腺病毒介导的hVEGF_(165)基因治疗对骨髓移植后骨髓造血干细胞恢复的影响及其意义

The Effects on the Recovery of Hematopoietic Stem Cells in the Femurs of Post-BMT mice by Adenovirus Mediated hVEGF_(165) Gene Treatment and its Significence

目的 :探讨重组腺病毒Ad EGFP/hVEGF165介导的血管内皮细胞生长因子在骨髓移植后小鼠体内的表达效率及对骨髓造血干细胞恢复的影响。方法 :利用细菌内同源重组法快速构建复制缺陷型腺病毒Ad EGFP/hVEGF165,经尾静脉注射给同基因骨髓移植BALB/c小鼠 ;在移植后不同时间 ,通过荧光显微镜、RT PCR、免疫组化染色和ELISA方法检测外源基因在小鼠体内的表达 ;取移植后 30d小鼠骨髓单个核细胞并计数 ,随后进行脾集落形成单位试验 (CFU S)以评价VEGF基因转移组小鼠骨髓造血干细胞数量恢复情况。结果 :重组腺病毒AdEGFP/hVEGF165介导的VEGF基因转移广泛分布在骨髓移植小鼠心、肺、肝、脾、肾、小肠和骨髓组织 ;小鼠血浆中VEGF浓度高峰出现在移植后 2d ;接受重组腺病毒AdEGFP/hVEGF165基因干预治疗组小鼠骨髓单个核细胞计数及其形成的脾集落数量明显高于其他对照组。结论 :腺病毒成功介导了hVEGF165基因在骨髓移植小鼠体内的稳定表达 ,明显促进骨髓移植小鼠骨髓造血干细胞数量的恢复 ,是骨髓单个核细胞数量增加的根本原因。

Objective:To investigate the expression of adenovirus mediated vascular endothelial growth factor gene transfer and the effects on the recovery of bone marrow hematopoietic stem cells in post-BMT mice.Method:By using homologous recombination in bacteria, the replicating deficiency adenovirus Ad-EGFP/hVEGF 165 was constructed and then injected into post-BMT BALB/c mice vial the tail vein. The expression of hVEGF 165 gene were monitored by fluorescent microscope, RT-PCR, immunohistochemistry and ELISA at different times post bone marrow transplantation. At +30 d, bone marrow mononuclear cells were collected and counted, after which the CFU-S assays were performed to determine the recovery of bone marrow hemotopoietic stem cells in group of hVEGF 165 gene transfer.Result:The expression of adenovirus mediated vascular endothelial growth factor165 gene transfer was significiently expressed in the heart, lung, liver, spleen, kidney, intestinal and bone marrow of mice. The maximum plasma level of hVEGF 165 was observed at +2 d post BMT. The counts of bone marrow MNCs and numbers of CFU-S formed by them were higher in the group treated with Ad-EGFP/hVEGF 165 than those of control groups.Conclusion:Adenovirus successfully mediated the stable expression of hVEGF 165 in post-BMT mice, which facilitated the recovery of bone marrow HSCs and seemed to be the main reason for increase of bone marrow MNCs.