Zhu N L, Wu L, Liu P X, Gordon E M, Anderson W F, Starnes V A, Hall F L
USC Gene Therapy Laboratories, Childrens Hospital of Los Angeles, and the University of Southern California School of Medicine, 90027, USA.
Circulation. 1997 Jul 15;96(2):628-35. doi: 10.1161/01.cir.96.2.628.
The contemporary treatment of coronary athero-occlusive disease by percutaneous transluminal coronary angioplasty is hampered by maladaptive wound healing, resulting in significant failure rates. Morbid sequelae include smooth muscle cell (SMC) hyperplasia and restenosis due to vascular neointima formation.
In this study, we examined the inhibitory effects of a concentrated retroviral vector bearing an antisense cyclin G1 gene on aortic SMC proliferation in vitro and on neointima formation in vivo in a rat carotid injury model of restenosis. Retroviral vectors bearing an antisense cyclin G1 construct inhibited the proliferation of transduced aortic SMCs in 2- to 6-day cultures, concomitant with down-regulation of cyclin G1 protein expression and decreased [3H]thymidine incorporation into DNA. Morphological examination showed evidence of cytolysis, giant syncytia formation, and apoptotic changes evidenced by overt cell shrinkage, nuclear fragmentation, and specific immunostaining of nascent 3'-OH DNA ends generated by endonuclease-mediated DNA fragmentation. Pronounced "bystander effects" including neighboring cells were noted in aortic SMCs transduced with the antisense cyclin G1 vector, as determined by quantitative assays and fluorescent labeling of nontransduced cells. In an in vitro tissue injury model, the proliferation and migration of antisense cyclin G1 vector-transduced aortic SMCs were inhibited. Moreover, in vivo delivery of high-titer antisense cyclin G1 vector supernatant to the balloon-injured rat carotid artery in vivo resulted in a significant reduction in neointima formation.
These findings represent the first demonstration of the inhibitory effects of an antisense cyclin G1 retroviral vector on nonneoplastic cell proliferation. Taken together, these data affirm the potential utility of antisense cyclin G1 constructs in the development of novel gene therapy approaches to vascular restenosis.
经皮腔内冠状动脉成形术对冠状动脉粥样硬化闭塞性疾病的当代治疗因适应性伤口愈合不良而受阻，导致显著的失败率。病态后遗症包括平滑肌细胞（SMC）增生和因血管内膜形成导致的再狭窄。
在本研究中，我们在大鼠颈动脉损伤再狭窄模型中，检测了携带反义细胞周期蛋白G1基因的浓缩逆转录病毒载体对体外主动脉平滑肌细胞增殖以及体内内膜形成的抑制作用。携带反义细胞周期蛋白G1构建体的逆转录病毒载体在2至6天的培养中抑制了转导的主动脉平滑肌细胞的增殖，同时细胞周期蛋白G1蛋白表达下调，且[3H]胸苷掺入DNA减少。形态学检查显示有细胞溶解、巨大多核细胞形成以及凋亡变化的证据，表现为明显的细胞皱缩、核碎裂以及由核酸内切酶介导的DNA片段化产生的新生3'-OH DNA末端的特异性免疫染色。通过定量分析和对未转导细胞的荧光标记确定，在用反义细胞周期蛋白G1载体转导的主动脉平滑肌细胞中观察到明显的“旁观者效应”，包括相邻细胞。在体外组织损伤模型中，反义细胞周期蛋白G1载体转导的主动脉平滑肌细胞的增殖和迁移受到抑制。此外，在体内将高滴度反义细胞周期蛋白G1载体上清液输送到球囊损伤的大鼠颈动脉，导致内膜形成显著减少。
这些发现首次证明了反义细胞周期蛋白G1逆转录病毒载体对非肿瘤细胞增殖的抑制作用。综上所述，这些数据证实了反义细胞周期蛋白G1构建体在开发血管再狭窄新基因治疗方法中的潜在效用。
