谷氨酸脱羧酶自身抗体对胰岛素分泌的影响

1、王建民周智广伍汉文 【摘要】目的探讨1型糖尿病胰岛素缺乏机理及GAD-Ab对胰岛细胞结构及功能的影响。方法以改良的放射配体法筛选1型糖尿病患者GAD-Ab阳性血清，以亲和层析法从该血清中分离纯化GAD-Ab，以免疫荧光法观察GAD-Ab与-细胞的结合；将纯化的GAD-Ab及/或补体加入人胎胰岛细胞培养体系。结果GAD-Ab能够与-细胞结合，并使培养胰岛细胞的胰岛素释放量减少；透射电镜观察，加GAD-Ab组-细胞胞浆颗粒减少；同时加入补体组-细胞的细胞膜被破坏。结论1型糖尿病患者血清GAD-Ab在体外能够抑制培养的人胎胰岛-细胞的胰岛素合成和释放，并且有补体依赖性细胞毒作用。 【关键词】1型糖尿

2、病发病机理谷氨酸脱羧酶自身抗体 Effectofglutamicaciddecarboxylaseautoantiboyon insulin secretionWang Jianmin,Zhou Zhiguang,Wu Hanwen.Institute of Metabolism & Endocrinology,Hunan Medical University,Changsha,410011 【Abstract】ObjectiveTo study the mechanism of insulin insufficiency in type 1 diabetes mellitus and to

3、investigate the effect of glutamic acid decarboxylase autoantiboy (GAD-Ab) on structure and function of pancreatic islet cells. MethodsRadioligand assay of GAD-Ab was used to select the positive sera of GAD-Ab in type 1 diabetes patients and affinity chromatography was used to purify GAD-Ab from the

4、 positive sera. Indirect immunofluorescent technique was used to observe the binding of GAD-Ab to islet beta cells. Cultured islet beta cells were incubated with GAD-Ab and/or complements and the released insulin was measured by radioimmunoassay. The structure of islet beta cells was observed with t

5、ransmission electron microscope. Results The amount of released insulin in group C (incubated with GAD-Ab) was lower than those of the group A (control) and B (incubated with complement; both P0.01); but it was higher than that of group D (incubated with GAD-Ab and complement) (P0.01). There were no

6、 differences between the values of groups A and B (P0.05). Granules in the cells of group C (incubated with GAD-Ab) were fewer than those in groups A (control) and group B (incubated with complement). In group D (incubated with GAD-Ab and complement) not only the granules were fewer, but also the ce

7、ll membrane was distroyed. ConclusionGAD-Ab had influences both on insulin synthesis and release, it also had the complement-dependent cytotoxicity to human fetal islet beta cells cultured in vitro. 【Key words】Tyep 1 diabetes mellitusPathogenesisGlutamic acid decarboxylase autoantibody (Chin J Endoc

8、rinol Metab, 1998,14：168-171) 1型糖尿病是一种器官特异性自身免疫性疾病。已经发现的自身抗体有胰岛素自身抗体(IAA)、胰岛细胞抗体(ICA)、胰岛细胞表面抗体(ICSA)和谷氨酸脱羧酶自身抗体(GAD-Ab)等二十多种；然而，这些抗体是胰岛细胞破坏的原因还是结果，至今仍无定论。GAD-Ab是近年来发现一种新的1型糖尿病自身抗体，可在绝大多数初发1型糖尿病患者和高危人群中检出，在1型糖尿病动物模型(非肥胖型糖尿病倾向小鼠，即NOD小鼠)的众多自身抗体中出现最早、对1型糖尿病的诊断及预报价值最高；因而，多数学者认为GAD是1型糖尿病的始动抗原，从而受到越来越多的关注1

9、-3。为了探讨人GAD-Ab对人胰岛细胞的作用，本文用纯化的人GAD-Ab(IgG)和人胎胰岛细胞进行研究，结果表明，GAD-Ab在体外能够破坏人胎胰岛细胞的结构和功能，造成胰岛素缺乏。现报告如下。 材料和方法 一、谷氨酸脱羧酶自身抗体检测 参考Petersen等4的方法并加以改良，简述如下。 1.试管内转录和翻译生产标记抗原(35S-GAD)。转录体系内主要含T7RNA多聚酶，GAD65 cDNA及NTP等，依照供货商(Promega)提供的反应条件进行试管内转录和翻译35S-GAD标记抗原。37孵育1小时，再加热至6710min，随后冷存备用。翻译体系含无蛋氨酸的兔网织红细胞裂解液，2g

10、DNA，35S-蛋氨酸0.8mCi/ml，总反应体积50l。用溶液含50mM NaH2PO4,3mM二巯基苏糖醇(DTT)，1mM磷酸吡哆醛，0.5%牛血清白蛋白，pH=7.4将翻译物35S-GAD65稀释至450l，过NAP5柱(Pharmacia)，100l一份，收集后用作GAD-Ab检测的示踪剂。 2.放射配体分析法(RLA)检测谷氨酸脱羧酶抗体(GAD-Ab)：将待测血清用TBST50mM Tris,150mM NaCl,1%(vol/vol)吐温20，pH=7.2稀释后与上述制备(试管内转录和翻译)的35S-GAD一起，在1.5ml Eppendorf管中4孵育过夜后，每管加入7.5

11、mg蛋白A琼脂糖凝胶(Pharmacia)，再4孵育，轻摇2小时。用TBST洗3次，弃上清留沉淀，加闪烁液1ml/管，液闪仪(Wallac1409)测cpm，每份样品均做2管，标准对照(阳性和阴性)做4管；阳性判断标准：样品cpm值正常对照均值+2个标准差被认为是阳性。 二、亲和层析分离纯化GAD-Ab(IgG) 从1型糖尿病患者中筛选出GAD-Ab阳性，胰岛素抗体和ICA阴性之血清样本作为GAD-Ab(IgG)的来源。酶免法测ICA，依照试剂盒(Biomerica, Inc；USA)的使用说明进行。放免法测胰岛素抗体(华西医科大学糖尿病科技开发研究所试剂盒)。自制蛋白A凝胶柱(1ml)，连接

12、于快速蛋白质液相层析仪(FPLC，Pharmacia)，进行亲合层析。将血清离心10000rpm,10min；用Buffer A(1.5M甘氨酸氢氧化钠，3.0M氯化钠pH=8.9)将样品稀释1倍。用PBS(Phosphate Buffered Saline)悬浮蛋白A凝胶(美国生命技术公司)，装柱。设定液体流速为0.5ml/min，检测波长280nm。用5倍柱体的Buffer A平衡柱体。每次上样500l。用Buffer B(0.1M枸橼酸氢氧化钠，pH=4.0)洗脱。每管收集3min，每支管内预先加150l Buffer D(含1.0M Tirs-HCL,pH=9.0)。用Buffer C

13、(含0.1M枸橼酸氢氧化钠，pH=3.0)将柱体清洗至基线回恢复零位(280nm,UV.)；用Buffer A平衡柱体，开始下一个层析周期。 国家自然科学基金资助课题 作者单位：410011 长沙湖南医科大学代谢内分泌研究所 参考文献 1王建民，周智广，伍汉文，等. 谷氨酸脱羧酶自身抗体检测与胰岛素依赖型糖尿病. 中华医学杂志，1996，76：868. 2Schmidli RS, Colman PG, Bonifacio E, et al. High level of concordence between assays for glutamic acid decarboxylase anti

14、bodies, the first international gluctamic acid decarboxylase antibody workshop. Diabetes, 1994,43:1005. 3Roll U, Christie MR, Standl E, et al. Associations of anti-GAD antibodies with islet cell antibodies and insulin autoantibodies in first-degree relatives of type I diabetic patients. Diabetes, 19

15、94,43:154. 4Petersen JS, Hejnaes KR, Moody A, et al. Detection of GAD65 antibodies and other autoimmune disease using a simple radioligand assay. Diabetes, 1994,43:459. 5胡远峰，王煜非，丁一明，等. 人胎胰胰岛细胞单层培养的研究. 中华器官移植杂志，1990，11：459. 6Kaufman DL, Clare-Saizier M, Tian J, et al. Spontaneous loss of T-cell toler

16、ance to glutamic acid decarboxylase in murine insulin-dependent diabetes. Nature, 1993,366:69. 7Lernmark A. Molecular biology of IDDM. Diabetologia 1994,377suppl.2：S73. 8Zlobina EN, Dubikin IV, Merkushov AV, et al. Pancreatic antigenic complex p64 69: involvement in regulation of insulin secretion and relation to glutamic acid decarboxylase. Immunol Lett, 1992,31:289. 9Harrison JC, Honeyman MC, Deaizpurua HJ, et al. Inverse rela