胰岛素样生长因子Ⅱ及雷帕霉素的联合干预在胰腺干细胞分化中的保护机制研究

IGF-Ⅱ restores rapamycin-induced suppression of β-cell differentiation and expansion of adult pancreas stem cells

目的研究免疫抑制剂雷帕霉素抑制新生猪胰腺成体干细胞（porcine neonatal pancreas cell clusters，NPCCs）分化和增殖的机制，探索能有效降低雷帕霉素不良反应的治疗方法。方法雷帕霉素/胰岛素样生长因子Ⅱ（insulin-like growth factor-Ⅱ，IGF-Ⅱ）处理NPCCs，检测caspase-3试验和[^3H]-胸腺嘧啶摄取试验分析细胞凋亡和增生，RT-PCR及Western blot分析胰岛细胞特定基因胰岛素、PDX-1、NeuroD/Beta2以及IGF-Ⅱ下游转录因子Foxo1的表达，评估胰腺成体干细胞的分化能力。结果NPCCs用雷帕霉素处理后，抑制β细胞的增殖、增加细胞凋亡，降低胰岛素分泌能力，抑制胰岛细胞特定基因PDX-1和NeuroD/Beta2的表达，明显降低IGF-Ⅱ的表达，增加Foxol的表达并诱导Foxol从胞质到细胞核的异位。雷帕霉素与IGF-Ⅱ的联合处理降低雷帕霉素的不良反应，抑制β细胞数量及胰岛素含量的减少，修复胰岛素、PDX-1、NeuroD/Beta2的表达，抑制Foxo1的表达及细胞内异位。结论IGF-Ⅱ及Foxol基因的异常表达是雷帕霉素抑制NPCCs的增殖和分化的重要诱因，且IGF-Ⅱ干预能有效降低雷帕霉素对NPCCs分化的不良反应。

ObjectiveTo investigate the mechanism of rapamycin inhibiting the differentiation and proliferation of newborn porcine pancreatic adult stem cells, and to explore the therapeutic methods that may effectively reduce the side effects of rapamycin.MethodPorcine NPCCs were treated with rapamycin alone or in combination with IGF-Ⅱ, and the caspase-3 and [^3H]-thymidine uptake assays were performed to detect apoptosis and proliferation. The expression of insulin, PDX-1, NeuroD/Beta2, and Foxo1, a downstream transcription factor of IGF-Ⅱ, were analyzed by RT-PCR and Western blot to evaluate the differentiation ability of pancreatic adult stem cells.ResultsThe NPCCs treated with rapamycin inhibited the proliferation of β-cells, increased apoptosis, reduced insulin secretion, inhibited the expression of PDX-1 and NeuroD/Beta2, and decreased the expression of IGF-Ⅱ. Foxo1 expression and induction of Foxo1 from the cytoplasm to the nucleus of the ectopic. The combined treatment of rapamycin and IGF-Ⅱ can reduce the side effects of rapamycin, inhibit the decrease of β-cell number and insulin content, repair the expression of insulin, PDX-1, NeuroD/Beta2, inhibit Foxo1 expression and intracellular ectopic.ConclusionAberrant expression of IGF-Ⅱ and Foxol genes is the key inducing factor of rapamycin inhibiting the proliferation and differentiation of NPCCs, and IGF-Ⅱ treatment can effectively reduce the side effects of rapamycin on NPCCs differentiation.