人降钙素基因相关肽重组腺相关病毒在原代培养大鼠阴茎海绵体平滑肌细胞中的表达及其作用

Expression and effect of hCGRP Recombinant Adeno-associated Virus in Primary Cultured Corporal Cavernosum Smooth Muscle Cells of the Rat in vitro

目的:观察腺相关病毒载体介导的人降钙素基因相关肽(hCGRP)基因在原代培养的大鼠阴茎海绵体平滑肌(CCSM)细胞的表达及其作用,探讨其应用于勃起功能障碍(ED)基因治疗的可行性。方法:原代培养的SD大鼠CCSM细胞随机分为4组:实验组、空病毒组、示踪剂组和对照组,分别以可分泌表达hCGRP重组腺相关病毒VssH-GCMV-hCGRP、空病毒VssHGCMV和表达绿色荧光蛋白(GFP)重组腺相关病毒VssCMV-GFP进行体外转染或不予任何处理。采用斑点印迹试验检测培养液中的hCGRP和放射免疫法检测转染细胞中的环磷酸腺苷(cAMP)和环磷酸鸟苷(cGMP)水平,观察hCGRP和示踪剂GFP的表达及其对CCSM细胞的影响。结果:重组腺相关病毒载体能有效地将外源基因hCGRP导入大鼠CCSM细胞并稳定表达,与空病毒组和对照组相比,该病毒明显刺激大鼠CCSM细胞中cAMP水平升高[(48.7±1.1)nmol/L对(12.3±1.2)nmol/L和(7.8±1.4)nmol/L,P均<0.01],培养液中可检测到免疫反应性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CCSM细胞过表达,并影响该细胞的cAMP水平,可被应用于ED的基因治疗。

Objective： To observe the expression and effect of human calcitonin gene-related peptide（hCGRP） gene mediated by recombinant adeno-assoeiated virus（rAAV） in primary cultured corporal cavernosum smooth muscle cells of the rat and explore the possi- bility of using CGRP gene for gene therapy in erectile dysfunction. Methods ： The primary cultured corporal cavernosum smooth muscle cells of the rat were randomly divided into 4 groups and infected with recombinant virus VssHGCMV-hCGRP, VssHGCMV, VssCMV-GFP and the untreated, respectively, CGRP-like immunoreactivity was measured by protein dot blot assay in the 24 b-culture medium, and intracellular cAMP and cGMP levels in the cultured cells were also determined using radioimmunoassay to ascertain bioactivity of transduced CGRP. Results. The exogenous gene was transferred into primary corporal cavernosum smooth muscle cells by VssHGCMV-hCGRP infection and efficiently expressed. Compared with the control group, intracellular cAMP level in the cell infected by VssHGCMV-hCGRP was significantly increased （48.7 ± 1.1 nmol/L vs 7.8 ± 1.4 nmol/L, P 〈 0.01 ） , whereas cGMP level remained unchanged in two groups, and CGRP-like immunoreactivity was also detected in the culture medium infected by VssHGCMVhCGRP. Conclusion ： The system of secretory expressing bioactive peptide rAAV mediated gene transfer may be used to express efficiently exogenous gene in corporal cavernosum smooth muscle cells and affect cAMP level in the corporal cavemosum smooth muscle cells of the rat. Natl J Androl,2005,11（12） ：918-921