Objective To investigate whether IL-10 gene combined with insulin-like growth factor-1 (IGF-1)gene transfer could attenuate pancreatic insulitis,increase the percentage of CD4 + CD25 + Foxp3 + regulatory T cells,and protect β cells from autoimmune destruction.Methods An adenoviral vector containing IL-10 gene (Ad-IL-10) or IGF-1 gene(Ad-IGF-1) was constructed separately.Forty female non-obese diabetic (NOD) mice were injected intraperitoneally with Ad-IL-10 and/or Ad-IGF-1,Ad-green fluorescent protein(GFP) and phosphate buffered saline(PBS)separately,repeated after 3 weeks.Blood glucose concentration was measured weekly.Serum insulin,cytokine production were tested by enzyme-linked immunosorbent assay.CD4 + CD25 + Foxp3 + Treg cells were determined by flow cytometry.Pancreatic histology was measured for determination of insulitis grades.Pancreatic insulin content and β-cell mass,proliferation were measured.Apoptosis was measured by using a terminal deoxynucleotidyl transferase dUTP nick end labeling assay.Results A significantly lower diabetes incidence (P ＜ 0.01) was observed in NOD mice treated with Ad-IL-10 and/or Ad-IGF-1,compared with mice treated with Ad-GFP or PBS alone,especially combined group.Lower insulitis score compared to control mice was found in Ad-IL-10 + Ad-IGF-1 group (all P ＜ 0.01).The serum level of TNF-α and IFN-γwere decreased and the level of IL-10 increased in combination therapy.The CD4 + CD25 +Foxp3 + cells was (7.17 ±0.38)％ in combined group,higher than that in the control groups.There was significantly less β-cell apoptosis(10.29 ±2.20)％ in combined group than that in other groups(all P ＜ 0.05).Conclusions Combination therapy with IL-10 and IGF-1 gene is able to increase the percentage of CD4 + CD25 + Foxp3 + regulatory T cells,reduce autoimmunity and increase pancreatic β-cell mass,indicating promising potential of these therapies as a new treatment strategy for diabetes mellitus.%目的 观察腺病毒介导的胰岛素样生长因子-1(Ad-IGF-1)和IL-10基因(Ad-IL-10)联合免疫治疗对非肥胖性糖尿病(NOD)小鼠胰岛炎、CD4+ CD25+ Foxp3+调节性T淋巴细胞(Treg)的影响及其对胰岛β细胞的保护作用.方法 雌性NOD小鼠40只随机分为Ad-IGF-1组、Ad-IL-10组、Ad-IGF-1+ Ad-IL-10组(联合组)、腺病毒空载体Ad-绿色荧光蛋白(GFP)对照组、PBS对照组.分别腹腔注射Ad-IGF-1、Ad-IL-10、Ad-IGF-1和Ad-IL-10、Ad-GFP、PBS各100 μL,3周后重复1次.每周监测血糖、体质量变化及糖尿病(DM)发病率.反转录(RT)-PCR检测胰腺IGF-1、IL-10表达,ELISA法检测血清Th1/Th2细胞因子及胰岛素水平,病理学检查胰岛炎程度,流式细胞术分析CD4+ CD25+ Foxp3+ Treg,TdT介导的dUTP缺口末端标记技术观察胰岛细胞凋亡并计数胰岛β细胞凋亡率.结果 腹腔注射Ad-IGF-1、Ad-IL-10可使IGF-1、IL-10在胰腺表达增强,IGF-1、IL-10单基因治疗可以减少DM的累积发病率,延缓DM的发生,增加体质量,联合组以上效果更加明显.联合组较单基因组降低胰岛炎积分效果更明显(P＜0.01)；联合基因干预明显降低TNF-α、IFN-γ水平,增加IL-10水平,调节Th1/Th2平衡；联合组CD4+ CD25+ Foxp3+ Treg细胞数为(7.17±0.38)％,与单基因组及对照组比较差异均有统计学意义(P均＜0.05)；联合组β细胞凋亡率为(10.29±2.20)％,低于单基因组及对照组,差异均有统计学意义(P均＜0.01).结论 IGF-1与IL-10基因联合治疗可增加CD4+ CD25+ Foxp3+ Treg细胞数,诱导免疫耐受,减少β细胞凋亡,促进胰岛β细胞重建,从而起到抗DM作用.
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