Gheflat Shivasadat, Sadeghi Abdolrahim, Bandehpour Mojgan, Ramezani Keyvan, Kazemi Bahram
Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Iran J Pharm Res. 2019 Fall;18(4):2111-2116. doi: 10.22037/ijpr.2019.14650.12567.
Diabetes is a common disorder worldwide, and exhaustive efforts have been made to cure this disease. Gene therapy has been considered as a potential curative method that has had more stability in comparison with other pharmaceutical methods. However, the application of gene therapy as a definitive treatment demands further investigation. This study is aimed to prepare a suitable high- performance vector for gene therapy in diabetes mellitus. The designed vector has had prominent characteristics, such as directed replacement, which makes it a suitable method for treating or preventing other genetic disorders. The whole rDNA sequence of the human genome was scanned. The 800 bp two homology arms were digested by EcoRI, synthesized and cloned into the pGEM-B1 plasmid (prokaryotic moiety). The carbohydrate sensitive promoter, L-pyruvate kinase, and insulin gene were sub-cloned between homologous arms (eukaryotic moiety). The PGEM-B1 plasmid was digested by EcoRI, and the eukaryotic fragments were purified and transfected into Hela cell and then cultured. Afterward, the 300 µg/mL of glucose were added to the culture medium. Insulin expression in the transfected cells with 200 and 400 ng of the construct in comparison with negative control was detected using western blot and ELISA methods. Results have shown insulin expression in different glucose concentrates.
糖尿病是一种全球常见的疾病，人们已经为治愈这种疾病付出了巨大努力。基因治疗被认为是一种潜在的治疗方法，与其他药物治疗方法相比，它具有更高的稳定性。然而，基因治疗作为一种确定性治疗方法的应用仍需要进一步研究。本研究旨在制备一种适合糖尿病基因治疗的高效载体。所设计的载体具有突出的特性，如定向置换，这使其成为治疗或预防其他遗传疾病的合适方法。对人类基因组的整个rDNA序列进行了扫描。用EcoRI酶切800 bp的两个同源臂，合成并克隆到pGEM - B1质粒（原核部分）中。将碳水化合物敏感启动子、L - 丙酮酸激酶和胰岛素基因亚克隆到同源臂之间（真核部分）。用EcoRI酶切PGEM - B1质粒，纯化真核片段并转染到Hela细胞中，然后进行培养。随后，向培养基中加入300 µg/mL的葡萄糖。使用蛋白质免疫印迹法和酶联免疫吸附测定法检测与阴性对照相比，转染200 ng和400 ng构建体的细胞中的胰岛素表达。结果显示了在不同葡萄糖浓度下的胰岛素表达。
