Department of Pediatrics, Shandong University Qilu Hospital, Jinan, Shandong 250012, China (Email:
Chin Med J (Engl). 2009 Sep 20;122(18):2159-64.
Islet beta-cells are almost completely destroyed when patients with type 1 diabete are diagnosed. To date, insulin substitute therapy is still one of the main treatments. The cure of type 1 diabetes requires beta-cell regeneration from islet cell precursors and prevention of recurring autoimmunity. Therefore, beta-cell regeneration and proliferation emerge as a new research focus on therapy for type 1 diabetes. Islet beta-cell regeneration and development are controlled by many growth factors, especially insulin-like growth factor-1 (IGF-1).
Recombinant adenovirus encoding rat IGF-1 (rIGF-1) was constructed and transduced into rat beta-cells, RINm5F cells. Western blotting analysis and ELISA were used to detect rIGF-1 protein. Streptozotocin (STZ) was used to induce RINm5F cell destruction. The level of nitric oxide (NO) was detected in cell culture supernatants by the Griess reaction. Islet cell function was evaluated by glucose-stimulated insulin production. Flow cytometry analysis was further used to investigate the apoptosis of RINm5F cells. Thiaoollyl blue viability assay was applied to determine cell viability.
The recombined adenovirus-rIGF-1 was successfully constructed and the titer was 4.0 x 10(8) pfu/ml. The rIGF-1 protein was effectively expressed in the RINm5F cells and cell culture supernatants. rIGF-1 expression remarkably inhibited STZ-induced islet cell apoptosis and significantly decreased the level of NO. Furthermore, IGF-1 expression also significantly protected insulin secretion and cell proliferation in a time-dependent manner.
Our study suggests that locally produced rIGF-I from RINm5F cells may be beneficial in maintaining beta-cell function, protecting beta-cells from the destruction of apoptosis factors and promoting beta-cell survival and proliferation. IGF-I might be considered as a candidate gene in gene therapy for type 1 diabetes. In addition, it appears that the apoptosis induced by STZ may be NO-dependent.
当 1 型糖尿病患者被诊断时，胰岛β细胞几乎完全被破坏。迄今为止，胰岛素替代疗法仍然是主要治疗方法之一。1 型糖尿病的治愈需要胰岛细胞前体细胞的β细胞再生和防止自身免疫的再次发生。因此，β细胞的再生和增殖成为 1 型糖尿病治疗的新研究焦点。胰岛β细胞的再生和发育受到许多生长因子的控制，特别是胰岛素样生长因子-1（IGF-1）。
构建并转染编码大鼠 IGF-1（rIGF-1）的重组腺病毒，转染大鼠β细胞 RINm5F 细胞。Western blot 分析和 ELISA 用于检测 rIGF-1 蛋白。链脲佐菌素（STZ）用于诱导 RINm5F 细胞破坏。通过格里斯反应检测细胞培养上清液中的一氧化氮（NO）水平。通过葡萄糖刺激胰岛素产生来评估胰岛细胞功能。进一步通过流式细胞术分析 RINm5F 细胞的凋亡。噻唑蓝比色法活力测定用于确定细胞活力。
成功构建了重组腺病毒-rIGF-1，滴度为 4.0×10(8)pfu/ml。rIGF-1 蛋白在 RINm5F 细胞及其细胞培养上清液中有效表达。rIGF-1 表达显著抑制 STZ 诱导的胰岛细胞凋亡，并显著降低 NO 水平。此外，IGF-1 表达还显著地以时间依赖性方式保护胰岛素分泌和细胞增殖。
我们的研究表明，从 RINm5F 细胞中局部产生的 rIGF-1 可能有益于维持β细胞功能，保护β细胞免受凋亡因子的破坏，并促进β细胞的存活和增殖。IGF-1 可被视为 1 型糖尿病基因治疗的候选基因。此外，STZ 诱导的凋亡似乎依赖于 NO。
