摘要
目的观察可分泌表达人降钙素基因相关肽(humancalcitoningene-relatedpeptide,hCGRP)重组腺相关病毒(rAAV)体外转染培养的血管内皮细胞(endothelialcellsofvessel,ECV)及其作用,探讨其应用于血管性勃起功能障碍基因治疗的可行性。方法分别以可分泌表达hCGRP的重组腺相关病毒VssHGCMV-hCGRP、表达报告基因GFP中的重组腺相关病毒VssCMV-GFP和空病毒pssHGCMV体外转染培养的ECV细胞,采用斑点印迹试验检测培养基中的hCGRP,以放射免疫法检测转染细胞中的cAMP和cGMP水平,观察GFP和hCGRP的表达及其对ECV细胞的影响。结果腺相关病毒载体能有效地将外源基因hCGRP导入血管内皮细胞并稳定表达,VssHGCMV-hCGRP病毒组细胞中cAMP水平(45.74±3.45)nmol/L较pssHGCMV空病毒组(14.84±0.65)nmol/L和对照组(12.74±0.65)nmol/L明显高(P<0.01),并可在实验组24h细胞培养液中检测到免疫反应性hCGRP。结论可分泌表达生物活性肽重组腺相关病毒基因转移系统可有效进行多肽类在血管内皮细胞过表达,并可选择性刺激该细胞中cAMP水平升高,提示hCGRP可作为血管性勃起功能障碍基因治疗的有效候选基因。
Objective To observe the effect of human calcitonin gene-related peptide （hCGRP） gene mediated by recombinant adeno-associated virus （rAAV） on cultured endothelial cells of vessel （ECV） and explore the possibility of using CGRP gene for gene therapy in vascular erectile dysfunction. Methods CGRP gene was transferred into cultured ECV cells by recombinant adeno-associated virus VssHGCMV-hCGRP in vitro. CGRP-like immunoreactivity was measured by protein dot blot assay in the 24h-culture medium, and the imtracellular cAMP and cGMP levels in the cultured cell were also determined using radioimmunoassay to ascertain bioactivity of transduced CGRP. Results The exogenous gene was transferred into cultured ECV cells by VssHGCMV- hCGRP infection and efficiently expressed. Compared with group rAAV and control, intracellular cAMP level in the cultured cell infected with VssHGCMV- hCGRP were significantly increased （45.7±3.45） nmol/L vs （14.8±0.65）nmol/L and （12.7±0.65）nmol/L （P 〈 0.01）, whereas there was no difference in the cGMP level between two groups, and CGRP-like immunoreactivity was also detected in the culture medium of cells infected by VssHGCMV- hCGRP. Conclusion The system secretory expressing bioactive peptide rAAV mediated gene transfer may be used to express efficiently exogenous gene in vascular endothelial cells. CGRP gene can serve as one of the promising candidate gene for gene therapy in vascular erectile dysfunction.
出处
《中国男科学杂志》
CAS
CSCD
2006年第1期17-21,共5页
Chinese Journal of Andrology
关键词
重组腺相关病毒
降钙素基因相关肽
转染
血管内皮细胞
recombinant adeno-associated virus
calcitonin gene-related peptide
transfection in vitro
vascular endothelial cells
