OBJECTIVE Type 1 diabetes onset is preceded by a pre-inflammatory stage leading to insulitis and followed by targeted destruction of the insulin-producing beta cells of the pancreas. Osteopontin (OPN) is a secreted phosphoprotein with cytokine properties, implicated in many physiological and pathological processes, including infection and autoimmunity. We have previously identified up-regulated osteopontin transcripts in the pancreatic lymph nodes of the NOD (Non-Obese Diabetic) mouse at the pre-diabetic stages. Investigating the underlined disease initiating mechanisms may well contribute to the development of novel preventive therapies. Our aim was to construct opn null mice in a NOD autoimmune-prone genetic background and address the pathogenic or protective role of the osteopontin molecule in the early stages of type 1 diabetes. METHODS We generated opn null mutant mice in a NOD genetic background by serial backcrossing to the existing C57BL/6 opn knockout strain. The presence of opn wild type or null alleles in the congenic lines was evaluated by PCR amplification. We used NOD opn-null mice to assess the phenotypic evolution of type 1 diabetes. The presence of OPN in the serum was evaluated by ELISA and by immunostaining on the mouse tissues. The primary gene structure of the NOD opn encoding gene and protein sequences were compared to the known alleles of other mouse strains. Evaluation of Single Nucleotide Polymorphisms (SNPs) variation between opn alleles of the opn gene is reported. RESULTS In the absence of OPN, type 1 diabetes is accelerated, suggesting a protective role of this cytokine on the insulin-producing cells of the pancreatic islets. Conversely, in the presence of the opn gene, an increase of the OPN protein in the serum of young NOD mice indicates that this molecule might be involved in the immune regulatory events taking place at early stages, prior to disease onset. Our data support that OPN acts as a positive regulator of the early islet autoimmune damage, possibly by a shift of the steady-state of T1D pathogenesis. We report that the OPN protein structure of the NOD/ShiLtJ strain corresponds to the a-type allele of the osteopontin gene. Comparative analysis of the single nucleotide polymorphisms between the a-type and b-type alleles indicates that the majority of variations are within the non-coding regions of the gene. CONCLUSIONS The construction of opn null mice in an autoimmune genetic background (NOD.B6.Cg-spp1-/-) provides important tools for the study of the implication of the OPN in type 1 diabetes, offering the possibility to address the significance of this molecule as an early marker of the disease and as a therapeutic agent in preclinical studies.

中文翻译：

---

NOD小鼠基因敲除骨桥蛋白基因的1型糖尿病研究。

目的1型糖尿病的发作是在发炎前导致胰岛素抵抗，然后有针对性地破坏胰腺中产生胰岛素的β细胞。骨桥蛋白（OPN）是一种具有细胞因子特性的分泌型磷蛋白，与许多生理和病理过程有关，包括感染和自身免疫。我们以前已经在糖尿病前期的NOD（非肥胖糖尿病）小鼠的胰腺淋巴结中发现了上调的骨桥蛋白转录物。研究强调疾病的起因机制可能很好地促进了新的预防疗法的发展。我们的目标是在易发NOD的自身免疫遗传背景下构建opn无效小鼠，并探讨骨桥蛋白分子在1型糖尿病早期阶段的致病性或保护性作用。方法我们通过与现有C57BL / 6 opn基因敲除菌株进行序列回交，在NOD遗传背景下产生了opn基因突变小鼠。通过PCR扩增评估在同基因系中opn野生型或无效等位基因的存在。我们使用NOD opn-null小鼠评估1型糖尿病的表型演变。通过ELISA和通过在小鼠组织上免疫染色来评估血清中OPN的存在。将NOD opn编码基因和蛋白质序列的一级基因结构与其他小鼠品系的已知等位基因进行了比较。报道了评价opn基因的opn等位基因之间的单核苷酸多态性（SNP）变异。结果在没有OPN的情况下，1型糖尿病会加速发展，提示该细胞因子对胰岛的胰岛素产生细胞具有保护作用。反过来，在存在opn基因的情况下，年轻NOD小鼠血清中OPN蛋白的增加表明该分子可能与疾病发作之前早期发生的免疫调节事件有关。我们的数据支持OPN可能是T1D发病机制稳态的转变，是早期胰岛自身免疫损伤的积极调节剂。我们报告说，NOD / ShiLtJ株的OPN蛋白结构对应于骨桥蛋白基因的a型等位基因。对a型和b型等位基因之间的单核苷酸多态性的比较分析表明，大多数变异都位于基因的非编码区内。结论在自身免疫遗传背景下构建了opn null小鼠（NOD.B6。