Ras相关C3肉毒杆菌毒素底物3在1型糖尿病和树突状细胞中的作用初探

Preliminary study on the role of Ras-related C3 botulinum toxin substrate 3 in type 1 diabetes mellitus and dendritic cells

目的初步探索ras相关C3肉毒杆菌毒素底物3(RAC3)在1型糖尿病(T1DM)和树突状细胞(DC)中的作用。方法选取来自广东省T1DM转化医学研究中2011至2016年入组后规律随访的T1DM患者作为病例组,选择2011至2016年来自中山大学附属第三医院就诊的正常糖耐量者作为健康对照组。病例组和对照组均在筛选阶段进行全外显子组测序,在验证阶段进行基因分型。使用CRISPR/Cas9基因编辑技术构建C57BL/6小鼠背景的RAC3全身敲除(KO)小鼠模型。使用聚合酶链反应(PCR)、实时荧光定量PCR(qRT-PCR)、Western blotting法分别验证RAC3在DNA、RNA和蛋白水平是否被敲除。通过流式细胞染色检测RAC3 KO小鼠和同窝野生对照(WT)小鼠(每组3只)的DC分化比例以及主要组织相容性复合体Ⅱ类分子(MHCⅡ)、CD86和CD80等成熟活化指标的表达水平。采用logistic回归分析法比较病例组和对照组之间等位基因频率分布的差异。组间比较采用独立样本t检验。结果筛选阶段共纳入72例T1DM患者和487名健康对照,验证阶段共纳入122例T1DM患者和577名健康对照。筛选阶段结果显示,T1DM患者中RAC3 rs4969478位点的等位基因T频率高于健康对照[分别为7.64%(11/144)和1.13%(11/974),OR=9.38,P<0.001]。验证阶段基因分型结果同样显示,T1DM患者中RAC3 rs4969478位点的等位基因T频率高于健康对照[分别为4.50%(11/244)和1.13%(13/1154),OR=4.14,P<0.01]。WT小鼠和RAC3 KO小鼠的DC分化比例差异无统计学意义(分别为85.6%±1.1%和83.3%±0.25%,P=0.08)。WT小鼠和RAC3 KO小鼠DC中MHCⅡ、CD86和CD80等成熟活化指标的表达水平差异均无统计学意义(P>0.05)。结论遗传学研究提示RAC3可能是人类T1DM的易感基因,但可能不通过DC参与T1DM的发生。

Objective To preliminarily investigate the role of the Ras-related C3 botulinum toxin substrate 3(RAC3)in type 1 diabetes mellitus(T1DM)and dendritic cells(DC).Methods Patients with T1DM were enrolled from the Guangdong Type 1 Diabetes Translational Medicine Study from 2011 to 2016.Healthy controls were volunteers with normal glucose tolerance who visited the Third Affiliated Hospital of Sun Yat-sen University from 2011 to 2016.Whole exome sequencing was performed in the screening stage,and genotyping was performed in the validation stage among all participants.CRISPR/Cas9 gene editing technology was used to construct the RAC3 whole-body knock-out(KO)mouse model on C57BL/6 mouse background.Polymerase chain reaction(PCR),quantitative real-time PCR(qRT-PCR),and Western blotting assay were used to verify that RAC3 was knocked out on DNA,RNA and protein level.Flow cytometry was used to compare the differentiation ratio of DC and the expression levels of maturation and activation indicators,such as major histocompatibility complex-Ⅱ(MHCⅡ),CD86 and CD80,between RAC3 KO mice and wild-type(WT)littermate control mice(3 mice in each group).Logistic regression was used to compare the frequency distributions of the alleles between case and control groups.Intergroup comparisons were conducted by independent-samples t-tests.Results A total of 72 patients with T1DM and 487 healthy controls were included in the screening stage,and 122 patients with T1DM and 577 healthy controls were included in the validation stage.The results in the screening stage showed that the frequencies of the T allele of RAC3 rs4969478 in patients with T1DM were higher than that in healthy controls[7.64%(11/144)and 1.13%(11/974),respectively,OR=9.38,P<0.001].The results in the validation stage also showed that the frequencies of the T allele of RAC3 rs4969478 in patients with T1DM were increased compared to the healthy controls[4.50%(11/244)vs.1.13%(13/1154),respectively,OR=4.14,P<0.01].There was no significant difference in the percentage of differentiated DC between WT mice and RAC3 KO mice(85.6%±1.1%vs.83.3%±0.25%,respectively,P=0.08).There was no significant difference in the expression levels of maturation and activation indicators(such as MHCⅡ,CD86 and CD80)in DC between WT mice and RAC3 KO mice(P>0.05).Conclusion Genetic studies suggest that RAC3 may be a susceptibility gene for human T1DM,but it may not be involved in the pathogenesis of T1DM via DC.