细胞因子对IgA肾病患者IgA1 O-糖基化的影响及相关机制研究
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摘要
目的探讨细胞因子对IgA肾病(IgAN)患者IgA1 O-糖基化的影响,分析相关影响机制。方法选取2017年4-10月于新疆军区总医院肾内科就诊的IgAN患者35例作为IgAN组,招募同期于医院就诊的健康志愿者15例作为对照组。对2组人群的IgA1分泌细胞与细胞因子共培养,观察细胞因子对IgA1 O-糖基化的影响及对C1GalT1和ST6GalNAc-Ⅱ酶活性和表达基因的影响;采用siRNA敲低技术,干扰IgA1分泌细胞中C1GALT1和(或) ST6GALNAC2的表达,验证细胞因子与之相关性;开发基于假设的体外测定,进一步诠释细胞因子对IgAN患者IgA1 O-糖基化的影响机制。结果 IL-6可显著促进IgA1的产生(P<0. 05),显著增加IgA1半乳糖缺乏(P<0. 05),对IgAN患者作用更明显(P<0. 05)。IL-6显著增加ST6GalNAc-II活性(P <0. 05),显著降低C1GalT1活性(P <0. 01); IL-6显著上调ST6GALNAC2基因表达,同时显著降低C1GALT1和COSMC基因表达(P<0. 01),与测定酶活性变化一致。siRNA干扰技术使每个基因mRNA水平降低了65%～75%。C1GALT1敲除显著促进IgAN细胞和对照组的IgA1半乳糖缺乏(P<0. 05); ST6GALNAC2敲减仅明显降低IgAN细胞分泌的IgA1半乳糖缺乏(P<0. 05),敲低后分泌的IgA1半乳糖含量与对照组相当。IgA1 O-聚糖的有效半乳糖基化可以通过过早的唾液酸化实质性降低。结论细胞因子IL-6通过对IgAN患者ST6GALNAC2和C1GALT1酶的活性及其基因表达调节,上调IgAN患者IgA1的半乳糖缺乏,进而增加免疫复合物形成和导致疾病恶化。
Objective To investigate the effects of cytokines on IgA1 O-glycosylation in IgAN patients and to analyze the related mechanisms. Methods 35 IgAN patients who were admitted to the Department of Nephrology,General Hospital of Xinjiang Military Region from April to October 2017 were selected as IgAN group,and 15 healthy volunteers who were admitted to our hospital at the same time were recruited as Controls group. The co-culture of IgA1-secreting cells from IgAN and Controls co-cultured with cytokines to observe the effects of cytokines on IgA1 O-glycosylation and the differences in cytokines on the activity of C1 GalT1 and ST6 GalNAc-II and the expression of genes; SiRNA knockdown technique was used to interfere with the expression of C1 GALT1 and/or ST6 GALNAC2 in IgA1-secreting cells from IgAN patients and healthy controls respectively to verify the correlation between cytokines;The hypothesis-based in vitro assay was developed to further elucidate the mechanism by which cytokines affect IgA1 O-glycosylation in IgAN patients. Results IL-6 significantly increased the production of IgA1( P < 0. 05),and promoted the expression of IgA1 in patients with IgAN( P<0. 05); IL-6 significantly increased the secretion of IgA1-galactose in both groups. The effect was more significant( P<0. 01). IL-6 significantly increased the activity of STGGal NAc-II( P<0. 05) and significantly decreased the activity of C1 GalT1( P<0. 01); IL-6 significantly increased the expression of ST6 GALNAC2 gene and significantly decreased the expression of C1 GALT1 and COSMC genes( P
Objective To investigate the effects of cytokines on IgA1 O-glycosylation in IgAN patients and to analyze the related mechanisms. Methods 35 IgAN patients who were admitted to the Department of Nephrology,General Hospital of Xinjiang Military Region from April to October 2017 were selected as IgAN group,and 15 healthy volunteers who were admitted to our hospital at the same time were recruited as Controls group. The co-culture of IgA1-secreting cells from IgAN and Controls co-cultured with cytokines to observe the effects of cytokines on IgA1 O-glycosylation and the differences in cytokines on the activity of C1 GalT1 and ST6 GalNAc-II and the expression of genes; SiRNA knockdown technique was used to interfere with the expression of C1 GALT1 and/or ST6 GALNAC2 in IgA1-secreting cells from IgAN patients and healthy controls respectively to verify the correlation between cytokines;The hypothesis-based in vitro assay was developed to further elucidate the mechanism by which cytokines affect IgA1 O-glycosylation in IgAN patients. Results IL-6 significantly increased the production of IgA1( P < 0. 05),and promoted the expression of IgA1 in patients with IgAN( P<0. 05); IL-6 significantly increased the secretion of IgA1-galactose in both groups. The effect was more significant( P<0. 01). IL-6 significantly increased the activity of STGGal NAc-II( P<0. 05) and significantly decreased the activity of C1 GalT1( P<0. 01); IL-6 significantly increased the expression of ST6 GALNAC2 gene and significantly decreased the expression of C1 GALT1 and COSMC genes( P
引文
[1]王心,陈铖.伴高尿酸血症的原发性IgA肾病的临床及病理特征分析[J].东南国防医药,2017,19(6):600-603.
[2]潘薇,张慧,樊均明.低糖基化IgA1与IgA肾病的研究进展[J].重庆医学,2016,45(4):548-551.
[3]孔荣珍,尹敏,王艺璇,等.IgA1分子糖基化异常在IgA肾病发病机制中的研究进展[J].中国实验诊断学,2016,20(3):513-516.
[4]张红,周楠,沈颖.异常糖基化IgA1抗体在IgA肾病诊治中的作用研究进展[J].中华实用儿科临床杂志,2016,31(5):392-394.
[5]向莉.Th1/Th2细胞因子与肾小球疾病的研究进展[J].中华实用医药杂志,2004,24(4):421-425.
[6]王缨,胡贵荣,李弼民.IgA肾病大鼠肾组织中TIM-1、IFN-γ、IL-4表达的变化及意义[J].广东医学,2017,38(4):505-508.
[7]Yamada K,Kobayashi N,Ikeda T,et al.Down-regulation of core 1 1,3-galactosyltransferase and Cosmc by Th2 cytokine alters O-glycosylation of IgA1[J].Nephrol Dial Transplant,2010,25(12):3890-3897.
[8]周楠,张红,刘小荣,等.抗糖基化异常免疫球蛋白A1抗体免疫球蛋白G在儿童IgA肾病临床的应用价值[J].中华儿科杂志,2017,55(9):663-667..
[9]Zhao N,Hou P,Lv J,et al.The level of galactose-deficient IgA1 in the sera of patients with IgA nephropathy is associated with disease progression[J].Kidney Int,2012,82(7):790-796.
[10]杨楠楠.肠道黏膜免疫功能异常与IgA肾病[J].肾脏病与透析肾移植杂志,2016,25(5):470-474.
[11]王金泉.IgA肾病的遗传易感性和黏膜免疫应答异常[J].医学研究生学报,2016,29(2):120-125.
[12]Smith AC,Molyneux K,Feehally J,et al.O-Glycosylation of serum IgA1 antibodies against mucosal and systemic antigens in IgAnephropathy[J].J Am Soc Nephrol,2006,17(12):3520-3528.
[13]朱合,徐道亮,刘昌华,等.IgA肾病发病机制-IgA1异常糖基化与免疫异常[J].中华肾病研究电子杂志,2017,6(4):182-185.
[14]孙红旭,卢嫣.IgA1异常糖基化致IgA肾病肾系膜细胞增殖[J].国际泌尿系统杂志,2017,37(5):783-786.
[2]潘薇,张慧,樊均明.低糖基化IgA1与IgA肾病的研究进展[J].重庆医学,2016,45(4):548-551.
[3]孔荣珍,尹敏,王艺璇,等.IgA1分子糖基化异常在IgA肾病发病机制中的研究进展[J].中国实验诊断学,2016,20(3):513-516.
[4]张红,周楠,沈颖.异常糖基化IgA1抗体在IgA肾病诊治中的作用研究进展[J].中华实用儿科临床杂志,2016,31(5):392-394.
[5]向莉.Th1/Th2细胞因子与肾小球疾病的研究进展[J].中华实用医药杂志,2004,24(4):421-425.
[6]王缨,胡贵荣,李弼民.IgA肾病大鼠肾组织中TIM-1、IFN-γ、IL-4表达的变化及意义[J].广东医学,2017,38(4):505-508.
[7]Yamada K,Kobayashi N,Ikeda T,et al.Down-regulation of core 1 1,3-galactosyltransferase and Cosmc by Th2 cytokine alters O-glycosylation of IgA1[J].Nephrol Dial Transplant,2010,25(12):3890-3897.
[8]周楠,张红,刘小荣,等.抗糖基化异常免疫球蛋白A1抗体免疫球蛋白G在儿童IgA肾病临床的应用价值[J].中华儿科杂志,2017,55(9):663-667..
[9]Zhao N,Hou P,Lv J,et al.The level of galactose-deficient IgA1 in the sera of patients with IgA nephropathy is associated with disease progression[J].Kidney Int,2012,82(7):790-796.
[10]杨楠楠.肠道黏膜免疫功能异常与IgA肾病[J].肾脏病与透析肾移植杂志,2016,25(5):470-474.
[11]王金泉.IgA肾病的遗传易感性和黏膜免疫应答异常[J].医学研究生学报,2016,29(2):120-125.
[12]Smith AC,Molyneux K,Feehally J,et al.O-Glycosylation of serum IgA1 antibodies against mucosal and systemic antigens in IgAnephropathy[J].J Am Soc Nephrol,2006,17(12):3520-3528.
[13]朱合,徐道亮,刘昌华,等.IgA肾病发病机制-IgA1异常糖基化与免疫异常[J].中华肾病研究电子杂志,2017,6(4):182-185.
[14]孙红旭,卢嫣.IgA1异常糖基化致IgA肾病肾系膜细胞增殖[J].国际泌尿系统杂志,2017,37(5):783-786.