undergo differentiation and lost tmorigenic potential.
+ O- Y$ x! s* wMutation of β-catenin is an early event in the transformation of colonic epithelial cells and established colorectal cancer depend critically on wnt signaling, but immunohistochemcal studies show that not all colorectal cancer cells is stained positively of nuclearβ-catenin. So , it maybe only expressed in CSC.
* s% P2 @1 |' V7 k: LColon cancer spheroidal cultures have heterogeneous Wnt activity levels: 1): Assess wnt signaling activity with TOP-GFP, control reporter(non-inducing firefly luciferase and constitutively expressed renilla luciferase), PGK-GFP and CMV-GFP(constitutive). To exclude variation in lentiviral intigration site and copy number, a single-clone was selected to do next.
" A4 Y" d. y! FCells in the single-cell-derived TOP-GFP culture show considerable heterogeneity in wnt signaling (about 100-fold ). 这个可以通过luciferase检测。 在CMV-GFP转染的细胞中没有出现这种差异。与干细胞有关的基因在TOP-GFPhigh细胞中表达，与上皮细胞有关的基因在TOP-GFPlow细胞中表达。Microassay， Real-time PCR和influorescence。上述data表示TOP-GFP细胞在培养后会自我更新和发生分化，wnt信号的活性不同。。2 O5 a) X1 w9 w* T8 V! k4 C$ ]
Clone CSCs are characterized by high expression levels of wnt activity. 1): 克隆形成潜能比较：单细胞来源的结肠癌细胞（球培养来源），单细胞来源的结肠癌细胞系，结肠癌细胞系，原代的结肠癌细胞（球培养来源）；wnt和其他基因如P53,K-ras之间的关系；wnt标志的结肠癌干细胞和其他结肠癌肿瘤干细胞标志之间的关系。2）移植实验。移植后显示TOP-GFPhigh细胞生长出肿瘤高于TOP-GFPlow。TOP-GFPhigh细胞种植后生成和原肿瘤一样的癌症，β-catenin和GFP也表现异质性分布。通过Ki-67检测，TOP-GFPhigh细胞包括cycling and non-cycling cell. Xenograft分离的结肠癌TOP-GFPhigh细胞克隆形成能力更强。TOP-GFPhigh细胞含有其他实验发现的结肠癌干细胞标志。3）自我更新实验。Xenograft分离的结肠癌TOP-GFPhigh细胞形成肿瘤的能力更强。TOP-GFPhigh细胞种植后生成和原肿瘤一样的癌症，β-catenin和GFP也表现异质性分布。我们可以分析多个乳腺癌细胞系和notch之间的关系，分析notch界定的干细胞和其他自我更新因子之间的关系和分析与其他肿瘤干细胞标志之间的关系。

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fjmedzengyue

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