【作者】 彭胜男；

【导师】 朱清仙；

【作者基本信息】 南昌大学医学院 ， 内科学， 2013， 博士

【摘要】 第一部分IgA肾病大鼠模型的构建及鉴定目的：用口服免疫致IgA肾病大鼠模型,并观察其肾脏病理变化,为之后IgA肾病发病机制的研究及探讨IgA肾病的防治提供基础。方法：20只sprague dawley(SD)雌性大鼠随机分成2组,即正常对照组和IgA肾病模型组(n=10)。利用牛血清白蛋白(BSA)+脂多糖(LPS)+四氯化碳(CCl4)联合构建IgA肾病大鼠模型。具体实施方案如下：BSA (400mg/kg,隔日灌胃)×6周+LPS(每只0.05mg/次,第6、8周尾静脉注射各1次)+CCl4(蓖麻油每只0.5ml/次+CCl4每只0.1ml/次,每周皮下注射1次)×9周。10周末处死动物,用免疫荧光法检测肾小球IgA的沉积,苏木素-伊红(HE)染色法观察各组大鼠肾脏的病理变化,动物处死前1天收集24小时尿液做尿沉渣红细胞计数和24小时尿蛋白量的检测。结果：正常对照组大鼠肾脏未见IgA免疫荧光；组织切片显示,肾小体大小正常,系膜区无明显增生,毛细血管袢开放良好,肾小管、肾间质均未见明显变化；尿液检测未见镜下血尿,24小时尿蛋白量正常。而IgA肾病模型组在肾小球系膜区可见亮绿色颗粒状IgA沉积荧光；肾脏组织病理学显示,肾小体增大,’肾小囊扩张,肾小球变小,肾小球血管稀疏,球内系膜增生,纤维化明显,肾小管肿胀阻塞；尿液检测可见镜下血尿,24小时尿蛋白量增加。结论：利用BSA+LPS+CC14能成功构建IgA肾病大鼠模型。模型构建的成功提示IgA肾病的发病可能与肠粘膜屏障功能破坏及粘膜免疫功能紊乱有关。第二部分IgA肾病模型大鼠肠粘膜屏障损伤及其与IgA肾病发病的关系目的：观察IgA肾病模型大鼠肠粘膜屏障的病理变化,并探讨其与IgA肾病发病的关系。方法：将14只SD雌性大鼠随机分成2组,即正常对照组和IgA肾病模型组(n=7)。用HE染色观察肠粘膜的一般组织病理变化,并用图像分析系统测量两组大鼠肠绒毛高度、肠隐窝深度及两者比值、肠绒毛中央乳糜管体密度及其直径；利用透射电镜观察两组大鼠小肠上皮紧密连接的变化；激光共聚焦显微镜观察小肠上皮紧密连接蛋白ZO-1和occludin的表达；RT-PCR和Western blot技术测其mRNA和蛋白表达的变化；用高效液相色谱法测量尿中乳果糖/甘露醇的比值以反映肠粘膜通透性的变化；用免疫荧光法检测肾小球IgA的沉积。结果：与正常对照组相比,IgA肾病模型鼠出现肠上皮脱落、肠绒毛变矮并不规整,肠绒毛数量减少并间距增宽,固有层充血、乳糜管扩张和炎症细胞浸润。图像分析结果显示,肠绒毛变短,肠隐窝加深,两者比值减小,肠绒毛中央乳糜管体密度增大,直径增大；肠上皮细胞紧密连接结构受到破坏,电子密度降低；小肠上皮紧密连接蛋白ZO-1(?)(?)occludin的mRNA及蛋白的表达均下降；尿中乳果糖/甘露醇的比值升高；肾小球系膜区有亮绿色颗粒状IgA沉积；肾小球IgA的平均光密度与其尿乳果糖/甘露醇的比值呈正相关。结论：IgA肾病模型组大鼠的肠粘膜屏障受损,肠粘膜通透性增加；肾小球IgA的相对表达量与肠粘膜的通透性呈正相关,提示肠粘膜屏障的受损与工gA肾病的发生和发展密切相关。第三部分大黄酸对IgA肾病大鼠肠粘膜屏障损伤的防治作用目的：观察大黄酸(RH)对IgA肾病大鼠肠粘膜屏障损伤的防治作用。方法：将28只SD雌性大鼠随机分成正常对照组、IgA肾病模型组、RH治疗组和RH预防组(n=7)。用HE染色和图像分析系统测量各组大鼠肠绒毛高度、肠隐窝深度、肠绒毛中央乳糜管体密度及其直径；利用透射电镜观察各组大鼠小肠上皮紧密连接的变化；激光共聚焦显微镜观察小肠上皮紧密连接蛋白ZO-1和occludin的表达；RT-PCR和Western blot技术测其mRNA和蛋白表达的变化；用高效液相色谱法测量尿中乳果糖/甘露醇的比值来反映肠粘膜通透性的变化。结果：正常对照组肠绒毛排列整齐,上皮细胞间的紧密连接位于细胞膜外侧面的顶端,呈致密的条带状结构,相邻细胞之间贴合紧密,无明显缝隙,紧密连接蛋白ZO-1和occludin均匀一致的分布在小肠上皮细胞连接处的顶端。与正常对照组相比,IgA肾病模型组肠绒毛排列不规整,绒毛高度缩短,肠隐窝深度加深,两者比值下降,中央乳糜管体密度和直径均增加,肠上皮细胞之间的紧密连接结构受到破坏,电子密度降低、模糊,肠绒毛近腔面上皮细胞之间的ZO-1和occludin分布减少,不均,荧光强度变弱,其mRNA和蛋白的表达水平均降低,尿乳果糖/甘露醇的比值增高。与IgA肾病模型组相比,RH预防组和RH治疗组大鼠肠粘膜病理改变明显减轻,肠绒毛高度增加,肠隐窝深度变浅,两者比值升高,中央乳糜管体密度和直径均下降,肠上皮紧密连接结构修复,电子密度增高,细胞之间连接紧密,ZO-1和occludin的免疫荧光均增强,其mRNA和蛋白的表达水平也增高,尿乳果糖/甘露醇的比值降低。结论：RH能促进肠上皮的生长,提高小肠上皮紧密连接蛋白ZO-1和occludin的表达,修复受损的紧密连接,保护肠粘膜和修复肠粘膜损伤。第四部分大黄酸对IgA肾病大鼠肾脏的防治作用目的：观察实验性工gA肾病肾脏各种病理变化和大黄酸(RH)预防工gA肾病形成的效果及对IgA肾病的修复作用。方法：28只SD雌性大鼠随机分成4组,即正常对照组、IgA肾病模型组、RH治疗组和RH预防组(n=7)。HE染色法观察各组大鼠肾脏的病理变化,并用图像分析系统测出各组动物肾小体、肾小囊和肾小球的体密度、球囊比、肾小体的数密度、肾小体和肾小球长径、近曲小管管腔和管壁的面积及其比值。免疫荧光法检测肾小球IgA的沉积,并测量各组大鼠尿沉渣红细胞数及24小时尿蛋白量。免疫组织化学法检测肾脏纤维连接蛋白(FN)和平滑肌α-肌动蛋白(α-SMA)的表达。结果：IgA肾病模型组和正常对照组相比,肾小体增大,肾小囊扩张,肾小球变小,肾小球血管稀疏,球内系膜增生,纤维化明显,肾小管肿胀阻塞；图像分析测量结果显示,肾小体体密度增大,肾小囊体密度增大,肾小球体密度减小,球囊比减小,肾小体长径增大,肾小球长径减小,近端肾小管管腔面积减小,管壁面积增大,腔壁比减小；肾小球系膜区出现IgA的沉积；尿沉渣红细胞数及24小时尿蛋白量增加；肾脏FN和α-SMA的表达增加。RH治疗组和RH预防组与IgA肾病模型组相比,肾小体肥大减轻,系膜增生减少,肾小囊扩张和近曲小管狭窄减轻；肾小球系膜区IgA的沉积减少,尿沉渣红细胞数及24小时尿蛋白量降低,肾脏FN和α-SMA的表达减少。结论：BSA+LPS+CCL4建模的IgA肾病肾脏出现肾小管肿胀阻塞、肾小球纤维化等病理变化,RH对其具有较好的防治作用,保护肠粘膜屏障可能是其重要的机制之一。更多还原

【Abstract】 Part One Establishment and identification of IgA nephropathy in rat modelObjective: To establish a rat model of IgA nephropathy by the oral immunization and observe the pathologic changes of the renal tissue for the study in the pathogenesis, prevention and treatment of IgA nephropathy.Methods: Twenty female sprague dawley (SD) rats were randomly divided into two groups (n=10), namely control group and IgA nephropathy group. Bovine serum albumin (BSA), endotoxin (LPS) and carbon tetrachloride (CC14) were jointly used to establish the rat model of IgA nephropathy and the specific implementation were as follows:BSA (400mg/kg, oral every other day) x6weeks+LPS (0.05mg, intravenous injection at the6th and8th weeks)+CC14(0.1ml dissolved in0.5ml castor oil, subcutaneous injection weekly) x9weeks. Animals were sacrificed in the tenth weekend, the glomerular deposition of IgA was detected by immunofluorescence, and the pathological changes of the kidney were observed by hematoxylin-eosin (HE) staining. Twenty-four-hour urine was collected to count the urinary red blood cells and to measure the24-hour urinary protein before animals were sacrificed.Results: In control group, no green fluorescence has been seen in the renal glomeruli, examination of kidney sections showed normal size of renal corpuscle, no significant proliferation of mesangial area, capillary loops were normal and no significant changes in the renal tubules and interstitial, microscopic hematuria has not been observed and the quantity of24-hour urine protein was normal. Compared with the control group, bright green fluorescence in a granular pattern in the mesangium has been seen in IgA nephropathy group. Examination of kidney sections from the IgA nephropathy group showed the structural lesions of glomeruli including glomerular hypertrophy, renal capsule dilation, renal glomerular smaller, glomerular vascular sparse, the mesangial proliferation, obvious fibrosis and the swelling of the renal tubule. Microscopic hematuria was observed and the quantity of24-hour urine protein increased in the IgA nephropathy group. Conclusion:Using bovine serum albumin (BSA)+endotoxin (LPS)+carbon tetrachloride (CC14) can successfully establish the rat model of IgA nephropathy. The success of establishment of rat IgA nephropathy model suggests that the intestinal mucosal barrier function damage and mucosal immune dysfunction may be involved in the pathogenesis of IgA nephropathy. Part Two The injury in intestinal mucosal barrier in the rat model of IgA nephropathy and its relationship with the pathogenesisObjective:To observe the pathologic changes in intestinal mucosal barrier in the rat model of IgA nephropathy, and to explore its relationship with the pathogenesis of IgA nephropathy.Fourteen female SD rats were randomly divided into two groups (n=7per group), namely control group and IgA nephropathy group. The pathologic changes in the intestinal mucosa were observed by hematoxylin-eosin staining, and combined with the image analysis system, the intestinal villus height, crypt depth, the ratio of them, volume density (Vv) and diameter of the central lacteals were measured. We observed the change of intestinal epithelial tight junctions of the rats using transmission electron microscope and the expression of intestinal epithelial tight junction proteins ZO-1and occludin using laser confocal microscope. The changes of the mRNA and protein expression of these proteins were measured by reverse transcription polymerase chain reaction (RT-PCR) and western blotting. The ratio of urinary lactulose/mannitol was measured by high performance liquid chromatography for assessing the intestinal permeability. The glomerular deposition of IgA was measured by immunofluorescence staining.Results:Compared with the control group, in IgA nephropathy group, intestinal epithelium sheded, intestinal villi became short and irregular, the number of intestinal villi reduced and their spacing widened. The congestive lamina propria, expanded lacteals and infiltration of inflammatory cells were observed in IgA nephropathy group. The results of image analysis showed that the intestinal villus height decreased, crypt depth deepened and the ratio decreased; Vv and diameter of the central lacteals increased; the density of tight junctions between intestinal epithelial cellls were reduced significantly; the mRNA and protein expression of intestinal epithelial tight junction protein ZO-1and occludin decreased; the ratio of urinary lactulose/mannitol increased; bright green fluorescence in a granular pattern in the mesangium was seen in IgA nephropathy group and the average optical density of glomerular IgA was positively correlated to the ratio of urinary lactulose/mannitol.Conclusions:In IgA nephropathy model group, the mucosal barriers were damaged, the permeability of the intestinal mucosa increased and the relative expression levels of the glomerular IgA was positively correlated to the permeability of the intestinal mucosa. These suggest that the mucosal barrier damage is closely related with the incidence and development of IgA nephropathy. Part Three Preventive and repairing effects of rhein on intestinal mucosal barrier injury in the rat model of IgA nephropathyObjective:To observe the preventive and repairing effects of rhein on intestinal mucosal barrier injury in the rat model of IgA nephropathy.Methods:Twenty-eight female SD rats were randomly divided into four groups (n=7per group), namely control group, IgA nephropathy group, rhein-treated group and rhein-prevented group. Using hematoxylin-eosin staining and image analysis system, the intestinal villus height, crypt depth, the ratio of them, volume density (Vv) and diameter of the central lacteals were measured. We observed the change of intestinal epithelial tight junctions of the rats using transmission electron microscope and the expression of intestinal epithelial tight junction proteins ZO-1and occludin using laser confocal microscope. The changes of the mRNA and protein expression of these proteins were measured by reverse transcription polymerase chain reaction (RT- PCR) and western blotting. The ratio of urinary lactulose/mannitol was measured by high performance liquid chromatography for assessing the intestinal permeability.Results:In control group, intestinal villi arranged in neat rows, the tight junctions between epithelial cells located on the outer side of the cell membrane to the top, banded structure was dense, close fit between neighboring cells, no significant gap, tight junction protein ZO-1and occludin uniformly distributed in the intestinal epithelial cells at the top of the junction. Compared with the control group, in IgA nephropathy model group, the arrangement of intestinal villi was not structured, the intestinal villus height decreased, crypt depth deepened and the ratio of them decreased; Vv and diameter of the central lacteals increased; the density of tight junctions was reduced significantly and blur; the fluorescence of ZO-1and occludin were weaker, reduced and uneven; the mRNA and protein expression of intestinal epithelial tight junction protein ZO-1and occludin decreased; the ratio of urinary lactulose/mannitol increased. Compared with the IgA nephropathy group, the intestinal villus height increased, crypt depth shallow and the ratio of them increased, volume density (Vv) and diameter of the central lacteals decreased, the intestinal epithelial tight junctions were repaired with respect to structural integration, with dense connection and high electron density; the fluorescence of ZO-1and occludin were stronger; the mRNA and protein expression of ZO-1and occludin increased; the ratio of urinary lactulose/mannitol decreased in the rhein-prevented group and rhein-treated group.Conclusions:The RH can promote the growth of intestinal epithelium, increase the expression level of intestinal epithelial tight junction protein ZO-1and occludin, repair the damaged tight junctions, protect the intestinal mucosal and repair the intestinal mucosal injury. Part Four Preventive and repairing effects of rhein on renal tissue in the rat model of IgA nephropathyObjective: To observe the pathological changes of renal tissue in the rat model of IgA nephropathy and the preventive and repairing effects of rhein on renal tissue.Methods:Twenty-eight female SD rats were divided randomly into four groups, namely control group, IgA nephropathy group, rhein-treated group and rhein-prevented group. The pathologic changes in renal tissue were observed by hematoxylin-eosin staining, and combined with the image analysis system, volume density (Vv) of the renal corpuscle, renal capsule and renal glomerulus, the volume rate of the renal glomerulus and the renal capsule, number density (Nv) of the renal corpuscle, the long diameter of the renal corpuscle and glomerulus, the area of the lumen and the wall of the proximal renal tubule and the rate of them were measured. The glomerular deposition of IgA was measured by immunofluorescence staining. The amount of urinary red blood cells and24-hour urinary protein excretion were measured. Fibronectin (FN) and a-smooth muscle actin (α-SMA) expression on renal tissue were measured via immunohistochemistry.Results:Compared with the control group, glomerular hypertrophy, renal capsule dilation, renal glomerular smaller, glomerular vascular sparse, the mesangial proliferation, obvious fibrosis and the swelling of the renal tubule in the IgA nephropathy group. The image analysis results showed that, in IgA nephropathy group, Vv of the renal corpuscle increased, Vv of the renal capsule increased, while Vv of the renal glomerulus decreased, and the volume rate of the renal glomerulus and the renal capsule also decreased. The long diameter of the renal corpuscle increased and that of the renal glomerular decreased. The area of the lumen of the proximal renal tubule reduced, and the area of the wall of that enlarged and the rate of them decreased. Bright green fluorescence in a granular pattern in the mesangium has been seen, the amount of the urine red blood cells and24-hour urine protein increased, the FN and a-SMA expression level increased in the renal tissue in the IgA nephropathy group. Histological examination of kidney sections from both rhein-treated group and rhein-prevented group showed that glomerular hypertrophy, mesangial proliferation, renal capsule dilation and renal tubule narrow were markedly ameliorated compared with IgA nephropathy group. Moreover, rhein treatment significantly reduced IgA deposition in glomerular, the amount of urinary red blood cells and24-hour urinary protein excretion. More importantly, increased FN expression in IgA nephropathy was backed to normal level in rhein-treated and rhein-prevented group, which was along with the reduction of a-SMA expression in renal tissues.Conclusions:The renal tissue of IgA nephropathy modeling by BSA+LPS+CC14occur the swelling of the renal tubule, glomerular fibrosis and other pathological changes. RH has better control effect, its role may be mainly achieved through the protection of intestinal mucosal barrier.更多还原