【作者】 张秋林；

【导师】 洪钦国；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2006， 博士

【摘要】 目的:（1）了解中西医对肾纤维化发病机制及其防治措施的研究进展;（2）观察升清泄浊法中药制剂尿毒清胶囊对慢性肾功能衰竭患者临床疗效的同时,重点观察其抗肾纤维化的疗效;（3）探讨尿毒清胶囊抗肾纤维化作用机制,为临床应用提供实验依据。 方法:（1）文献研究:通过查阅大量古代和现代中、西医国内外文献,总结和评价有关肾纤维化的中医、西医发病机制和防治措施研究现状。（2）临床研究:用简单随机化、前瞻性的研究方法,将78例慢性肾功能衰竭患者按1∶1分为治疗组和对照组。治疗组用基本治疗加尿毒清胶囊,对照组为基本治疗加包醛氧淀粉,观察患者主要症状、证候（纳差腹胀,恶心呕吐,神疲乏力,腰膝酸痛,皮肤瘙痒,颜面唇甲少华,肌肤甲错,形寒肢冷,手足心热,浮肿,头晕）和实验室检查指标:血红蛋白（Hb）、红细胞总数（RBC）、血清肌酐（Scr）、尿素氮（BUN）、内生肌酐清除率（Ccr）、血清白蛋白（ALB）、血清总胆固醇（Tch）、甘油三酯（TG）、低密度脂蛋白胆固醇（LDL-C）、高密度脂蛋白胆固醇（HDL-C）、血清层粘蛋白（LN）、Ⅲ型前胶原（PC-Ⅲ）、透明质酸（HA）等,4周为一疗程,连续治疗两个疗程统计结果。继续追踪治疗至6个月时统计各组因病情加重放弃内科治疗,而改用透析治疗的病例数。（3）实验研究:将体外培养人肾小管上皮细胞（HK-2）,分为6组:正常对照组、5%尿毒症血清组、10%尿毒症血清组、5%尿毒清胶囊含药血清组、10%尿毒清胶囊含药血清组和蒙诺组(蒙诺浓度为10^-6mmol/L),用MTT法检测细胞增殖、用流式细胞术检测细胞周期、a-平滑肌肌动蛋白（α-SMA）阳性细胞的百分率、用逆转录—聚合酶链反应（RT-PCR）测定结缔组织生长因子mRNA（CTGFmRNA）。 结果:（1）文献研究:中医学没有肾纤维化的表述,但根据其临床表现可归于“水肿”、“关格”、“癃闭”、“虚劳”等病症范畴;在病因病机方面,认为脾肾亏虚是本,湿热壅滞三焦,浊毒内盛、瘀血阻络等为标;治疗上多采用通腑泄浊为主,兼用益气补肾、活血化瘀等法。药物有复方制剂、也有单味药物,还有提取的有效单体成分;作用机制有:抑制参与纤维化细胞的增殖和转分化,抑制致纤维化因子的产生、减少ECM的沉积等,表现出较好的疗效。现代医学认为,肾纤维化涉及到肾固有细胞的激活、增殖,单核巨噬细胞的浸润、炎症介质、细胞因子的产生和分泌,最终导致肾脏正常结构的破坏、细胞外基质的沉积,形成纤维化。上皮—间充质细胞转分化（EMT）在肾纤维化中起重要作用,转化生长因子-β1（TGF-β1）和CTGF是强致肾纤维化因子。从防治措施来讲,寻找抑制参与纤维化细胞的增殖和转分化、抑制致纤维化因子的分泌或拮抗其作用的药物,虽然有些用于临床,但疗效不理想。（2）临床研究:治疗组主要症状总积分、大部分症状单项积分均少于对照组（p<0.05或p<0.01）,症状有效率和总体疗效高于对照组（p<0.05或p<0.01）;尿毒清胶囊能提高慢性肾功能衰竭更多还原

【Abstract】 Objective: (1)To review the research advancement on the pathogenesy and the therapy of prevention and cure in renal fiabrosis. (2)To observe the clinical efficacy on chronic renal failure(CRF) and the anti-renal fibrosis effect of Chinese traditional medicine Niaoduqing Capsules(NDQC) by the therapy of tonifying and lifting clear QI and excreting pathogenic QI. (3)To explore the mechanism of action of NDQJN on renal fibrosis.Methods:(1)Literature study:To look up a great deal of internal and foreign research documents about the pathogenesy and the therapy of prevention and cure in TCM and western medicine on renal fiabrosis, and summarize and assess these datas. (2)Clinical research:Seventy-eight patients with CRF were randomly and equally divided into subject group and control group .The subject group was administered by symptomatic treatment medicines and NDQC, and the control group was administered by symptomatic treatment medicines and Oxyamyli tectus Aldehydum(OTA). The cardinal symptoms and laboratory examination indexes were observed . The cardinal symptoms included bad appetite, abdominal distension, nausea and vomiting, lassitude, sore waist or knee, skin pruritus, pale face or lip and nails, squamous and dry skin, cold body and limbs, feverish palms and soles, edema, and dizziness. The laboratory examination items contained haematoglobin(HB), red blood cell number(RBC), serum creatinine(Scr), serum urea nitrogen(BUN), creatinine clearance(Ccr), seralbumin(ALB), total cholesterol(Teh), triglyceride(TG), low density lipoprotein cholesterol(LDL-C), high-density lipoprotein cholesterol(HDL-C), serum laminin(LN), precollagen-III(PC-III) , and hyaluronic acid (HA). These items was respectively determined before administration and after eight weeks. The number of patients with dialysis treatment, because of aggravated patient’s condition, was recorded in follow-up four months. (3)Experimental study:Human renal tubular epithelial cells (HK-2) were respectively cultured in six different media. Theywere normal control group , 5% uremic serum group, 10% uremic serum group, 5% NDQC serum group, 10% NDQC serum group and Monopril geoup(fosinopril density was 10~6mmol/L). Cell proliferation was measured by methylene blue assay (MTT method). Cell life cycle and the positive cell percentage of a-smooth muscle actin(a-SMA) in every group were evaluated by flow cytometry. Connective tissue growth factor messenger RNA (CTGFmRNA) was detected by reverse transcript ion-polymerase chain reaction(RT-PCR) method.Results: (DReview of publications:There was no the nomenclature ’renal fibrosis’ in TCM, but the clinical manifestation of renal fibrosis or CRF was discovered in these diseases such as ’edema’ , ’frequent vomiting and dysuria’ , ’anuria’ , ’asthenia’ , ’lumbago’ and so on. The etiopathogenisis and pathogenesis of renal fibrosis was summarized as asthenia in origin and state of evil domination in superficiality. The asthenia was mainly due to the deficiency of spleen and kidney, and the state of evil domination mainly included stagnation of damp-heat in triple warmer , turbid toxin in body, obstruction of collaterals by blood stasis. The therapeutic methods of cleaning out FU-organ and excreting turbid pathogen were mainly taken , and tonifying QI and invigorating the kidney were also used at the same time in CRF. The materia medica for CRF contained compound preparation, single pharmaceutical product , and simple substance of effective ingredients extracted. Thier mechanisms of action on renal fibrosis were that they inhibited cell proliferation and transdifferentiation , restrained the production of fibrotic factors, and reduced the deposition of extracellular matrix (ECM) in kidney. TCM had satisfactory curative effect on renal fibrosis. At the viewpoint , the pathogenesy of renal fibrosis related to activation and proliferation of renal cells, infiltration of mononuclear macrophage, production and secretion of mediators of inflammation and cytokines. These resulted in destruction of normal kidney structure and deposition of ECM, and fibrous degeneration finally. Epithelium- mesenchymal transdifferentiation(EMT) was one of important pathogenesies in renal fibrosis. Transforming growth factor-P 1(TGF-0 1 ) and CTGF are important growth factor inducing renal fibrosis. People are researching new methods or medicines to prevent and cure renal fibrosis. They may inhibit cell proliferation and EMT , or prevent these cells from secreting or synthesizing cytokines that results in renal fibrosis. Cytokines antagon are also being studied. Some of these medicines have beenadministered in some patients, but their therapeutic effect are dissatisfactory. ?The clinical research:The total score of all symptoms and the single score of most of symptoms were both remarkable fewer in subject group than in control group (p<0. 05 UK p<0. 01) .The curative effect rate of symptom and total effective power were both more higher in subject group than in control group(p<0. 05 or p<0. 01) .The levels of ALB and HDL-C were more higher , and the levels of Tchu TG> BUN> Scr> Ccr> LI^ PC-IIL HA were much lower in subject group than in control group(p<0. 05 or p<0. 01) .NDQC remarkably decreased the number of patients who accepted dialytic medication in six months (p<0. 05). ?Experimental study: (DAbsorbance 490(A490) was remarkably higher in 5% uremic serum group and 10% uremic serum group than in normal control group with use of MTT(p<0.01) ,and that was without significant difference between 5% NDQC serum group and 5% uremic serum group (p>0. 05) , and that was much lower both in 10% NDQC serum group and in monopril group than in 5% uremic serum group (both p<0. 01) .No significant difference of A490 was found among 5% NDQC serum group, 10% NDQC serum group, monopril group and normal control group (p>0. 05) .?The percentage of cells in GO/G1 phase was remarkably lower in 5% uremic serum group and 10% uremic serum group than in normal control group with use of flow cytometry(p<0. 01), and that was without significant difference between 5% uremic serum group and 10% uremic serum group (p>0. 05) .It was increased significantly in 5% NDQC serum group, 10% NDQC serum group, and monopril group compared with that in 5% uremic serum group (all p<0.01) , and it was not showed significant difference among the fist three group (p>0. 05) .The percentage was lower significantly in 5% NDQC serum group than that in normal control group (p<0.05) ,but No significant difference of the percentage was found among 10% NDQC serum group, monopril group and normal control group (p>0.05) . ?Proliferation index(PI) of HK-2 was increased significantly in 5% uremic serum group , 10% uremic serum group and 5% NDQC serum group than that in normal control group with use of flow cytometry (p<0.01) ,but the PI was without significant difference among 10% NDQC serum group, monopril group and normal control group (p>0. 05) . It was decreased significantly in 5% NDQC serum group, 10% NDQC serum group, and monopril group compared with that in 5% uremic serum group (all p<0.01) .No significant difference of the PI was found among 5% NDQC serum group, 10% NDQC serum group, and monopril group (p>0. 05) . ?The positive percentage of a -SMAcells was increased significantly in 5% uremic serum group and 10% uremic serum group than that in normal control group with use of flow cytometry (p<0. 01) , but no significant difference between the first two groups (p>0. 05) .It wasn’ t decreased significantly in 5% NDQC serum group compared with that in 5% uremic serum group (p>0. 05) , but it was significantly higher in this group than in normal control group (p<0. 01) . The positive percentage was remarkably lower in 10% NDQC serum group and monopril group than in 5% uremic serum group (both p<0. 01) , but higher than in normal control group (p<0. 05) . No significant difference of the positive percentage was found among 5% NDQC serum group, 10% NDQC serum group and monopril group (p>0. 05) . ?The expression of CTGFmRNA was much lower in normal control group than in others (all p<0. 01) , The expression in 10% uremic serum group was highest in all groups (p<0. 01) . No significant difference was found in 5% NDQC serum group compared with that in 5% uremic serum group (p>0. 05) . CTGFmRNA expression was remarkably lower in 10% NDQC serum group and monopril group than in 5% uremic serum group (both p<0.05) . No significant difference of CTGFmRNA expression was found among 5% NDQC serum group, 10% NDQC serum group and monopril group (p>0. 05) . Conclusion: (DThe pathogenesy of renal firosis in CRF is a complicated and interacting network mechanism. Many component elements in the mechanism are not distinct. Most of prevention and cure methods of renal firosis are still in research , and their therapeutic effects are yet dissatisfactory in clinic. TCM has manifested satisfactory anti-renal fibrosis effect and favourable clinical application perspective . (2) NDQC implied ’tonifying and lifting clear QI and excreting turbid pathogen’ not only improved clinical symptoms , but also protected renal function, and decreased the fibrotic indexes in CRF patients.All these results show NDQC is a satisfactory inhibitor of renal fibrosis. (3)The mechanism of action of NDQC on renal fibrosis possibly relates to inhibiting cell proliferation and EMT, and preventing cells from synthetizing or secreting fibrotic cytokines. This research result will provide experimental evidence for NDQC s clinical application.更多还原