【作者】 王延栋；

【导师】 孙书珍；

【作者基本信息】 山东大学 ， 儿科学， 2010， 硕士

【摘要】 研究背景与研究目的：过敏性紫癜(Henoch-Schonlein purpura, HSP)是一种以变态反应性炎症为主要病理改变的系统性小血管炎,肾脏受累与否是决定其预后的关健因素,由紫癜性肾炎(Henoch-Schonlein purpura nephropathy, HSPN)导致的急慢性肾功能衰竭是HSP患儿死亡的主要原因。但是,HSPN的确切发病机制至今仍不清楚,关于其治疗也尚无统一有效的药物。已知细胞外基质(Extracellular matrix, ECM)的产生过多而降解减少导致ECM过度沉积是肾小球硬化、肾脏纤维化的主要原因。而HSPN的主要病理特点为系膜细胞增生,大量基质堆积,系膜区变宽、基底膜增厚,随着病情进展逐步发展为肾小球硬化,最终导致整个肾脏功能的丧失。近年研究发现肾小管上皮细胞转分化(epithelial-mesenchymal transition, EMT)是肾间质纤维化的关键。α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA),作为肌成纤维细胞的标志蛋白,现被普遍用于检测肾小管上皮细胞向肌成纤维细胞的表型转化。已有研究证明中性粒细胞明胶酶相关运载蛋白(neutrophil gelatinase-associated lipocalin, NGAL)可以通过多种途径影响到肾脏纤维化,促进肾小管细胞的增生和修复。本实验将通过观察HSP及HSPN患儿肾组织α-SMA、NGAL的表达及变化以及NGAL在这些患儿血清和尿中的变化,探讨其在HSPN发生、发展中的作用,为HSPN的早期诊断和估计病情提供新的思路。研究方法：选择临床诊断为HSP的患儿81例,根据HSP患儿急性期尿微量白蛋白排泄率(urinary albumin excretion rate, UAER)的不同将其分为：正常白蛋白尿组(A组,38例)：UAER<20μg／min；微量白蛋白尿组(B组,26例)：UAER 20-200μg／min；大量白蛋白尿组(C组,17例)：UAER>200μg／min或总蛋白>50mg／kg·d。以年龄、性别相匹配的正常健康体检儿童35例(男20例,女15例,平均年龄7.6岁)作为健康对照组(D组)。81例HSP患儿中共有22例于急性期行经皮肾穿刺活检,其中,A组5例,B组9例；C组8例,以2例无肾脏病史因外伤而行肾切除患者的肾组织作为对照。2.血清和尿NGAL的检测：采用酶联免疫吸附法(ELISA)3.肾组织病理形态学的观察各组肾组织石蜡切片分别进行苏木精-伊红(HE)和过碘酸-希夫氏(PAS)染色,光镜下观察,对肾脏病理损害程度进行分级。4.肾组织α-SMA、NGAL和Ⅳ型胶原表达的检测采用免疫组织化学SABC法检测肾组织α-SMA、NGAL和Ⅳ型胶原的表达,应用Image-Pro plus图像分析软件测定α-SMA、NGAL和Ⅳ型胶原的累积光密度值(IOD)。5.统计学处理统计学处理采用SPSS 16.0软件进行统计学处理,所有实验数据数据均用x±s表示,各组间比较采用最小显著差(LSD)t检验。部分指标进行直线相关分析,P<0.05为差异有统计学意义。研究结果：1.HSP急性期各组患儿血清和尿液NGAL的变化：与正常对照组比较,A、B组患儿血清NGAL浓度变化无统计学意义,C组明显增高(P<0.05)；与正常对照组比较,A、B、C组患儿尿NGAL水平均明显升高(A组P<0.05,B组P<0.01,C组P<0.01),且随尿白蛋白排泄率的增加而呈递增趋势(P<0.05)：2.HSP患儿肾组织α-SMA的表达：在正常的肾组织中仅有少量或无α-SMA的表达,HSP患儿肾组织α-SMA的表达较之有明显的增加,且在A、B、C组呈递增趋势(P<0.05)。3.HSP患儿肾组织Ⅳ型胶原的表达：在正常的肾组织中仅有少量或无Ⅳ型胶原的表达,HSP患儿肾组织Ⅳ型胶原的表达较之有明显的增加,且在A、B、C组呈递增趋势(P<0.05)。4.HSP患儿肾组织NGAL的表达：在正常的肾组织中仅有少量或无NGAL的表达,HSP患儿肾组织NGAL的表达较之有明显的增加,且在A、B、C组呈递增趋势(P<0.05)。5.相关分析结果显示肾组织NGAL与Ⅳ型胶原表达呈正相关, (r=0.823,P<0.01)；HSP患儿肾组织NGAL的表达与尿NGAL水平呈正相关(r=0.604,P=0.03)；HSP患儿肾组织NGAL表达与α-SMA表达也呈正相关,(r=0.792,P<0.01)。研究结论：1.随着尿白蛋白的升高和肾组织病理损害的加重,HSP患儿尿NGAL水平和肾组织NGAL也呈递增趋势,提示NGAL与HSPN的发生和发展有关。2.HSP患儿肾组织存在α-SMA的异常表达,表明在HSPN的发生和发展中存在肾小管上皮细胞转分化,并且α-SMA在正常白蛋白尿组就已表达,表明这种转化在正常白蛋白尿组就已发生。3.肾组织NGAL与α-SMA呈正相关,提示NGAL可能对EMT起调节作用。4.HSP患儿的尿NGAL水平与肾组织NGAL的表达呈正相关,提示尿NGAL的水平可以反映肾组织中NGAL的表达水平。5.HSP患儿在正常白蛋白尿组尿NGAL水平已升高,提示其可作为早于尿白蛋白的早期诊断HSPN的敏感指标。更多还原

【Abstract】 Background and objective:It is well known that Henoch-Schonlein purpura (HSP) is a systemic abnormal reactive vasculitis, and renal function failure induced by progressive Henoch-Schonlein purpura nephropathy (HSPN) is the primary cause of the mortality. However, the exact pathogenesis of HSPN remains unknown, and no unified and effective medicine have been found to heal HSPN. The pathological hallmarks of HSPN are mesangial cell proliferation and extracellular matrix(ECM) accumulation, which lead to increased thickness of the glomerular and tubular basement membrane(GBM) and mesangial expansion, and finally result in glomerulosclerosis and loss of the renal function. These mainly result from an imbalance between the synthesis and degradation of ECM. Recent study show that epithelial-mesenchymal transition is the key point of renal fibrosis.α-Smooth Muscle Actin, as the marker of myofibroblast, is generally used to detect the phenotype transformation from renal tubular epithelial cell to myofibroblast. Most study have indicate that neutrophil gelatinase associated lipocalin can decrease the degree of renal fibrosis by many ways and promote a certain proliferative effect on renal tubular cell. In this study, we examined the expression of a-SMA and NGAL in renal tissue of HSP children and HSPN children, also detected serum and urinary changes of NGAL in these cases, discussed their function in the development and progression of HSPN. Our results suggest novel approaches for the early diagnosis and accurate evaluation of HSPN progression.Methods:1. Subjects:Our study included 81 patients who had been diagnosed with HSP and 35 healthy children as the control. Based on the urinary albumin excretion rate (UAER) in the acute phase, the HSP children were divided into three groups. Group A (the normoalbuminuria group, N= 38), group B (the microalbuminuria group, N=26), and group C (the macroalbuminuria group, N=17). Among the above 81 patients, 22 received renal biopsy in the acute phase, including five children from group A, nine children from group B, and eight children from group C. The control kidney samples were taken from the normal portions of nephrectomy specimens of patients who underwent surgery due to trauma.2.Blood serum and urinary NGAL excretion was determined by ELISA.3. Renal pathology observation:The nephridial tissues were stained by HE and PAS seperately, then observed by a light microscope.4.α-SMA/ NGAL andⅣ-collagen expression in renal tissues: Immunoenzymic-SABC method was applied, then film reading was viewed by LEICA Qwin V3 image processing system and Image-Pro plus software was used to determine the integral optical density (IOD) ofα-SMA/NGAL andⅣ-collagen exression.5. Statistical methods:Software used:SPSS 16.0Data representation:x±SDComparison among control group and HSP groups:the least significant difference, LSD (P＜0.05)Association between the urinary NGAL level and the renal NGAL expression and the renal NGAL expression with a-SMA expression:linear correlation analysisResults:1.The blood serum and urinary NGAL level in acute phase Compared with the control group, the serum NGAL level in group A and B were not found statistical difference(P＞0.05), howerve, the serum NGAL level in group C was increased obviously(P＜0.05). The urinary NGAL level was increased in group A (P＜0.05)、group B (P＜0.01) and group C(P＜0.01).2. The a-SMA expression in kidneyNone or little a-SMA was expressed in the renal tissues of the controls while much more a-SMA was apparently expressed in the HSP patients. The IOD ofα-SMA staining was increased with the aggravate of renal damage(P＜0.05).3. TheⅣ-collagen expression in kidneyNone or littleⅣ-collagen was expressed in the renal tissues of the controls while much moreⅣ-collagen was apparently expressed in the HSP patients. The IOD ofⅣ-collagen staining was increased with the aggravate of renal damage(P＜0.05).4. The NGAL expression in kidneyNone or little NGAL was expressed in the renal tissues of the controls while much more NGAL was apparently expressed in the HSP patients. The IOD of NGAL staining was increased with the aggravate of renal damage(P＜0.05).5. The correlationThe linear correlation analysis revealed a tight correlation between NGAL expression andⅣ-collagen expression (r=0.823, P＜0.01)The linear correlation analysis revealed a tight correlation between urinry NGAL level and renal NGAL expression (r=0.604, P=0.03).The linear correlation analysis revealed a tight correlation between NGAL expression and a-SMA expression (r=0.792, P＜0.01)Conclusion:1. NGAL is related to the development and progression of HSPN, expression of NGAL will increase along with the aggravation of pathological damages in HSPN cases.2. Children with HSP has a-SMA expression, which show there exists EMT of renal tubular epithelial cells in Children with HSPN, and a-SMA has expressed in normal albuminuria group, which can indicate this change has appeared in normal albuminuria group.3. The linear correlation between NGAL expression andα-SMA expression is positive, which prompt that NGAL can accommodate the process of EMT.4. In children with HSP, NGAL levels in urine is significantly positively related to expression of NGAL, this result reveals NGAL level in urine can reflect levels of NGAL expression in renal tissue.5. The urinary NGAL is appeared in normal albuminuria group which indicate NGAL could be an early marker for predicting HSPN.更多还原