Li Rongying, Oka Kazuhiro, Yechoor Vijay
Department of Medicine, Baylor College of Medicine.
J Vis Exp. 2012 Oct 10(68):4321. doi: 10.3791/4321.
Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes. Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression(1). They lack chronic toxicity because they lack viral coding sequences(2-5) and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes. In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice.
1型糖尿病是由T细胞介导的胰腺中胰岛素生成细胞的自身免疫性破坏引起的。到目前为止，胰岛素替代疗法仍然是主要治疗方法，因为胰岛移植受到供体可用性以及长期免疫抑制需求的限制。通过胰岛谱系定义特异性转录因子神经源素3（Ngn3）和胰岛生长因子β细胞ulin（Btc）的基因转移诱导胰岛新生有治愈1型糖尿病的潜力。腺病毒载体（Ads）是高效的基因转移载体；然而，早期一代的腺病毒载体在体内使用时有几个缺点。辅助依赖型腺病毒载体（HDAds）是为改善早期一代腺病毒载体的安全性和延长转基因表达而开发的最先进的腺病毒载体（1）。它们缺乏慢性毒性，因为它们缺乏病毒编码序列（2 - 5），并且仅保留载体复制和包装所需的腺病毒顺式元件。这允许克隆长达36 kb的基因。在本方案中，我们描述了生成HDAd - Ngn3和HDAd - Btc并将这些载体递送至链脲佐菌素诱导的糖尿病小鼠体内的方法。我们的结果表明，HDAd - Ngn3和HDAd - Btc的共注射可在肝脏中诱导“新胰岛”并逆转糖尿病小鼠的高血糖症。
