【作者】 钟云良；

【导师】 谢桂权；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2009， 博士

【摘要】 目的:本课题选择原发性肾病综合征湿热证为对象,先系统研究湿热因素对肾病综合征临床病情的影响,全面了解湿热因素对肾病综合征影响的重点环节,为指导肾病综合征临床治疗,提高临床疗效提供理论依据。再进行原发性肾病综合征湿热证的蛋白质组学研究,希望通过对肾病综合征湿热证的蛋白质组学研究,可能会发现特异蛋白。通过对特异蛋白的相关蛋白的分析,能够揭示中医肾病综合征湿热证的实质及湿热致病机理。方法:本课题主要采用横断面、前瞻、回顾性调查研究方法。通过调查收集符合纳入标准的原发性肾病综合征病例的一般情况、症状、体征、以及部分实验室检查指标,利用SPSS统计软件进行统计分析,全面系统分析湿热因素对原发性肾病综合征病程、病情、复发、实验室指标、疗效等影响。再采用最前沿的蛋白质组学方法,对原发性肾病综合征湿热证与非湿热证及健康对照组利用双向电泳进行差异蛋白分析,再利用质谱技术进行差异蛋白的鉴定。探讨原发性肾病综合征湿热证的分子机制,研究湿热证的本质。结果:（一）湿热因素对原发性肾病综合征临床病情的影响本研究入选病例共120例,根据中医辨证分为湿热证组60例,非湿热证组60例。对两组病例的一般情况、症状、体征、实验室指标、疗效等进行分析比较,全面系统分析湿热因素对原发性肾病综合征病程、病情、复发、实验室指标、疗效等影响。结果如下:1、一般资料分析湿热证组60例,其中男性47例,女性13例,年龄28.77±16.13岁;非湿热证组60例,其中男性为39例,女性为21例,年龄26.72±15.93岁;经统计学检验,两组在性别、年龄等方面无统计学差异（P＞0.05）。2、湿热对原发性肾病综合征患者住院时间的影响湿热证组平均住院天数为23.90±11.75天,非湿热证组平均住院天数为16.00±7.47天,湿热证原发性肾病综合征患者住院时间明显长于非湿热证患者。经统计学检验,有统计学意义（P＜0.05）。3、湿热对原发性肾病综合征患者病程的影响湿热证组平均病程为42.13±46.49月,非湿热证组平均为29.18±29.27月,湿热证原发性肾病综合征患者病程明显长于非湿热证患者。经统计学检验,有统计学意义（P＜0.05）。4、湿热与伴发感染的关系湿热证组与非湿热证组感染情况比较,湿热证组伴发感染比例（38.3%）叫显高于非湿热证组（16.7%）,经统计学检验后,有显著性差异（P＜0.01）。其中湿热证组伴有呼吸系感染者11（47.8%）例,泌尿系感染者5（21.7%）例,胃肠道感染4（17.4%）例,其他3（13.1%）例;非湿热证组伴有呼吸系感染者5（50.0%）例,泌尿系感染者3（30.0%）例,胃肠道感染1（10.0%）例,其他1（10.0%）例。两组在感染类别情况中并无差异,经统计学检验,无统计学意义（P＞0.05）。5、湿热因素对肾病综合征病情复发的影响湿热证组与非湿热证组复发率比较,原发性肾病综合征湿热证组的复发率（65.0%）明显高于非湿热证组复发率（46.7%）,经统计学检验后,有统计学意义（P＜0.05）6、湿热对临床实验室指标的影响湿热证组与非湿热证组实验室指标比较中,湿热证组血白细胞总数（11.33±6.51*10e9/L）明显高于非湿热证血白细胞总数（8.77±4.50*10e9/L）;湿热证组血清总胆固醇（10.99±4.00mmol/L）亦高于非湿热证组（8.19±2.53 mmol/L）:湿热证组24小时尿蛋白定量（7.74±4.42 g/24h）大于非湿热证组（5.05±3.55 g/24h）。经统计学检验后,有统计学意义（P＜0.05）。而其他指标比较虽在数值上有一定的差异,但经统计学检验,并无统计学意义（P＞0.05）。7、湿热与病理类型的关系在收集的120例肾病综合征病例中有39例有病理报告,占总病例的32.5%,其中湿热证组20例,非湿热证组19例。湿热证组病理类型中:微小病变肾病6例（30%）;系膜增生性肾炎5例（25.0%）;局灶性节段性肾小球硬化0例（0.0%）;膜性肾病8例（40.0%）;系膜毛细血管性肾小球肾炎1例（5.0%）。非湿热证组病理类型中:微小病变肾病4例（21.1%）;系膜增生性肾炎10例（52.6%）;局灶性节段性肾小球硬化3例（15.8%）;膜性肾病2例10.5%);系膜毛细血管性肾小球肾炎0例（0.0%）。结果显示:原发性肾病综合征湿热证组病理类型中的膜性肾病明显多于非湿热证组,经统计学检验后,有统计学意义（P＜0.05）,其它类型比较无明显差异,无统计学意义（P＞0.05）。8、湿热对疗效的影响湿热证组完全缓解19例（31.7%）,显著缓解12例（20.0%）,部分缓解10例（16.7%）,无效19例（31.7%）,总有效率68.3%。非湿热证组完全缓解24例（40.0%）,显著缓解17例（28.3%）,部分缓解9例（15.0%）,无效10例（16.7%）,总有效率83.3%。可见湿热证组有效率明显低于非湿热证组。采用等级资料的秩和检验统计分析,有统计学意义（P＜0.05）。（二）原发性肾病综合征湿热证的蛋白质组学研究在蛋白质组学研究中,选取5例正常对照组、10例原发性肾病综合征湿热证组和10例非湿热证组进行血清双向凝胶电泳和质谱分析。实验结果:图像清晰,没有背景条纹的干扰,蛋白质斑点分离完全,在相同条件下,反复3次重复的实验结果图大致相同,三组图谱图象分析后平均检测到1108个蛋白质斑点。选取正常对照组图谱作为参考胶,其它两组胶与参考胶匹配,使各凝胶中的蛋白质斑点与参考胶中的对应起来,与参考胶中同一蛋白质斑点相匹配的点为同一种蛋白质。匹配结果显示三组胶蛋白质斑点匹配百分率约为80.12%。用PDQuest软件将正常对照组、肾病综合征湿热证组和肾病综合征非湿热证组凝胶图像进行定性定量差异表达分析。结合肉眼分辨,我们发现有19个蛋白质斑点在三组凝胶中有显著差异表达。结合软件分析和手工筛选,我们从表达差异蛋白质点中选取6个点清晰且表达水平改变明显的蛋白质点作为最终质谱鉴定的对象。鉴定的结果为:2号蛋白为载脂蛋白C-Ⅲ（Apolipoprotein C-Ⅲ）;5号蛋白为α₂-HS-糖蛋白（Alpha-2-HS-glycoprotein）;8号蛋白为免疫球蛋白轻链k（Immunoglobulin kappa1-5）;10号蛋白为间α-胰蛋白酶抑制因子重链的H2（Inter-alpha-trypsin inhibitor heavy chain H2）;15号蛋白为α₁-抗胰蛋白酶（Isoform 1 of Alpha-1-antitrypsin）;16号蛋白为血清转铁蛋白（Serotransferrin）。结合临床和蛋白质功能分析,其中载脂蛋白C-Ⅲ和α₂-HS-糖蛋白可作为原发性肾病综合征湿热证的特异表达蛋白,涉及功能主要与脂蛋白代谢、抗炎、抗胰岛素等有关。结论:1、由湿热与肾病综合征感染并发症、复发研究的资料分析提示:湿热是引起肾病综合征病情复发的重要因素。2、由湿热与肾病综合征病程、住院时间、血脂、尿蛋白、疗效等资料分析提示:湿热是引起肾病综合征病情加重、迁延、难治的重要原因。3、通过对健康对照组和原发性肾病综合征湿热证组及非湿热证组的血清蛋白银染2-DE图谱分析,我们发现确实存在差异表达蛋白。4、通过对差异蛋白的鉴定分析,初步认为载脂蛋白C-Ⅲ和α₂-HS-糖蛋白可作为原发性肾病综合征湿热证的特异表达蛋白,涉及功能主要与脂蛋白代谢、抗炎、抗胰岛素等有关。更多还原

【Abstract】 Objective:To guide clinical treatment of nephrotic syndrome,it was started with primary nephrotic Damp-heat and the first systematic research on the nephrotic syndrome of the impact of clinical conditions which is a comprehensive understanding of heat nephrotic syndrome on the impact of key links.With the purpous of further research in damp-heat syndrome with primary nephrotic syndrome and the possibility of the discovery of that specific protein,we analyzed the specific protein that could reveal the damp-heat syndrome of TCM nephrotic syndrome and the pathogenesis of the connotation.Methods:To explore the molecular mechanism of Primary nephrotic syndrome Damp-heat,and the nature of Damp-heat.It was worked in the methods of cross-section,forward-looking and retrospective survey.To standard in line with the primary nephrotic syndrome,we investgated the patients in general,symptoms,signs,and some indicators of laboratory tests by using SPSS statistical software for statistical analysis to explore the Damp-heat factor in the onset primary nephrotic syndrome and the role in the course of its relevance.Systematically-analyzed the effects of the Damp-heat factors in each course of disease,condition, relapse,laboratory indicators,efficacy,etc.This study use the most cutting-edge proteomics approach,with primary nephrotic syndrome of damp-heat syndrome with non-heat Syndrome and healthy controls for differences in the use of two-dimensional gel electrophoresis protein analysis,and then carried out using mass spectrometry to identify protein differences.Result:(一) The impact of Damp-heat on the clinical condition of Primary nephrotic syndromeIn this study,a total of 120 cases of selected cases,including 60 cases of Damp-heat syndrome group and 60 cases of non Damp-heat syndrome group.Through analyzed and compared between the two groups in general, symptoms,signs,laboratory indicators,effects,this study systematically analysis the effects of the Damp-heat factor in the Drimary nephrotic syndrome patient’s course of disease,condition,relapse, laboratory indicators,efficacy,etc.The results are as follows.1.General data analysisThere are 60 cases of Damp-heat syndrome group,Including 47 males and 13 females,and their average age is 28.77±16.13 years old.There are 60 cases of non Damp-heat syndrome group,Including 39 males and 21 females,and their average age is 26.72±15.93 years old.By statistical testing,there is not significant difference between the two groups in gender,age and so on(P＞0.05).2.The impact of Damp-heat on the days staying in hospital of PNS patientsThe average days staying in hospital of Damp-heat syndrome group are 23.90±11.75 days;and the average days staying in hospital of non Damp-heat syndrome group are 16.00±7.47 days.The days staying in hospital of primary nephrotic syndrome Damp-heat syndrome group are significantly longer than the non Damp-heat syndrome group.By statistical testing,there is difference between the two groups(P＜0.05).3.The impact of Damp-heat on the course of PNS patientsThe average duration of Damp-heat syndrome group is 42.13±46.49 months;and the average duration of non Damp-heat syndrome group is 29.18±29.27 months.The duration of PNS Damp-heat syndrome group are significantly longer than the non Damp-heat syndrome group.By statistical testing,there is difference between the two groups(P＜0.05).4.The relationship between Damp-heat and infectionThe percent of infection cases in the Damp-heat group(38.3%)is higher than the non Damp-heat group(16.7%).By statistical testing,there is significant difference between the two groups(P＜0.01).The Damp-heat group includes 11 cases(47.8%) of respiratory infections,5 cases (21.7%)of urinary tract infections,4 cases(17.4%) of gastrointestinal infection and other 3 cases(13.1%);The non Damp-heat group includes 5 cases(50.0%) of respiratory infections,3 cases(30%)of urinary tract infections,1 cases(10.0%) of gastrointestinal infection and other 1 cases(10.0%).By statistical testing,there is not difference between the two groups on the type of infection.5.The impact of Damp-heat on the relapse of PNS patientsThe percent of relapse in the Damp-heat group(65.0%) is higher than the non Damp-heat group(46.7%).By statistical testing,there is difference between the two groups(P＜0.05).6.The impact of Damp-heat on the laboratory indicators of PNS patientsThe total white blood cell of Damp-heat group(11.33±6.51*10e9/L) is significantly higher than the Damp-heat group’ s(8.77±4.50*10e9/L).The Total cholesterol of Damp-heat group(10.99±4.00mmol/L) is higher than the Damp-heat group’ s(8.19±2.53mmol/L). The 24-hour urinary protein of Damp-heat group(7.74±4.42 g/24h) is higher than the Damp-heat group’ s(5.05±3.55 g/24h).By statistical testing,there is difference between the two groups on total white blood cell,Total cholesterol and 24-hour urinary protein(P＜0.05).But,by statistical testing,there is not difference between the two groups on Other laboratory indicator(P＞0.05).7.The relationship between Damp-heat and Pathological typesIn the collection of 120cases of PNS,there are 39 cases of patients having pathology report,accounted for 32.5 percent of total cases, including 20 cases of Damp-heat syndrome group and 19 cases of non Damp-heat syndrome group.The Damp-heat group includes 6 cases(30%) of Minimal change nephropathy,5 cases(25.0%) of Mesangial proliferative glomerulonephritis,8 cases(40.0%) of Membranous nephropathy and 1 case(5.0%)of Mesangial capillary glomerulonephritis.And the non Damp-heat group includes 4 cases(21.1%) of Minimal change nephropathy, 10 cases(52.6%) of Mesangial proliferative glomerulonephritis,3 case(15.8%) of Focal segmental glomerulosclerosis and 2 cases(10.5%) of Membranous nephropathy.The results showed that the membranous nephropathy patients of PNS Damp-heat Syndrome group are more than the non Damp-heat syndrome group’ s.By statistical testing,there is difference between the two groups on membranous nephropathy(P＜0.05). But,by statistical testing,there is not difference between the two groups on other pathological types(P＞0.05).8.The impact of Damp-heat on curative effectThe Damp-heat group includes 19 cases(31.7%) of complete remission, 12 cases(20.0%) of significant remission,10 cases(16.7%) of partial remission and 19 cases(31.7%) of invalid.The total effective rate of the Damp-heat group is 68.3 percent.The non Damp-heat group includes 24 cases(40.0%) of complete remission,17 cases(28.3%) of significant remission,9 cases(15.0%) of partial remission and 10 cases(16.7%) of invalid.The total effective rate of the non Damp-heat group is 83.3 percent.This shows the effective rate of Damp-heat syndrome group was lower than the non Damp-heat syndrome group.By statistical testing,there is difference between the two groups on effective rate(P＜0.05).(二) Proteome research of Primary nephrotic syndrome Damp-heat syndromeIn proteomics research,That select 5 cases of normal control group, 10 cases of PNS Damp-heat syndrome group and 10 cases of non Damp-heat syndrome group to do serum Two-dimensional gel electrophoresis and mass spectrometry analysis.The experimental results show:The pictures are clear;There is no background interference stripes;The protein spots are completely separated;Under the same conditions and repeated 3 times,the experimental results similar to Figure;Through analysising the three sets of maps,there are finded out 1108 protein spots.Select the normal map as a reference gel,the other two groups with reference to glue rubber match,so that the gel of protein spots with reference to the corresponding glue together with the reference gel protein spots in the same match with the point of a protein.The results showed that the percentage of protein spots matched on the three sets of gel is about 80.12%.The study use PDQuest software to analysis the differentially expressed protein in the two-dimensional gel images ofthe normal control group,PNS Damp-heat syndrome group and non Damp-heat group.Combined with the naked eye to distinguish,we found 19 protein spots in the gel three groups were significantly different in expression.By software analysis and manual selection,we selected six protein spots that are clear and obvious changes in expression levels from the differentially expressed protein to do mass spectrometry.The results of identified show that the No.2 protein is Apolipoprotein C-Ⅲ,the No.5 protein is Alpha-2-HS-glycoprotein,the No.8 protein is Immunoglobulin kappa 1-5, the No.10 protein is Inter-alpha-trypsin inhibitor heavy chain H2,the No.15 protein is Isoform 1 of Alpha-1-antitrypsin and the No.5 protein is Serotransferrin.Combined clinical and protein function,that the Apolipoprotein C-Ⅲandα2-HS-glycoprotein protein can be used as the specific expressed protein of PNS Damp-heat Syndrome.Conclusion:1.Form the data of the relationship between PNS Damp-heat and infection and recurrence,the study came to the conclusion that Damp-heat is an an important factor that Leads to recurrence of PNS patients.2.Form the data of the relationship among PNS Damp-heat,course of disease,the days staying in hospital,blood lipids and urinary protein, efficacy,etc,;The study came to the conclusion that Damp-heat is an important factor that Leads to the condition of PNS patients exacerbate, procrastinate and Refractory.3.Through analysising Serum protein silver staining 2-DE map of Normal control group,PNS Damp-heat syndrome group and non Damp-heat Syndrome, we found the differentially expressed proteins.4.Through the identification of differences in protein,the study reached a preliminary view that Apolipoprotein C-Ⅲandα2-HS-glycoprotein protein can be used as evidence of the specific expressed protein of PNS Damp-heat.更多还原