【作者】 潘燕；

【导师】 赵家军；

【作者基本信息】 山东大学 ， 内分泌与代谢， 2007， 硕士

【摘要】 研究目的：肾小管酸中毒(renal tubular acidosis，RTA)是由于近端及(或)远端肾小管功能障碍所致的代谢性酸中毒，而肾小球功能正常或损害轻微。本征主要特征有高氯性代谢性酸中毒、电解质失衡，临床上表现为烦渴、多饮、多尿、肾性佝偻病或骨软化症、肾钙化症等。目前大都将RTA分为4型：即Ⅰ型(远端肾小管酸中毒，distal renal tubular acidosis，dRTA)、Ⅱ型(近端肾小管性酸中毒，proximal renal tubular acidosis，pRTA)、Ⅲ型(混合型肾小管性酸中毒)、Ⅳ型(高血钾型肾小管性酸中毒)。既往肾小管酸中毒曾被认为是一种常染色体显性或者隐性遗传性疾病。但是近年来越来越多的研究发现肾小管酸中毒常伴发着某些自身免疫性疾病，提示RTA的发病可能与自身免疫因素有某些内在联系。其中以Ⅰ型RTA常见。Ⅰ型RTA在成人往往表现为散在发病，在儿童多为家族发病。散在发病可以是原发的(几乎都发生在女性)也可以是继发性，如继发于自身免疫性疾病，尤其是干燥综合征。干燥综合征的肾损害较常见，不同的研究结果报告有所差异，但是多数认为其发生率为40％-50％，而肾损害主要累及远端肾小管出现肾小管酸中毒。免疫荧光技术(immunofluorescence assay，IFA)是根据抗原抗体反应具有高度特异性，以异硫氰酸荧光素(FITC)、罗丹明(RB200)等荧光色素标记物与已知的抗原或抗体结合，然后测定未知抗原或抗体的技术。荧光素与抗体的结合物称为荧光抗体，荧光抗体与相应抗原的结合在荧光显微镜下显示不同程度的荧光，由此可对抗原进行定性、定量或定位的检测。本实验即是采用间接免疫荧光技术，以FITC标记的抗人IgG定性检测肾小管酸中毒病人血清抗肾小管抗体的存在，从免疫学角度初步探讨其发病的自身免疫性。研究方法：1．研究对象：确诊为肾小管酸中毒患者血清11份，诊断依据为①临床上有代谢性酸中毒，而尿PH值在6.0以上；②低血钠、低血钾及低钙血症，血氯明显增高；③骨痛及骨软化症。④辅助检查发现骨质疏松，病理性骨折或尿路结石等。健康对照者空腹血血清10份，采血前3月内均无感冒、服药史，性别、年龄与RTA患者匹配。干燥综合症患者血清10份，干燥综合症患者确诊依据为①干燥性角膜炎；②口干燥症；③血清中有下列一种抗体阳性：抗SS-A抗体、抗SS-B抗体、抗核抗体(ANA)＞1：20、类风湿因子(RF)＞1：20，具备以上3条并除外其它结缔组织病、结节病、移植物抗宿主病、淋巴瘤等疾病者可以确诊。空白对照使用PBS缓冲液代替血清反应。2．研究方法：①抗肾小管基底膜抗体的检测：采用欧蒙公司提供的猴肾间接免疫荧光法检测试剂盒。第一次温育时，已稀释的血清与固定在载片反应区中生物薄片上的猴肾冰冻切片反应。如果标本阳性，特异性IgG、IgA、IgM抗体与相应抗原结合。在第二次温育时，结合的抗体与荧光素标记的抗人抗体反应，然后在荧光显微镜下观察特异的荧光模型。②抗肾小管细胞胞浆抗体的检测：采用人体肾组织石腊切片，利用传统的湿盒，使用10％山羊血清封闭非特异性结合位点，起始采用血清原液。均使用共聚焦显微镜观察并采集图像。③肾小管酸中毒患者血清均测空腹血生化、血清蛋白电泳、血沉、ANA、ENA系列、尿常规、24小时尿蛋白定量。部分检测甲状腺功能，行肾脏B超、骨密度检查。3．统计分析：所有数据均以均数±标准差((?)±s)表示。各组数据组间均数比较采用两样本t检验或t’检验(方差齐采用两样本t检验，方差不齐采用t’检验)。检验水平α=0.05。结果：肾小管酸中毒患者血清抗肾小管抗体阳性率为6/11，明显高于干燥综合症患者组(1/10)和健康对照患者组。结论：肾小管酸中毒患者体内存在抗肾小管抗体，主要为抗肾小管细胞胞浆抗体，提示肾小管酸中毒发病与自身免疫因素有关。意义：关于肾小管酸中毒免疫学发病机制有不少的研究报道，但是尚无充分的实验室证据。该病在治疗上也是一直沿用传统的纠正酸中毒及电解质紊乱、补钾补钙等对症治疗。本实验说明肾小管酸中毒患者体内存在自身抗体，提示该病具有自身免疫性，这为该疾病发病机制的研究及治疗提供了一定的帮助。更多还原

【Abstract】 Objective:Renal tubular acidosis(RTA) is a form of metabolic acidosis due to dysfunction of proximal and (or)distal renal tubular,while the function of glomerulus is normal or slightly damaged.RTA is charactered as hyperchloraemic metabolic acidosis and electroly disorders.RTA patients often present as dipsesis.polydipsia,polyuria,renal rickets/osteomalacia,nephrocal cinosis and so on. Renal tubular acidosis is often classified by four types:type I (distal renal tubular acidosis, dRTA) ,type II (proximal renal tubular acidosis,pRTA) ,typeIII (mixed renal tubular acidosis ) as well as type IV (hyperkaliemia renal tubular acidosis) . Renal tubular acidosis(RTA) was once defined as a type of heritage disease.But more researches indicate that RTA is associated with various immunological diseases which demonstrates that there are possibly internal association between RTA invasion and autoimmune factors. Of the total,type I RTA is the most common.Type I RTA is often a kind of sporadic disease in adult and familial disease in children.Sporadic diseases may be primary(almost in women) or secondary.For example.it can be secondary to some autoimmune diseases combined with hypergammaglobulinemia,especially sjogren syndrome(SS).The kidney damage is common in sjogren syndrome. The incidence rate is different according to different reports and most consider 40%-50%. The kidney damage mainly involves in distal renal tubular and occurres renal tubular acidosis. Immunofluorescence assay is based on antigen-antibody reaction and has high degree of specificity,by which tumer markers with fluorchromes such as FITC,RB200 and so on react with antigens or antibodies known,and then determine the unknown antigens or antibodies. What makes up of fluorchromes and antibodies are called fluorescent antibodies (FA).Fluorescent antibodies conjugated with corresponding antigens can manifest different degree fluorescent light.Therefore, we can detect antigens qualitatively, quantitatively or site-specificly.The experiment is to detect anti-renal tubular antibodies in sera of renal tubular acidosis qualitatively with anti-human IgG marked by FITC. Methods:1. Objects:sera of 11 patients diagnosed renal tubular acidosis.Diagnosis standard: ①metabolic acidosis and PH value of urine>6.0;②hyponatremia, hypopotassaemia, hypocalcemia and hyperchloraemia; ③ostalgia and osteomalacia; ④osteoporosis, pathologic fracture or lithangiuria and so on.Ten shares of fasting sera of healthy people that did not have a cold or anything medicine three months ago,and match with the renal tubular acidosis patients in gender and age. Diagnosis standard of SS: ① aridity keratitis.②oral xerosis.③anyone of these antibodies is positive:anti-SSA,anti-SSB,ANA>1:20,RF>1:20.Anybody that accords with three standards above and remove other connective tissue diseases, sarcoidosis, lymphadenoma and so on can be diagnosed SS. Phosphate-buffered saline takes place of sera in blank control.2 Methods: ①anti-TBM:Monkey-kidney indirect immunofluorescence kit was bought from Euroimmun Company.At the first incubation, diluted sera_reacted with monkey-kidney frozen sections fixed in slide reaction zones.If the sera are positive,specific IgG,IgA,IgM antibodies will combine with corresponding antigens.Combined antibodies reacted with anti-human antibodies marked by FITC,and then we observed specific fluorescence models under fluorescence microscope. ②anti-renal tubular cytoplasm antibody :human kidney par. sections,traditionary wet box, 10% goat sera to block nonspecific combining sites,stock solution used in the first time,observation and collection pictures by microscope. ③Sera of RTA patients were detected biochemistry,serin electrophoresis, blood sedimentation,ANA,ENA,urine routine,24-hour-urine protein quantitation. Some was proceeding thyroid function, kidney B ultrasound and bone density examination.3. Statistical analysis:All data would be represented by mean ±standard deviation (x|-±s) .Means of each group were compared by t-test or t’-test.Analysis level isα=0.05. Results: The positive rate of RTA patients sera is 6/11,highly larger than that in SS groups (1/10) and healthy controls. Conclusions:The RTA patients have anti-renal tubular antibodies and mainly anti-renal tubular cytoplasm.So we konw the morbidity of RTA is associated with autoimmune factors. Significance:There are many research reports about immune pathogenesis of RTA but still no sufficient experiment evidences.The treatment of RTA continues to use traditional methods:retrieve acidosis and electrolyte disturbances and so on. In our experiment,we found that RTA patients had auto-antibodies.It is important for us to be provided help in RTA pathogenesis and treatment research.更多还原