【作者】 范焕芳；

【导师】 陈志强； 赵玉庸；

【作者基本信息】 河北医科大学 ， 中西医结合临床， 2004， 硕士

【摘要】 目的： 局灶节段性肾小球硬化（FSGS）是指病理学上累及部分肾小球中部分毛细血管袢的非炎症性硬化性病变，是各种肾小球疾病进展到终末期肾衰的共同病理途径。病理表现为系膜基质增多，肾小球基底膜上皮下透明物质积聚以及毛细血管袢与包氏囊粘连等改变。FSGS为难治性肾病的主要病理类型，一般在临床症状发作后10年左右进入终末期肾功能衰竭，导致肾小球硬化及间质纤维化。大多数表现为肾病综合症的FSGS患者对激素和细胞毒类药物治疗反应差，且有较大毒副作用。血管紧张素转换酶抑制剂（angiotensin converting enzyme inhibitor ACEI）和血管紧张素受体拮抗剂(angiotensin II receptor blocker ARB)对肾脏具有一定的保护作用，但肾脏疾病是由于多种因素共同作用的结果，单一的治疗难以有效地控制疾病，并且ACEI和ARB有一定副作用，使用受到限制，故寻找无毒或低毒的安全药物对肾脏病的治疗具有很大意义。大量临床资料已证实，FSGS与凝血纤溶系统障碍有关。凝血与纤溶平衡紊乱是肾小球硬化及间质纤维化病变发展过程中的重要病理生理改变，凝血纤溶系统的平衡依靠组织型纤溶酶原激活物（Plasminogen activator, t-PA）及其抑制物（Plasminogen antivator inhibition, PAI-1）来调节。研究表明，t-PA是肾脏的保护因子之一。PAI-1是t-PA特异性抑制因子，<WP=4>不仅能抑制纤溶酶原转化为纤溶酶,从而抑制纤维蛋白的水解,而且能抑制细胞外基质（ECM）降解,从而导致ECM积聚。故纠正凝血纤溶系统功能紊乱对于延缓肾小球硬化及间质纤维化的发展具有重要意义。研究认为，肾小球硬化，球囊粘连，毛细血管腔塌陷或狭窄，以及肾小管间质纤维化和萎缩等病理改变，都属于中医学瘀血内阻的范畴。瘀血内阻当用活血化瘀法，现代研究证实活血化瘀中药红花具有抗凝作用，可防止血栓形成，已经广泛应用于心、脑血管疾病。有关红花对FSGS凝血纤溶系统及病理形态学的影响尚无报道。本实验用红花对阿霉素诱导的FSGS大鼠模型进行干预性治疗，采用免疫组织化学和原位杂交的方法观察肾组织中t-PA及其抑制物PAI-1和PAI-1mRNA表达，并进行肾脏组织形态学观察，同时以血管紧张素受体拮抗剂缬沙坦（valsartan）为对照，探讨红花对FSGS纤溶系统的影响及作用机理。方法 ：选用健康雄性SD大鼠40只，适应性喂养1周后，分别置于代谢笼里取尿，检测尿蛋白和尿红细胞，结果全部为阴性。随机分为假手术组、模型组、缬沙坦组、红花组，每组10只。除假手术组外，各组动物采用右侧肾切除，并在术后第7天及30天时分别尾静脉注射0.1%阿霉素5mg/kg和3mg/kg。第1次尾静脉注射1周后缬沙坦组和红花组开始灌药，假手术组与模型组分别灌入等量生理盐水。实验第8周末全部检杀，取血、尿进行常规及生化检查，取左侧肾脏进行病理学观察，采用免疫组化及原位杂交方法检测t-PA、PAI-1在蛋白及基因水平的表达。结果：⑴ 实验第2、4、6、8周末各组24h尿蛋白的定8周量检测结果显示：第2周末各组间无明显差异（P＞0.05）；<WP=5>第4周末模型组与假手术组比较有显著性差异（P<0.01），第6周及第8周末模型组明显高于假手术组（P<0.01）。两治疗组明显低于模型组（P<0.01），两治疗组之间无明显的差异（P＞0.05）。⑵ 实验第8周末各组生化检测结果显示：TP、Alb的含量模型组显著低于假手术组（P<0.01），而TC、TG的含量明显升高（P<0.01）；两个治疗组TP、Alb的含量高于模型组（P<0.01或 P<0.05），而TC、TG的含量低于模型组（P<0.01）；两个治疗组之间TP、Alb、TC的含量无明显差异（P＞0.05），而TG存在明显差异（P<0.05），红花组优于缬沙坦组。BUN、SCr的含量模型组显著高于假手术组（P<0.01）；两治疗组与模型组比较，BUN、SCr的含量明显下降（P<0.01），两治疗组之间无显著性差异（P＞0.05）。模型组的血β2-微球蛋白、尿β2-微球蛋白明显高于假手术组（P<0.01），两治疗组则明显低于模型组（P<0.01）。两治疗组之间无明显差异（P＞0.05）。⑶ 光镜观察结果显示：模型组约15%的肾小球伴有节段性硬化，约3%呈球性硬化，，受累肾小球系膜区呈局灶性或弥漫性增宽，毛细血管塌陷，球囊粘连明显，部分肾小球代偿性肥大。病变肾小球周围近曲小管上皮细胞变性肿胀，部分小管腔内可见蛋白管型。间质有局灶性萎缩和纤维组织增生。未受累肾小球呈阴性表现。红花组约10%肾小球伴有节段性硬化，约2%呈球性硬化受累肾小球毛细血管塌陷，系膜区轻度增宽，偶见球囊粘连。有少数近曲小管上皮细胞变性，间质有少量的纤维组织增生及炎性细胞浸润。缬沙坦组的病理改变与红花组基本一致，但小管及间质损伤明显。模型组与假手术组比较，肾小球面积增加（P<0.05），而两治疗组与模型组比较及两治疗组之间无明显差异（P＞0.05）。⑷肾组织<WP=6>t-PA、PAI-1免疫组化分析结果：模型组与假手术组比较，t-PA相对含量及积分光密度表达显著降低（P<0.01），PAI-1的表达明显增高（P<0.01）。两治疗组与模型组相比，t-PA相对含量及积分光密度表达升高（P<0.01或<0.05），而PAI-1则明显降低（P<0.01）。两治疗组中t-PA表达无明显差异（P＞0.05）。PAI-1含量存在明显差异（P<0.01更多还原

【Abstract】 Objective: Focal Segmental Glomerulosclerosis (FSGS) is a common non-inflammation sclerosis pathological change involved in the capillary and glomeruli , which is a common pathologic process of most kidney disease leading to renal failure. The pathological character shows that messegium increased, transparency material of the glomerulus basement membrane epithelium accumulates, capillary and capsular adheres. FSGS is the main pathologic type of nephropathy, which leads to end-stage renal failure 10 years after discovered, and induces glomerulosclerosis and interstitial fibrosis. Most FSG patients with nephritic syndrome react weakly to steroid and the cytotoxic drug treatment, with heavy side effect. Angiotensin converting enzyme inhibitor (ACEI) and Angiotensin II receptor blocker (ARB) can protect the renal function, but the treatment of ACEI and ARB is only one way to kidney disease caused by different pathogenesis. Furthermore, ACEI and ARB have invariably renal damage. So looking for safe drugs is of great significance. Various clinical studies has confirmed that the FSGS has something to do with blood clotting and dissolving system <WP=8>obstacle. Unbalance of blood coagulation and hemolysis system is the important pathophysiologic changes in glomerulosclerosis and interstitial fibrosis. The system depends on Plasminogen activator (t-PA) and Plasminogen antivator inhibition (PAI-1) to adjustment. The key enzyme in the blood coagulation process — zymoplasm, can promote the inherent cell proliferation and secrete various growth factors and the expression of the cell factor, among them including PAI-1 etc., and downregulated the express of t-PA . The research shows that the t-PA is one of the protection factors of the kidney. PAI-1, a specific inhibition factor of t-PA, not only represses in the plasminogen conversion to fibrinolysin so as to inhibit fibrious hydrolysis, but also inhibits extracellular matrix (ECM) accumulation. The plasmase zymoplasm can make fibre proteinosis and inhibit matrix degradation through the up-regulation of PAI-1, lead to glomerulosclerosis. It is important to regulate the balance of blood coagulation and hemolysis system in treatment. It is believed that glomerulosclerosis and ECM accumulation, glomeruli and capsular adhesion, the microthrombus, capillary luman collapse or narrow, interstitial fibrosis with atrophy etc. all of the pathology changes are the category of the stagnant blood internal resistance. So the method of activating blood circulation to dissipate blood stasis must be used. Hong Hua, a Chinese herbal medicine, with the role to activate blood circulation to dissipate blood stasis, has the <WP=9>anticogulation function, keeping blood from bolting formation which is already extensive applied in the type of the cardiovascular and cerebrovascular disease. There is no report about the effect of Hong Hua on blood blood coagulation and hemolysis system and pathomorphology in renal disease. Hong Hua was used in this experiment to treat the FSGS rats model induced by Adriamycin , to observe the expressions of t- PA and its inhibitor PAI-1 with immunohistochemistry and mRNA PAI-1 with in situ hybridization, combining with the renal histochemistry observation, meanwhile the mechanism of Hong Hua in kidney disease was discussed compared to Valsartan.Method: Forty male Sprague-Dawley rats were put into metabolism cage to obtain urine, and urine protein and red blood cell were detected. The result was negative. Then they were randomly divided into four groups (n=10), Sham-operation, Model, Valsartan, Hong Hua. Each animal adopted right side nephrectomy, tail intravenous injection of 0.1% A driamycin respectively after 7 and 30 days of operation except Sham group. Drugs were administered from seventh day after operation but the saline were used in Sham and Model groups orally. Animals were sacrificed after 8 weeks, proceeded by the routine and biochemistry check, and the left side was obtained for pathology observing, expressions of t-PA and PAI-1 were detected by更多还原