Maeda Y, Ikeda U, Ogasawara Y, Urabe M, Takizawa T, Saito T, Colosi P, Kurtzman G, Shimada K, Ozawa K
Department of Cardiology, Jichi Medical School, Tochigi, Japan.
Cardiovasc Res. 1997 Sep;35(3):514-21. doi: 10.1016/s0008-6363(97)00163-6.
Recombinant viral vectors based on the nonpathogenic parvovirus, adeno-associated virus (AAV), have a number of attractive features for gene therapy, including the ability to transduce non-dividing cells and its long-term transgene expression. In this study, an AAV vector containing bacterial beta-galactosidase gene (lacZ) was used to transduce cultured rat vascular smooth muscle cells (VSMC) in vitro and rat thoracic aortas ex vivo.
VSMC were transduced with AAV-lacZ at multiplicities of infection (MOI) ranging from 5.0 x 10(5) to 1.0 x 10(7). Expression of beta-galactosidase (beta-gal) in VSMC was evaluated by X-gal staining and a beta-gal ELISA method. Excised rat aortas were incubated with medium containing AAV-lacZ. Expression of beta-gal in the aortic segments was evaluated by X-gal staining.
With increasing MOI, up to 50% of cultured VSMC were positive by X-gal staining and the beta-gal expression increased up to 15 ng/mg protein. The expression gradually decreased during the culture but was detectable for at least 1 month. In the ex vivo study, AAV vectors transduced endothelial and adventitial cells in rat aortic segments, while no expression was seen in medial VSMC.
AAV vectors can efficiently transduce rat VSMC in vitro. AAV-mediated ex vivo gene transfer into the normal aorta resulted in efficient gene transfer into endothelial and adventitial cells but not into medial VSMC. These findings suggest that AAV-based vectors are promising for use in cardiovascular gene therapy.
基于无致病性细小病毒腺相关病毒（AAV）构建的重组病毒载体，在基因治疗方面具有诸多吸引人的特性，包括能够转导非分裂细胞以及实现长期转基因表达。在本研究中，使用携带细菌β-半乳糖苷酶基因（lacZ）的AAV载体，在体外转导培养的大鼠血管平滑肌细胞（VSMC），并在离体状态下转导大鼠胸主动脉。
用感染复数（MOI）范围为5.0×10⁵至1.0×10⁷的AAV-lacZ转导VSMC。通过X-gal染色和β-半乳糖苷酶ELISA法评估VSMC中β-半乳糖苷酶（β-gal）的表达。将切除的大鼠主动脉与含有AAV-lacZ的培养基一起孵育。通过X-gal染色评估主动脉段中β-gal的表达。
随着MOI增加，高达50%的培养VSMC经X-gal染色呈阳性，β-gal表达增加至15 ng/mg蛋白。在培养过程中表达逐渐下降，但至少1个月内仍可检测到。在离体研究中，AAV载体转导大鼠主动脉段的内皮细胞和外膜细胞，而中膜VSMC未见表达。
AAV载体可在体外有效转导大鼠VSMC。AAV介导的离体基因转移至正常主动脉可导致基因有效转移至内皮细胞和外膜细胞，但不会转移至中膜VSMC。这些发现表明基于AAV的载体在心血管基因治疗中具有应用前景。
