反义Smad3腺病毒表达载体的构建及体外表达

Construction of recombinant antisense Smad3 adenoviral vector and its expression in vitro

目的 构建在体外细胞中高效表达的反义Smad3腺病毒载体,探讨应用于基因治疗病理性瘢痕的可行性。方法 用DNA直接克隆连接的方法,构建反义Smad3腺病毒重组质粒,再用293细胞包装。重组腺病毒经扩增、纯化后,感染体外培养的瘢痕疙瘩成纤维细胞,RT-PCR检测细胞中反义Smad3 mRNA的转录。结果 经酶切和PCR鉴定证实反义Smad3重组腺病毒质粒构建成功,RT-PCR证实瘢痕疙瘩细胞中有腺病毒载体介导的反义Smad3 mRNA的表达。结论 所构建的反义Smad3腺病毒表达载体可在瘢痕疙瘩成纤维细胞中表达,可进一步应用于瘢痕疙瘩基因治疗的研究。

Objective To construct the recombinant antisense Smad3 adenoviral vector with efficient expression in cuhured cells in vitro, and to evaluate the possibility of gene therapy for pathologic scar. Methods The recombinant antisense Smad3 adenoviral vector was constructed by the direct DNA cloning protocol and then transfected into 293 cells for virus packaging. After amplification and purification, the recombinant adenovirus was used to infect the keloid fibroblasts. The antisense Smad3 mRNA transcription of the infected cells was detected by RT-PCR. Results The recombinant adeno-antiSmad3 was correctly constructed and confirmed by both restriction analysis and PCR analysis. RT-PCR showed the expression of adenovirus mediated antiSmad3 mRNA in keloid cells. Conclusion These results demonstrate that the recombinant antisense Smad3 adenoviral vector can be expressed in cuhured keloid fibroblasts in vitro, and it may provide a new therapeutic strategy for keloid gene therapy.