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背景:IgA肾病发病机制迄今不明,目前研究证实IgA肾病患者IgA1分子铰链区β1,3-半乳糖基转移酶的活性降低引起IgA1分子O-糖基化异常是该病发生的重要途径。作者前期研究推测IgA肾病患者外周血B淋巴细胞β1,3-半乳糖基转移酶特性伴侣蛋白Cosmc表达降低可能是IgA肾病糖基化异常原因的中心环节。目的:检测IgA肾病患者β1,3-半乳糖基转移酶特异性伴侣蛋白Cosmc基因编码区的DNA序列,并与Gene Bank公布的序列比对。设计:病例-对照观察。单位:四川大学华西医院肾内科。对象:选择2005-11/2006-08四川大学华西医院肾内科收治的肾病患者37例,包括IgA肾病患者27例和非IgA肾病患者10例,另选择5例健康自愿者,所有观察对象均知情同意。方法:实验于四川大学生物治疗国家重点实验室完成。采集所有观察对象的肝素钠抗凝外周静脉血2mL,采用经典酚氯仿法提取外周血基因组DNA,应用紫外分光光度仪定量DNA的含量,应用聚合酶链反应扩增所有观察对象的β1,3-半乳糖基转移酶特异性伴侣蛋白Cosmc基因编码区,并对每个观察对象的聚合酶链反应产物进行直接测序,将所有测序结果分别与GeneBank公布序列一一进行比对。主要观察指标:β1,3-半乳糖基转移酶特异性伴侣蛋白Cosmc基因编码区的聚合酶链式反应产物扩增结果及测序序列。结果:①Cosmc基因的编码区位于Cosmc基因的257-1213位,扩增后的Cosmc基因大小为1247bp。②IgA肾病患者、非IgA肾病患者以及健康自愿者之间的β1,3-半乳糖基转移酶特异性伴侣蛋白Cosmc基因编码区序列均一致,并与GeneBank公布的序列无差异。结论:未发现IgA肾病患者有Cosmc基因编码区序列异常,提示Cosmc基因编码区序列与IgA肾病IgA1分子的糖基化异常可能无关。
BACKGROUND: The pathogenesis of IgA nephropathy has not been elucidated until now. Studies have confirmed that IgA1 molecular O-glycosylation abnormality is an important pathway for the pathogenesis of IgA nephropathy due to the reduced activity of β1,3-galactosyltransferase in the hinge region of IgA1 molecule. The author’s preliminary study presumed IgA nephropathy patients with peripheral blood B lymphocytes decreased expression of chaperone protein Cosmc β1,3-galactosyltransferase may be the cause of IgA nephropathy abnormalities in the center of the link. OBJECTIVE: To detect the DNA sequence of the coding region of Cosmc gene of β1,3-galactosyltransferase chaperone in patients with IgA nephropathy and to compare with the sequences published by Gene Bank. Design: Case-control observation. Unit: Department of Nephrology, West China Hospital, Sichuan University. PARTICIPANTS: Thirty-seven patients with nephropathy were enrolled from Department of Nephrology, West China Hospital of Sichuan University between November 2005 and August 2006, including 27 patients with IgA nephropathy and 10 patients with non-IgA nephropathy. Five other healthy volunteers were selected and all the subjects were informed agree. Methods: The experiment was completed in State Key Laboratory of Biotherapy, Sichuan University. Peripheral blood DNA was extracted from all the observed heparin anticoagulated peripheral venous blood using classical phenol-chloroform method. The DNA content was quantified by UV spectrophotometer. PCR was used to amplify all the observed β1,3- Galactosyltransferase specific chaperone Cosmc gene coding region, and each observation of the PCR products were sequenced directly, and all the sequencing results were published GeneBank sequence one by one for comparison. MAIN OUTCOME MEASURES: Amplification results of polymerase chain reaction products from the coding region of Cosmc gene of β1,3-galactosyltransferase specific chaperonin and sequencing. Results: ① The coding region of Cosmc gene was located at positions 257-1213 of Cosmc gene, and the amplified Cosmc gene size was 1247 bp. ② The coding region of Cosmc gene of β1,3-galactosyltransferase chaperone between IgA nephropathy patients, non-IgA nephropathy patients and healthy volunteers were consistent and there was no difference with the sequences published by GeneBank. Conclusion: No abnormalities in the coding region of Cosmc gene were found in patients with IgA nephropathy, suggesting that there may be no correlation between the coding region of Cosmc gene and the abnormal glycosylation of IgA1 in IgA nephropathy.