eNOS基因重组腺病毒载体在大鼠骨髓来源内皮祖细胞中的表达分析

Construction of recombinant adenovirus containing eNOS gene and analysis of its expression and location in endothelial progenitor cells from rat bone marrow

目的探讨eNOS基因重组腺病毒载体在大鼠骨髓来源内皮祖细胞(EPCs)中的表达与作用。方法将eNOS基因插入载体PSUCMV中构建重组质粒PSUCMVeNOS,经酶切、插入、转染后包装扩增成腺病毒颗粒;大鼠胫骨获取骨髓,制备EPCs,鉴定正确后传代纯化,将三代细胞随机分为PBS对照组(C组)、Lac基因转染组(L组)和eNOS基因转染组(N组)。分别于干预后第1天、第3天、第7天观察各组细胞形态的变化,检测培养液中NO含量和各组细胞eNOS表达的变化。结果 AdCMVeNOS病毒DNA成功包装成完整的病毒颗粒,测其滴度为1.58×1010PFU/mL;将其转染鉴定正确的EPCs,结果于体外转染后1 d内,即可检测到eNOS转基因产物的表达增加;N组同一时点eNOS含量明显高于L组、C组,组间比较差异显著;组内比较,N7、N3与N1组比较差异显著。随时间延长NO生成量递增,第3天与第7天结果差异有显著性。结论 eNOS基因可在大鼠EPCs中高效表达,并可促使培养液中NO含量明显增高。

Objective To construct an adenovirus vector containing eNOS gene,and analyze the expression and location in endothelial progenitor cells （EPC） from rat bone marrow. Methods To establish the vector for recombinant adenovirus： the recombinant plasmid PSUCMV-heNOS was established by insertion of heNOS gene into the vector PSUCMV. It was transferred into the cell line 293 cells with the plasmid including the right arm of 5-type adenovirus by Lipofectamine 2000. 18 male Wistar rats, aged 4 -6 w, were selected for EPCs culture. EPCs from the rat bone marrow were transfected by recombinant adenovirus with eNOS gene. The cells in the third passages were randomly allocated to 3 groups： PBS control group （group C） , Lac gene transfection group （group L） and eNOS gene transfection group （group N）. Each group was separated into 3 subgroups following 1 d, 3 d and 7 d after treatment. The morphological changes, NO product in the culture medium and eNOS expression was monitored. Results The DNA of AdCMVheNOS was successfully packaged inside and transferred into the 293 cells by liposome. After confirmed by PCR analysis, the titer was 1.58 ×10^10 PFU/mL after amplification. The culture of EPCs from rat bone marrow was observed, and the expression of eNOS product was increased after transfection for one day. At the same time point, the content of eNOS in the group N was significantly higher than that of the groups L and C. The N7 and N3 subgroups showed a significant difference compared with the N1 subgroup. The NO production was increased following the time and the difference was statistically significant between the 3 d and 7 d subgroups. Conclusion Adenovirus can mediate a high expression of eNOS gene in the EPCs and induce an increase of NO content in the culture medium.