Daxx is found in the nucleus where it localizes to PML oncogenic domains (PODs). Its multiple domains can interact proteins involved in transcriptional regulation and apoptotic signal transduction. In addition, Daxx i...Daxx is found in the nucleus where it localizes to PML oncogenic domains (PODs). Its multiple domains can interact proteins involved in transcriptional regulation and apoptotic signal transduction. In addition, Daxx is associated with viral infection、tumorigenesis and embryonic development.展开更多
目的构建重组慢病毒表达载体pCDH-Daxx-EGFP,并探讨Daxx对血管紧张素Ⅱ(AngⅡ)诱导VSMCs增殖的影响。方法基于PAS(PCR-based Accurate Synthesis)的方法构建重组慢病毒表达载体pCDH-Daxx-EGFP。经测序和酶切验证后,用重组慢病毒表达载体...目的构建重组慢病毒表达载体pCDH-Daxx-EGFP,并探讨Daxx对血管紧张素Ⅱ(AngⅡ)诱导VSMCs增殖的影响。方法基于PAS(PCR-based Accurate Synthesis)的方法构建重组慢病毒表达载体pCDH-Daxx-EGFP。经测序和酶切验证后,用重组慢病毒表达载体pCDH-Daxx-EGFP与包装辅助载体共转染293T细胞进行慢病毒包装,收集病毒液纯化后感染VSMCs,Western blot法鉴定过表达Daxx的VSMCs株。然后将pCDH-EGFP病毒液感染的空载体细胞和pCDH-Daxx-EGFP病毒液的感染的过表达Daxx细胞都分成无血清培养基孵育组和AngⅡ孵育组。用MTT法检测细胞活性、流式细胞术观察细胞周期、划痕法检测细胞迁移、Western blot法检测细胞中p-Akt蛋白表达。结果基因测序与双酶切鉴定表明Daxx基因慢病毒表达载体构建成功;与Vector组比,转染Daxx组、蛋白表达水平明显增加(P<0.05);经AngII处理后,转染Daxx组细胞活性、S期细胞比率和与细胞迁移率与Vector组比较显著降低(P<0.05);转染Daxx组细胞中p-Akt蛋白表达显著降低(P<0.05)。结论成功构建了重组慢病毒表达载体pCDH-Daxx-EGFP和过表达Daxx的VSMCs株;Daxx能显著抑制AngII诱导的VSMCs增殖和迁移,其机制可能与p-Akt蛋白有关。展开更多
基金The National Natural Science Foundationof China (30470719, 30600249)The Education Department of HunanProvince (99B11) The Sanitarian Research Foundation of HunanProvince (B2004-078)~~
文摘Daxx is found in the nucleus where it localizes to PML oncogenic domains (PODs). Its multiple domains can interact proteins involved in transcriptional regulation and apoptotic signal transduction. In addition, Daxx is associated with viral infection、tumorigenesis and embryonic development.
基金supported by grants from Hi-Tech Research and Development Program of China(2009ZX09503-020)National Basic Research Program of China(2010CB529903)The National Natural Science Foundation of China(30971617)~~
文摘目的构建重组慢病毒表达载体pCDH-Daxx-EGFP,并探讨Daxx对血管紧张素Ⅱ(AngⅡ)诱导VSMCs增殖的影响。方法基于PAS(PCR-based Accurate Synthesis)的方法构建重组慢病毒表达载体pCDH-Daxx-EGFP。经测序和酶切验证后,用重组慢病毒表达载体pCDH-Daxx-EGFP与包装辅助载体共转染293T细胞进行慢病毒包装,收集病毒液纯化后感染VSMCs,Western blot法鉴定过表达Daxx的VSMCs株。然后将pCDH-EGFP病毒液感染的空载体细胞和pCDH-Daxx-EGFP病毒液的感染的过表达Daxx细胞都分成无血清培养基孵育组和AngⅡ孵育组。用MTT法检测细胞活性、流式细胞术观察细胞周期、划痕法检测细胞迁移、Western blot法检测细胞中p-Akt蛋白表达。结果基因测序与双酶切鉴定表明Daxx基因慢病毒表达载体构建成功;与Vector组比,转染Daxx组、蛋白表达水平明显增加(P<0.05);经AngII处理后,转染Daxx组细胞活性、S期细胞比率和与细胞迁移率与Vector组比较显著降低(P<0.05);转染Daxx组细胞中p-Akt蛋白表达显著降低(P<0.05)。结论成功构建了重组慢病毒表达载体pCDH-Daxx-EGFP和过表达Daxx的VSMCs株;Daxx能显著抑制AngII诱导的VSMCs增殖和迁移,其机制可能与p-Akt蛋白有关。
