MCF10A-Er-Src诱导转化模型的构建

目的 构建能够被他莫西芬（tamoxifen,TAM）激活的MCF10A-Er-Src诱导转化模型.方法 利用PCR扩增法分别获得雌激素受体配体结合结构域序列（hbER）和Src序列,并将其分别插入到改造后的反转录病毒表达载体pMSCV-FLAG-HA中,构建ER-Src融合蛋白表达质粒.包装pMSCV-ER-Src-FLAG-HA重组反转录病毒,感染MCF10A,利用基因组PCR和Western blot法筛选能够稳定表达外源融合蛋白ER-Src的细胞系.利用免疫沉淀（IP）和Western blot法验证TAM对稳转细胞系的激活,并观察细胞形态变化.结果 构建的MCF10A-Er-Src细胞系能够稳定表达ER-Src融合蛋白.TAM能诱使Src第416位酪氨酸发生磷酸化,从而激活Src,使MCF10A-Er-Src发生转化.结论 成功获得能够被TAM诱导激活的MCF10A-Er-Src转化模型.

Construction of an Inducible Transformation Model Consisting of Nontransformed MCF-10A Containing Er-Src

Objective To construct an transformation model consisting of non - transformed MCF - 10A containing Er - Src, which can be induced to transform by tamoxifen （TAM）. Methods To construct ER - Src, DNA sequences encoding the hormone binding do- main of the HE14 allele of the human estrogen receptor （hbER） were fused to the Schmidt Ruppin A form of v - Src and cloned into the pMSCV - FLAG - HA retrovirus expression vector. MCF - 10A were infected with retrovirus expressing ER - Src, and cells were tested for expression of ER - Src by genome PCR and Western blot. The activation of ER - Src was examined by immunoprecipitation and Western blot, and the changes of morphological transformation of MCF10A - Er - Src after induction of TAM was observed. Results MCF10A can stably express ER - Src. Src Y416 was phosphorylated after induction of TAM, and the activation of ER - Sre lead to the transformation of MCF10A - Er - Src. Conclusion The inducible transformation model of MCF10A - Er - Src was successfully constructed.