Objective To construct AWP1 (associated with protein kinase C related kinase 1) recombinant adenovirus as the tool of transferring the gene and investigate its expression and localization in human vascular endothelial cell ECV304. Methods Cloned AWP1 cDNA was inserted into the multiply clone sites (MCS) of plasmid pcDNA3 for adding flag tag, and the flag-AWP1 gene was subcloned into shuttle vector pAdTrack-CMV. After identified with restrictional enzymes, plasmid pAdTrack-flag-AWP1 was linearized by digestion with restriction endonuclease PmeⅠ, and subsequently cotransformed into E.coli BJ5183 cells with adenoviral backbone plasmid pAdEasy-1 to make homologous recombination. After linearized by PacⅠ, the homologous recombinant adenovirus plasmid transfected into 293 cells with Lipofectamine to pack recombinant adenovirus. After PCR assay of recombinant adenovirus granules, recombinant adenoviruses infected 293 cells repeatedly for obtaining the high-level adenoviruses solution. And then, the recombinant adenoviruses infected human ECV304 cells for observing the expression and localization of AWP1 under laser scanning confocal microscope (LSCM). Results PCR assay showed that recombinant adenovirus Ad-flag-AWP1 was obtained successfully; and ECV304 cells were infected high-efficiently by the homologous recombinant virus. Then, it was observed that flag-AWP1 protein expressed in ECV304 cells and distributed in the leading edges of the cell membrane. Conclusion The vectors of flag-AWP1 recombinant adenovirus are constructed, and the localization of AWP1 protein in ECV304 cells might show that AWP1 may be a potential role on the cell signal transduction.
目的:研究丹参酮ⅡA(Tan ⅡA)对急性早幼粒细胞白血病(APL)细胞株NB4细胞诱导的血管内皮细胞株(ECV304)促凝活性(PCA)的影响,并对其机制作初步探讨。方法:(1)分别用1.0μg/mL TanⅡA、0.3μg/mLATRA、0.01%DMSO、PRMI1640处理NB4细胞24、48和72h,取其上清液作为条件培养基(hNB4-CM)。将这些CM分别与ECV304细胞在37oC共同孵育0、4、8和12h,用反复冻融法制备ECV304细胞裂解液,采用一期凝血法测定其PCA;采用ELISA法测定条件培养基中的TNF-α 。(2)ECV304细胞与1.0μg/mL TanⅡA及TanⅡA 72h-NB4-CM 在37oC共同分别孵育6、12、24和48h,并以ATRA和DMSO分别作为阳性和阴性对照,用上述相同方法测定ECV304细胞裂解液的PCA。结果:(1)1.0 μg/mL Tan ⅡA可以诱导NB4细胞分化,其作用NB4细胞的培养基有一定的升高ECV304细胞PCA的作用,该作用在孵育4h时达高峰,之后ECV304细胞PCA逐渐下降。与0.3μg/mL ATRA的作用无统计学差异(Pgt;0.05)。(2)1.0 μg/mL的TanⅡA对TanⅡA72h-NB4-CM促ECV304细胞PCA有抑制作用,其强度随作用时间增加而增加,与1.0μmol/L ATRA比较,Pgt;0.05。(3)TanⅡA作用NB4细胞的培养基中TNF-α浓度,在作用前7h内随作用时间增加而增加,与0.3μg/mL ATRA比较无差异(Pgt;0.05)。结论:Tan ⅡA能诱导NB4细胞分化,后者在分化过程中释放的TNF-α可能与ECV304细胞PCA活性升高有关;Tan-ⅡA又能抑制Tan-ⅡA-NB4-CM增强ECV304细胞PCA的作用。