重组腺病毒介导自杀基因HSV1-tk转染内皮祖细胞的实验研究

Experimental Study on Recombinant Adenovirus Vector-me-diated Herpes Simplex Virus Type 1 Thymidine Kinase(HSV1-tk) Gene Transfecting to Endothelial Progenitor Cells

[目的]构建含有自杀基因单纯疱疹病毒胸腺激酶基因(HSV1-tk)的重组腺病毒载体,并感染大鼠骨髓来源的内皮祖细胞(EPCs),以期用于抗肿瘤血管生成的基因治疗及核素报告基因显像。[方法]从大鼠骨髓中分离、培养EPCs,利用流式细胞术进行鉴定。构建重组腺病毒载体Ad5-HSV1-tk-EGFP,并感染EPCs,以腺病毒Ad5-EGFP为对照。利用RT-PCR法、Westernblot免疫印迹法检测HSV1-tk的表达,利用MTT法检测更昔洛韦(GCV)对病毒感染后EPCs的杀伤作用。[结果]流式细胞术结果显示从大鼠骨髓中分离出的EPCs阳性表达CD34(80.09%)和CD133(81.75%),RT-PCR及Westernblot免疫印迹结果显示HSV1-tk基因在转录及蛋白水平可以正确表达,MTT结果显示HSV-tk/GCV自杀基因系统对EPCs细胞具有明显的杀伤作用。[结论]重组腺病毒Ad5-HSV1-tk-EGFP感染EPCs后可以在细胞中成功表达,并且感染后自杀基因具有生物学活性,从而为利用EPCs作为载体进行肿瘤靶向基因治疗、报告基因显像提供实验依据。

[Purpose] To construct the recombinant adenovirus vector containing herpes simplex virus type 1 thymidine kinase（HSV1-tk） gene and to transfect rat bone marrow derived endothelial progenitor cells（EPCs）,which will be used in antiangiogenic therapy and report gene imaging.[Methods] EPCs were isolated from rat bone marrow,cultured and then identified by flow cytome-try.The recombinant adenovirus vector Ad5-HSV1-tk-EGFP containing HSV1-tk was constructed,purified,and characterized by the gene technique.A replication-defective adenovirus carrying the enhanced green fluorescent protein gene（Ad5-EGFP） was used as a control.EPCs were infected with the recombinant adenovirus vector Ad5-HSV1-tk-EGFP,the expression of HSV1-tk was de-tected by RT-PCR and Western blot.The cytotoxicity of GCV（Ganciclovir） on EPCs after infec-tion with adenovirus vector was observed by MTT assay.[Results] Flow cytometry results showed that 80.09% cells expressed CD34 and 81.75% cells expressed CD133.RT-PCR and Western blot results displayed that the HSV1-tk gene expressed both at transcription and protein level after EPCs were infected by Ad5-HSV1-tk-EGFP.The MTT assay demonstrated an obvious lethal effect of ganciclovir（GCV） on EPCs expressed HSV1-tk.[Conclusions] Recombinant adenovirus Ad5-HSV1-tk-EGFP can infect EPCs successfully and the HSV1-tk gene can kill the EPCs after infec-tion.This provides a possibility for target gene therapy and reporter gene imaging of carcinoma using EPCs transfected by HSV1-tk gene.