摘要
目的 制备含人Fas基因的重组腺病毒 ,感染瘢痕疙瘩成纤维细胞后 ,使其稳定高效地表达以替换原有无功能的Fas蛋白 ,并恢复正常的重建后Fas信号传导通道。方法 应用高效细菌内同源重组系统Ad Easy ,自PMD T Fas质粒中切取Fas基因 ,将Fas基因克隆到穿梭质粒 ,在BJ5 183细菌内重组 ,最后在 2 93细胞中构建携带Fas基因的重组腺病毒 .将腺病毒感染瘢痕疙瘩成纤维细胞 ,检测转染前后Fas蛋白的表达的变化及蛋白的功能。结果 通过Ad Easy系统成功构建高滴度的携带Fas基因的重组腺病毒Ad Fas ,Ad Fas感染瘢痕疙瘩成纤维细胞后能稳定提高Fas蛋白的表达 ,并且在Fas单克隆抗体的诱导下可以发现明显的细胞凋亡。结论 (1)构建的重组腺病毒Ad Fas在体外实验中能有效地提高瘢痕疙瘩成纤维细胞蛋白的表达 ,并且可以重建原来阻断的死亡信号传导通道。 (2 )再次证实了Fas基因突变与瘢痕疙瘩之间的联系 ,为瘢痕疙瘩基因治疗开辟了一条新途径。
Objective To generate recombinant adenovirus with human Fas gene and transfect the Fas gene into keloid-derived fibroblasts to take place of the dysfunctional Fas gene, reconstruct the blocked Fas signal. Methods We used a new bacterial homologous recombination system(Ad-Easy system) to construct recombinant adenovirus vector. Fas gene was cut down from the PMD-T-Fas plasmid and then transferred to track plasmid. Recombination was successfully completed in bacteria BJ5183 and recombinant adenovirus was produced in 293 cells. Then we infected keloid derived fibroblasts and compared the expression of Fas protein. Finally, we detected the function of newly produced Fas protein. Results We successfully constructed the recombinant adenovirus with Fas gene and detected its highly expressed Fas protein in infected keloid derived fibroblasts. Obvious apoptosis was also detected in infected keloid derived fibroblasts exposed to FasMcab. Conclusion (1)The recombinant adenovirus with Fas gene can transfect the Fas gene into keloid-derived fibroblasts and highly improved the expression of Fas protein. The newly expressed Fas gene can reconstruct the blocked Fas signal. (2) The correlation between keloid and Fas gene was further proved and it may paves a sound foundation for further gene therapy in keloid.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2004年第7期559-563,共5页
National Medical Journal of China
