视黄酸依赖反义cyclinD1 RNA表达载体的构建与鉴定

Construction of retinoic acid-dependent anti-sense RNA expression vector of human cyclinD1

目的 构建人细胞周期素D1(CyclinD1)基因的视黄酸 (Retinoicacid ,RA)依赖反义RNA表达载体 ,为进一步在细胞内应用视黄酸诱导其表达从而发挥效应的肿瘤基因治疗奠定基础。方法 运用DNA重组技术构建含有RARE3 TK启动子的反义cyclinD1RNA真核表达载体 (pCI neo RARE3 TK AScyclinD1) ,经酶切、PCR以及DNA测序确认后 ,用脂质体方法转染HL 6 0细胞 (人早幼粒白血病细胞系 ) ,RA处理后 ,利用免疫组织化学方法检测细胞内靶基因cyclinD1的表达情况。结果 成功构建重组反义cyclinD1RNA表达载体 ,与预期设计路线相符 ;检测DNA序列与原始片段互补 ,且方向相反。ATRA处理转染和未转染HL 6 0细胞后 ,cyclinD1的表达均有所下降 ,其中转染组细胞的表达量要明显低于未转染组。结论 本实验成功构建了视黄酸依赖反义cyclinD1RNA表达载体 ;转染HL 6 0细胞后 ,反义cyclinD1的表达可受到RA的诱导调控。

Objective To construct an anti sense RNA expression vector of human cyclinD1 for the study of inducing in retinoic acid dependent manner in the gene therapy of tumor. Methods An anti sense cyclinD1 RNA eukar yotic expression vector (pCI neo/RARE3 TK/AScyclinD1) containing RARE3 TK was constructed with DNA recombination technique, identified with restriction endonuclease digestion and PCR and DNA sequence analysis, then transfected into the cells of HL 60 with liposome DOTAP, and observed the expressions of cyclin D1 by immunohistochemistry in the cells treated with RA. Results Anti sense cyclin D1 fragment and RARE3 TK fragment were amplified and cloned into pCI neo expression vector successfully. Nucleotide sequence analysis showed that the constructed fragment was complemented and opposice to human original gene fragment . After transfected or non transfected cells were treated with RA, the expressions of cyclin D1 were both decreased and cyclin D1 expression was lower in transfected cells than nontransfected. Conclusion An anti sense cyclinD1 RNA eukar yotic expression vector (pCI neo/RARE3 TK/AScyclinD1) containing RARE3 TK was successfully constructed. After transfected into the cells of HL 60, anti sense cyclin D1 was expressed in the retinoic acid dependent manner.