重组β-半乳糖苷酶腺病毒载体的构建及其在血管平滑肌细胞中的表达

The construction of recombinant β-galactosidase adenovirus and its expression in transduced rat aortic smooth muscle cells

目的 :构建表达 β -半乳糖苷酶 (β -gal)的重组腺病毒 ,以期为再狭窄的基因治疗研究提供一个对照载体和为腺病毒介导的基因转移的安全性、可行性、转染效率的研究提供一有用的工具。方法 :采用基因重组方法构建穿梭质粒pAd - β -gal,通过同源重组法制备出重组腺病毒Ad - β -gal,转染的 2 93细胞用X -gal染色鉴定Ad - β-gal的正确与否。通过测定 2 6 0nm的紫外光吸收值估计病毒高度。原代大鼠血管平滑肌细胞采用组织块培养法。Ad - β -gal以 10 0moi感染VSMCs并行X -gal染色 ,以观察 β -gal在VSMCs中的表达情况。 结果 :成功构建了pAd - β -gal穿梭质粒和Ad - β -gal重组腺病毒 ,转染的 2 93细胞和VSMCs经X -gal染为蓝色 ,病毒浓度为 9× 10 11pfu/ml,以 10 0moiAd - β-gal转染VSMCs,其转染效率接近 10 0 %。结论 :表达 β -gal的重组腺病毒构建成功 ,并在VSMCs中得到有效表达 ,本研究为将来再狭窄基因治疗研究提供了一对照载体 ,也为腺病毒介导的基因转移的安全性、可行性、有效性的研究提供了一有用的工具。

Objective To construct a replication-defective recombinant adenovirus expressing β-galactosidase for providing a control vector in future study of gene therapy for restenosis, and of safety, feasibility, and efficiency of gene transfer mediated by adenovirus. Methods The shuttle vector pAd-β-gal was constructed by recombinant DNA technology and characterized by endonuclease digestion and agarose gel electrophoresis. Ad-β-gal was generated by homologous recombination and characterized by staining the transfected 293 cells with X-gal. The virus titer was determined by measuring the absorbance at 260 nm.Rat aortic vascular smooth muscle cells (VSMCs) were cultured by explant methods and characterized by specific smooth muscle α-actin immunohistochemical staining. VSMCs were transfected with Ad-β-gal at 100 moi and stained with X-gal to observe the expression of β-galactosidase. Results (1)pAd-β-gal shuttle vector and Ad-β-gal were constructed successfully;(2)The transfected 293 cells and VSMCs were stained blue with X-gal;(3)The virus titer is 9×10 11 pfu/ml;(4)The efficiency of gene transfer in VSMCs at 100 moi was nearly 100%. Conclusions (1)The recombinant adenovirus expressing β-galactosidase, Ad-β-gal, was constructed, successfully;(2) This investigation provides an excellent control vector for future study of gene therapy in restenosis;(3)Ad-β-gal is a useful tool in the research of safety, feasibility and the efficiency of gene transfer mediated by adenovirus.