重庆医科大学检验医学院临床血液学教研室临床检验诊断学教育部重点实验室
Published:2017,
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Wang Teng, Huang Zhenglan, Gao Miao, et al. Effect of FABD on Bcr/Abl cytoplasm localization and function in 293T cells. [J]. Journal of Chongqing Medical University 42(11):1477-1483(2017)
Wang Teng, Huang Zhenglan, Gao Miao, et al. Effect of FABD on Bcr/Abl cytoplasm localization and function in 293T cells. [J]. Journal of Chongqing Medical University 42(11):1477-1483(2017) DOI: 10.13406/j.cnki.cyxb.001405.
目的:构建F-actin结合结构域(F-actin binding domain
FABD)缺失腺病毒载体Ad-Bcr/Abl-ΔFABD和野生型Bcr/Abl腺病毒载体Ad-Bcr/Abl
探讨FABD结构域对Bcr/Abl蛋白定位及功能的影响。方法:PCR扩增bcr/abl和bcr/abl-ΔFABD片段
双酶切后克隆至穿梭质粒p Ad Track-CMV
经Ad Easy构建系统获得腺病毒Ad-Bcr/Abl-ΔFABD和Ad-Bcr/Abl
并在HEK293细胞中进行包装。将细胞分为3组
Ad-GFP组、Ad-Bcr/Abl组以及Ad-Bcr/Abl-ΔFABD组
分别加入对应的腺病毒。Western blot验证Bcr/Abl和Bcr/Abl-ΔFABD在293T细胞中的表达水平
间接免疫荧光法检测Bcr/Abl和Bcr/Abl-ΔFABD在293T细胞中的定位。流式细胞术、MTT法分别检测Bcr/Abl和Bcr/Abl-ΔFABD对293T细胞凋亡、增殖的影响。结果:双酶切以及测序验证穿梭质粒构建正确;PacⅠ酶切验证重组腺病毒质粒重组成功;绿色荧光显示重组腺病毒包装成功;Western blot验证Bcr/Abl和Bcr/Abl-ΔFABD可在293T细胞中表达;间接免疫荧光发现Bcr-Abl蛋白在细胞内的分布依赖于F-actin;FABD缺失后虽然不能使Bcr/Abl入核
但使其在细胞内的分布方式由均匀变为点状或块状聚集;FABD缺失后
Bcr/Abl在细胞内呈块状聚集的方式与IM处理后的结果相似。流式细胞术和MTT结果显示
与Bcr/Abl相比
Bcr/Abl-ΔFABD组293T细胞的凋亡比例增加(P<0.05)
增殖能力降低(P<0.05)。结论:成功构建Ad-Bcr/Abl和Ad-Bcr/Abl-ΔFABD重组腺病毒
FABD缺失后使Bcr/Abl蛋白在细胞质中的分布方式发生改变
并能抑制Bcr/Abl的抗凋亡能力和促增殖能力。
Objective:To construct the recombination adenoviruses Ad-Bcr/Abl-ΔFABD and Ad-Bcr/Abl
and to explore the effect of FABD on the cytoplasm localization and function of Bcr/Abl oncoprotein. Methods:The fragments of bcr/abl and bcr/abl-ΔFABD gene were amplified by PCR and cloned into shuttle vector p Ad Track-CMV. The adenoviruses Ad-Bcr/Abl-ΔFABD and Ad-Bcr/Abl were constructed by Adeasy system and amplified in human embryonic kidney HEK293 cells. The expressions of Bcr/Abl-ΔFABD and Bcr/Abl in 293 T cells were detected by Western blot. The cytoplasm localization of Bcr/Abl-ΔFABD and Bcr/Abl in 293 T cells was detected by indirect immunofluorescence. The effect of Bcr/Abl and Bcr/Abl-ΔFABD on apoptosis and proliferation in 293 T cell was detected by flow cytometry and MTT. Results:The shuttle plasmids of adenovirus were confirmed to be correctly constructed by double digestion and gene sequencing. The recombination adenovirus plasmids was verified by PacⅠ digestion. The expression of GFP indicated the adenoviruses were packed. The constructed adenoviruses were expressed in 293 T cells by Western blot. Bcr/Abl-ΔFABD protein located in the cytoplasm not in nuclear as form of particles and did not depend on the distribution of the F-actin? After treating by IM
the distribution of Bcr/Abl is similar with that of Bcr/Abl-ΔFABD protein. As results of flow cytometry and MTT
effect of Bcr/Abl-ΔFABD on apoptosis in 293 T cell was promoted(P<0.05)and effect on proliferation was reduced(P<0.05)compared with that of Bcr/Abl. Conclusion:The adenoviruses Ad-Bcr/Abl-ΔFABD and Ad-Bcr/Abl are constructed successfully. The distribution of Bcr/Abl-ΔFABD mutation protein has changed. The deletion of FABD inhibit the anti-apoptosis ability and proliferation of Bcr/Abl.
Bcr/AblFABD细胞定位来普霉素伊马替尼
Bcr/AblFABDcytoplasm localizationLMBimatinib
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