| 张国鹏,沈艳霞,朱小华.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[J].肿瘤学杂志,2012,18(10):724-728. |
| 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-mediated Herpes Simplex Virus Type 1 Thymidine Kinase (HSV1-tk) Gene Transfecting to Endothelial Progenitor Cells |
| 投稿时间：2012-09-03 |
| DOI：10.11735/j.issn.1671-170X.2012.10.B002 |
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| 中文关键词: 肿瘤血管生成 内皮祖细胞 基因治疗 单纯疱疹病毒胸腺激酶 腺病毒 载体 |
| 英文关键词:tumor angiogenesis endothelial progenitor cells gene therapy herpes simplex virus type 1 thymidine kinase (HSV1-tk) adenovirus vector |
| 基金项目:中国教育部博士点新教师基金（20070487139）；华中科技大学自主创新研究基金（2011JC060） |
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| 中文摘要: |
| 摘 要:［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、Western blot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Western blot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。 |
| 英文摘要: |
| Abstract: ［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 cytometry. 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 detected by RT-PCR and Western blot. The cytotoxicity of GCV (Ganciclovir) on EPCs after infection 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 infection. This provides a possibility for target gene therapy and reporter gene imaging of carcinoma using EPCs transfected by HSV1-tk gene. |
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