狼疮性肾炎中医证型与临床病理的相关性及其蛋白质组学研究
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摘要
目的
1、临床研究
本课题通过搜集狼疮性肾炎患者的一般情况、中医证候、理化指标及肾脏病理结果,建立狼疮性肾炎中医证候要素和理化指标、病理分型的观察表和数据库,利用统计软件分析狼疮性肾炎中医证型和理化指标、病理分型的关系,实现中医宏观辨证与微观辨证的有机结合,为中医临床辨证提供客观依据。
2、实验研究
通过分析正常人与狼疮性肾炎患者及狼疮性肾炎患者热毒炽盛型和脾肾阳虚型的血清蛋白质组学,筛选出差异性表达蛋白,发现狼疮性肾炎的相关蛋白和具有重要应用前景的生物标志物,以及从分子生物学水平揭示与中医某一证候形成相关的蛋白质及其特征,探索蛋白质表达图谱与疾病中医分型之间系统的有规律的联系,为狼疮性肾炎的中医辨证分型提供客观化依据。
方法
1、临床研究
将120例狼疮性肾炎患者进行中医辨证分型,分为本证(热毒炽盛、脾肾气虚、脾肾阳虚、肝肾阴虚、气阴两虚、风湿内扰)、兼证(血瘀、湿热),并进行临床理化指标及肾脏病理资料的采集,利用SPSS统计软件进行统计分析,全面系统分析狼疮性肾炎患者中医证型与理化指标、肾脏病理分型的相关性。
2、实验研究
对狼疮性肾炎患者与正常人及狼疮性肾炎热毒炽盛型与脾肾阳虚型的血清利用双向电泳技术分离蛋白质,凝胶用银染显色,扫描仪获取凝胶图像并对其进行软件分析,找出差异蛋白点,对差异蛋白点进行胶内酶切、质谱分析,获得肽指纹图谱,通过蛋白质数据库进行检索,鉴定蛋白质。
结果
1、临床研究
本研究共选病例120例,根据患者的一般情况、症状、体征、理化指标及肾脏病理等进行对比分析,全面系统分析中医证型与理化指标、肾脏病理的相关性,具体如下：
(1)一般资料分析：120例狼疮性肾炎患者,其中男性14例,年龄20岁-38岁,平均年龄27.6±5.2岁,病程最短1周,最长5年,平均28.3±6.5月；女性106例,年龄16岁-58岁,平均年龄32.5±8.6岁,病程最短3天,最长12年,平均36.7±9.3月；
(2)中医证候分布：通过频数分析结果显示120例狼疮性肾炎患者,中医辨证分型主证以脾肾气虚型最多见,共35例(29.2%)；肝肾阴虚、热毒炽盛、气阴两虚型、脾肾阳虚分别有26例(21.7%)、16例(13.3%)、14例(11.7%)、13例(10.8%),风湿内扰型16例(13.3%)；兼证包括血瘀型和湿热型,分别为56例(46.7%)和41例(34.2%)。
(3)病理分型：通过频数分析结果显示120例狼疮性肾炎患者,最常见的病理分型是Ⅳ型,共87例(72.5%),其次是Ⅴ+Ⅳ型、Ⅴ型和Ⅲ型,分别有10例(8.3%)、8例(6.7%)、8例(6.7%)；Ⅴ+Ⅲ型有6例(5%),Ⅱ型5例(4.2%),Ⅰ型和Ⅵ型分别有4例(3.3%)和2例(1.7%)。采用Spearman秩相关分析,结果提示中医证型与肾脏病理分型无相关性。
(4)临床分型：通过频数分析结果显示120例狼疮性肾炎患者中,临床表现为肾病综合征最多72例(60%),慢性肾炎型36例(30%),隐匿性肾炎型和急进性肾炎型分别6例(5%)、4例(3.3%)。采用Spearman秩相关分析,结果提示中医证型与临床分型无相关性。
(5)免疫指标：通过频数分析结果显示120例狼疮性肾炎患者ANA全部阳性(100%),血清补体C3降低者104例(86.7%),抗ds-DNA|阳性者101例(84.2%),抗ssA阳性者55例(45.8%),抗Sm阳性者41例(34.2%),抗ssB阳性者33例(27.5%), ANCA阳性者4例(3.3%)。采用Spearman秩相关分析,结果提示中医证型与免疫指标无相关性。
(6)狼疮病情活动度分期：通过频数分析结果显示120例狼疮性肾炎患者中,SLE-DAI积分最低为4分,最高为28分,平均(16.28±5.46)分。以狼疮重度活动(SLE-DAI≥15分)为主,共70例(58.3%),其次为中度活动33例(27.5%),轻度活动者14例(11.7%),无活动者仅有3例(2.5%)。采用Spearman秩相关分析,结果提示中医证型与狼疮病情活动分期无相关性。
采用单因素方差分析对中医证型与SLE-DAI积分进行统计分析,结果提示：气阴两虚型的SLE-DAI积分最低(12.69±5.06分),明显小于脾肾阳虚和热毒炽盛型的SLE-DAI积分(P<0.05)；风湿内扰型的SLE-DAI积分低于脾肾阳虚型(P<0.05)。表明气阴两虚与脾肾阳虚及热毒炽盛型间的SLE-DAI积分、风湿内扰型与脾肾阳虚型的SLE-DAI积分差异有统计学意义。其它各证型之间的SLE-DAI积分无明显差异。
(7)临床表现：通过频数分析结果显示120例狼疮性肾炎患者中,肾外脏器的表现以血液系统损害最常见共92例(76.7%),发热36例(30%),关节炎49例(40.8%),面部红斑44例(36.7%),口腔溃疡25例(20.8%),光敏感25例(20.8%),浆膜炎18例(15%),神经系统损害6例(5.0%)；采用Spearman秩相关分析,结果提示中医证型与临床表现无相关性。
(8)理化指标：理化指标WBC、TG、LDL-C和HDL-C各证型组间对比无明显差异；热毒炽盛组LYM明显低于肝肾阴虚组和脾肾气虚组,脾肾阳虚组LYM低于肝肾阴虚组,其余各组间对比无明显差异；脾肾阳虚组RBC明显低于肝肾阴虚组和气阴两虚组,其它组间对比无明显差异；脾肾阳虚组HGB明显低于肝肾阴虚组,其它组间对比无明显差异；气阴两虚组PLT明显高于湿热组,其它组间对比无明显差异；血瘀组FIB水平明显高于其它各组；脾肾阳虚组ALB水平明显低于肝肾阴虚、脾肾气虚、风湿内扰和湿热组,血瘀组ALB水平明显低于肝肾阴虚,其它组间对比无明显差异；脾肾阳虚组BUN、CREA、UA和Cys水平明显高于其余各组,其它组间对比无明显差异；肝肾阴虚组24小时尿蛋白定量明显低于脾肾阳虚组和血瘀组,其它组间对比无明显差异；说明中医证型与LYM、RBC、HGB、PLT、FIB、BUN、CREA、UA和Cys水平可能有关。
2、实验研究
在蛋白质组学研究中,选取4例正常对照组、4例热毒炽盛型LN患者和4例脾肾阳虚型LN患者进行血清双向凝胶电泳和质谱分析。实验结果显示：LN患者热毒炽盛型与脾肾阳虚型比较表达的差异点有5个,热毒炽盛型高表达的蛋白质是免疫球蛋白亩链C和间a-胰蛋白酶抑制因子重链H4,脾肾阳虚型高表达的蛋白质是细胞角蛋白10；正常人与狼疮性肾炎患者的差异点有4个,狼疮性肾炎高表达的蛋白质是α1-抗胰蛋白酶和α1-抗糜蛋白酶,正常人高表达的是二硫键异构酶A6蛋白。
结论
1、临床研究
(1)狼疮性肾炎的中医证型与肾脏病理分型、临床分型、免疫指标及临床表现之间的相关性无统计学意义。
(2)狼疮性肾炎的中医证型与SLE-DAI存在相关性。
(3)狼疮性肾炎的中医证型与实验室指标LYM、RBC、HGB、PLT、FIB、BUN、CREA、 UA、CysC水平和24小时尿蛋白定量存在相关性；而与WBC、TG、LDL-C和HDL-C无相关性。
2、实验研究
正常人与狼疮性肾炎患者的血清蛋白质表达有差异,这些差异蛋白可能有助于揭示狼疮性肾炎的发病机制或作为其新的生物标记物,对狼疮性肾炎的诊断及治疗有重要意义；热毒炽盛型与脾肾阳虚型LN患者的血清蛋白质表达有差异,提示同一疾病不同中医证型间的蛋白质表达不同,利用蛋白质组学技术有望对中医证候的发生机制进行阐释。
Objectives
1. Clinical Study
The study established observation tables and databases of lupus nephritis with TCM syndrome elements、physicochemical index and pathological types etc through the collection of lupus nephritis patients in general、TCM syndromes、 physicochemical index and renal pathology, then analysed the relationship of TCM syndromes and physicochemical index、pathological types of lupus nephritis by using statistical analysis software, to provide evidence for microcosmic differentiation of TCM syndromes,which was beneficial to guide us for the diagnosis and treatment of LN with Integrated Traditional Chinese and Western Medicine.
2. Experimental Study
The study analysed serum proteomics of normal human and patients with lupus nephritis and lupus nephritis patients with excessive heat-toxic and yang deficiency of spleen and kidney, screening differential expression proteins, and try to found proteins that associated with lupus nephritis and important application perspective biomarkers, as well as to found proteins that related with TCM syndromes,then to explore the protein expression profiles and relationship between proteins and TCM types. To try to provide objective basis for syndrome differentiation and treatment of lupus nephritis.
Methods
1. Clinical studies
120cases of patients with lupus nephritis were divided into eight different TCM syndromes including (excessive heat-toxic、yin deficiency of liver and kidney、Qi deficiency of spleen and kidney、yang deficiency of spleen and kidney、deficiency of both Qi and Yin、wind-dampnes internal disturbance、 blood stasis and Damp-heat), and clinical physicochemical index and renal pathology collection, and then used SPSS statistical analysis software to analyse the relationship between TCM types and physicochemical index、renal pathological types of patients with lupus nephritis.
2. Experimental Study
To separate serum proteins by using two-dimensional gel electrophoresis for protein separation on lupus nephritis patients and normal persons and lupus nephritis with excessive heat-toxic and Yang deficiency of spleen and kidney, the gels were dyed with silver staining and scanned for gel pictures, to find out the differential proteins with software. The differential proteins were cut and digested with trypsin and identificated them with mass spectrographic analysis, and to obtain the finger print map of peptide, then to serch them through Protein Data Bank and identificate them.
Results
1. Clinical studies
The study selected120cases, according to the general condition of patients, symptoms, signs, physicochemical index and renal pathology were compared and analysed the relationship of TCM types and physicochemical index, renal pathology, as follows:
(1) General data analysis:120cases of patients with lupus nephritis,14cases were male, aged20years to38years, mean age was27.6±5.2years old, average disease course was28.3±6.5months;106cases were female, aged16years to58years, mean age was32.5±8.6years, average disease course was36.7±9.3months.
(2) The distribution of TCM types:The frequency analysis showed that Qi deficiency of spleen and kidney was the major type, there was35cases (29.2%); yin deficiency of liver and kidney、excessive heat-toxic、deficiency of both Qi and Yin、yang deficiency of spleen and kidney, respectively there was26cases (21.7%)、16cases (13.3%)、14cases (11.7%)、13cases (10.8%), wind-dampness internal distrubance was16cases (13.3%); blood stasis and damp-heat types, respectively was56cases (46.7%) and41cases (34.2%).
(3) Pathology types:The frequency analysis showed that type Ⅳ was the major type, there was87cases (72.5%), followed by the type Ⅴ+Ⅳ, type Ⅴ and type Ⅲ, there were10cases (8.3%)、8cases (6.7%)、8cases (6.7%); the type Ⅴ+Ⅲ was6cases (5%), type Ⅱ was5cases (4.2%), type Ⅰ and type Ⅵ each had4cases (3.3%) and2cases (1.7%). Using Spearman rank correlation analysis, results suggested that TCM types and renal pathology types without correlation.
(4)Clinical types:The frequency analysis showed that nephrotic syndrome was the major type,72cases (60%), chronic nephritis there was36cases (30%), latent nephritis and rapidly progressive glomerulonephritis respectively was6cases (5%) and4cases (3.3%). Using Spearman rank correlation analysis, results suggested that TCM types and clinical types without correlation.
(5) Immune index:The frequency analysis results showed that120cases of lupus nephritis patients with ANA positive (100%); Anti ds-DNA positive was101cases (84.2%), anti-Sm positive was41cases(34.2%), anti-ssA positive was55cases (45.8%), the anti-ssB positive was33cases(27.5%), serum C3decreased was104cases (86.7%), the ANCA positive only had4cases(3.3%). Using Spearman rank correlation analysis, results suggested no correlation between TCM types and immune index.
(6) Lupus disease activity level:The frequency analysis results showed that the lowest score of SLE-DAI integral was4points, the highest was28points, the average score was (16.28±5.46) points. Severe activity lupus (SLE-DAI>15)there was70cases (58.3%), followed by moderate activity was33cases (27.5%), mild activity was14cases (11.7%), no activity only had3cases (2.5%). Using Spearman rank correlation analysis, results suggested no correlation between TCM types and disease activity types.
Using single factor statistical analysis of variance on TCM types and SLE-DAI integrals, the results suggested:the SLE-DAI integral of deficiency of both Qi and Yin type was the lowest (12.69±5.06), significantly less than excessive heat-toxic and Yang deficiency of spleen and kidney (P<0.05). the SLE-DAI integral of wind-dampness internal distrubance less than Yang deficiency of spleen and kidney (P<0.05). The SLE-DAI integral between other TCM types had no statistical significance (P>0.05).
(7) Clinical manifestations:The frequency analysis results showed that the most common damage of the extra-renal organs was the blood system, there was92cases (76.7%), fever in36cases (30%),49cases of arthritis (40.8%), facial erythema was44cases (36.7%), oral ulcers was25cases (20.8%), light-sensitive was25cases (20.8%), serositis was18cases (15%), nervous system damage was6cases (5.0%). Using Spearmanrank correlation analysis, results suggested that TCM types and clinical manifestations without correlation.
(8)Physicochemical indicators:The results showed that physicochemical indexes of WBC, TG, LDL-C and HDL-C in groups of differential TCM types have no difference;the LYM of Excessive heat-toxic type was significantly lower than Yin deficiency of liver and kidney and Qi deficiency of spleen and kidney, LYM of yang deficiency of spleen and kidney group was significantly lower than Yin deficiency of liver and kidney, other groups showed no differences;the RBC of yang deficiency of spleen and kidney group was significantly lower than Yin deficiency of liver and kidney and deficiency of both Qi and Yin, other groups showed no significant difference; the HGB of yang deficiency of spleen and kidney group was significantly lower than Yin deficiency of liver and kidney, other groups showed no significant difference;the PLT of deficiency of both Qi and Yin group was significantly higher than damp-heat group, other groups had no significant difference; the FIB level of blood stasis type was significantly higher than the other groups;the ALB level of Yang deficiency of spleen and kidney group significantly lower than the types of yin deficiency of liver and kidney, Qi deficiency of spleen and kidney, wind-dampness internal disturbance and damp-heat,and the ALB level of blood stasis group significantly lower than yin deficiency of liver and kidney, the other groups had no obvious difference; the levels of BUN, CREA, UA and Cys C of Yang deficiency of spleen and kidney were significantly higher than those of other groups, the other groups there were not significantly different; the quantitates of24hour urine protein of Yin deficiency of liver and kidney group was significantly lower than that yang deficiency of spleen and kidney and blood stasis groups, other groups had no significant difference. These results may suggest that TCM types have relavent with LYM、RBC、HGB、PLT、FIB、BUN、CREA、UA、Cys C and the quanti tates of24hour urine protein.
2.Experimental Study
In the study we selected4cases of the normal persons,4cases of excessive heat-toxin type of LN patients and4cases of Yang deficiency of spleen and kidney type of LN patients, by using two-dimensional gel electrophoresis for protein separation. The experimental results showed:the excessive heat-toxin type of LN patients had5differential expression spots contrast to Yang deficiency of spleen and kidney of LN patients, the excessive heat-toxin type of LN patients high expressed of the proteins were Ig mu chain C and Inter-alpha-trypsin inhibitor heavy chain H4, yang deficiency of spleen and kidney type of LN patients high expressed of the protein was Keratin type1cytoskeletal10. the LN patients had4differential expression spots contrast to normal persons, LN patients high expressed of the proteins were Alpha-1-antitrypsin and Alpha-1-antichymotrypsin, normal persons high expressed of the protein was Protein disulfide-isomerase A6.
Conclusion
1. Clinical research
(1) The TCM types have no correlation with renal pathology, clinical classification, immune indicators and clinical manifestations in lupus nephritis patients.
(2)The SLE-DAI is correlated with the TCM types in Lupus Nephritis.
(3)The TCM types have relevant to physichemical indicators such as blood LYM、RBC、HGB、PLT、FIB、BUN、CREA、UA、Cys C and urinary protein; and have no significant correlation with blood WBC、TG、LDL-C and HDL-C.
2. Experimental Study
There are some difference of proteins expressing between normal persons and Lupus Nephritis patients, the different proteins may help to reveal the pathogenesis of lupus nephritis or as new biomarkers for lupus nephritis, it is important in the diagnosis and treatment of lupus nephritis;and also there are some difference of proteins expressing between excessive heat-toxic and yang deficiency of spleen and kidney in Lupus Nephritis patients, we may can use proteomics technology to reveal the mechanism of TCM.
1、临床研究
本课题通过搜集狼疮性肾炎患者的一般情况、中医证候、理化指标及肾脏病理结果,建立狼疮性肾炎中医证候要素和理化指标、病理分型的观察表和数据库,利用统计软件分析狼疮性肾炎中医证型和理化指标、病理分型的关系,实现中医宏观辨证与微观辨证的有机结合,为中医临床辨证提供客观依据。
2、实验研究
通过分析正常人与狼疮性肾炎患者及狼疮性肾炎患者热毒炽盛型和脾肾阳虚型的血清蛋白质组学,筛选出差异性表达蛋白,发现狼疮性肾炎的相关蛋白和具有重要应用前景的生物标志物,以及从分子生物学水平揭示与中医某一证候形成相关的蛋白质及其特征,探索蛋白质表达图谱与疾病中医分型之间系统的有规律的联系,为狼疮性肾炎的中医辨证分型提供客观化依据。
方法
1、临床研究
将120例狼疮性肾炎患者进行中医辨证分型,分为本证(热毒炽盛、脾肾气虚、脾肾阳虚、肝肾阴虚、气阴两虚、风湿内扰)、兼证(血瘀、湿热),并进行临床理化指标及肾脏病理资料的采集,利用SPSS统计软件进行统计分析,全面系统分析狼疮性肾炎患者中医证型与理化指标、肾脏病理分型的相关性。
2、实验研究
对狼疮性肾炎患者与正常人及狼疮性肾炎热毒炽盛型与脾肾阳虚型的血清利用双向电泳技术分离蛋白质,凝胶用银染显色,扫描仪获取凝胶图像并对其进行软件分析,找出差异蛋白点,对差异蛋白点进行胶内酶切、质谱分析,获得肽指纹图谱,通过蛋白质数据库进行检索,鉴定蛋白质。
结果
1、临床研究
本研究共选病例120例,根据患者的一般情况、症状、体征、理化指标及肾脏病理等进行对比分析,全面系统分析中医证型与理化指标、肾脏病理的相关性,具体如下：
(1)一般资料分析：120例狼疮性肾炎患者,其中男性14例,年龄20岁-38岁,平均年龄27.6±5.2岁,病程最短1周,最长5年,平均28.3±6.5月；女性106例,年龄16岁-58岁,平均年龄32.5±8.6岁,病程最短3天,最长12年,平均36.7±9.3月；
(2)中医证候分布：通过频数分析结果显示120例狼疮性肾炎患者,中医辨证分型主证以脾肾气虚型最多见,共35例(29.2%)；肝肾阴虚、热毒炽盛、气阴两虚型、脾肾阳虚分别有26例(21.7%)、16例(13.3%)、14例(11.7%)、13例(10.8%),风湿内扰型16例(13.3%)；兼证包括血瘀型和湿热型,分别为56例(46.7%)和41例(34.2%)。
(3)病理分型：通过频数分析结果显示120例狼疮性肾炎患者,最常见的病理分型是Ⅳ型,共87例(72.5%),其次是Ⅴ+Ⅳ型、Ⅴ型和Ⅲ型,分别有10例(8.3%)、8例(6.7%)、8例(6.7%)；Ⅴ+Ⅲ型有6例(5%),Ⅱ型5例(4.2%),Ⅰ型和Ⅵ型分别有4例(3.3%)和2例(1.7%)。采用Spearman秩相关分析,结果提示中医证型与肾脏病理分型无相关性。
(4)临床分型：通过频数分析结果显示120例狼疮性肾炎患者中,临床表现为肾病综合征最多72例(60%),慢性肾炎型36例(30%),隐匿性肾炎型和急进性肾炎型分别6例(5%)、4例(3.3%)。采用Spearman秩相关分析,结果提示中医证型与临床分型无相关性。
(5)免疫指标：通过频数分析结果显示120例狼疮性肾炎患者ANA全部阳性(100%),血清补体C3降低者104例(86.7%),抗ds-DNA|阳性者101例(84.2%),抗ssA阳性者55例(45.8%),抗Sm阳性者41例(34.2%),抗ssB阳性者33例(27.5%), ANCA阳性者4例(3.3%)。采用Spearman秩相关分析,结果提示中医证型与免疫指标无相关性。
(6)狼疮病情活动度分期：通过频数分析结果显示120例狼疮性肾炎患者中,SLE-DAI积分最低为4分,最高为28分,平均(16.28±5.46)分。以狼疮重度活动(SLE-DAI≥15分)为主,共70例(58.3%),其次为中度活动33例(27.5%),轻度活动者14例(11.7%),无活动者仅有3例(2.5%)。采用Spearman秩相关分析,结果提示中医证型与狼疮病情活动分期无相关性。
采用单因素方差分析对中医证型与SLE-DAI积分进行统计分析,结果提示：气阴两虚型的SLE-DAI积分最低(12.69±5.06分),明显小于脾肾阳虚和热毒炽盛型的SLE-DAI积分(P<0.05)；风湿内扰型的SLE-DAI积分低于脾肾阳虚型(P<0.05)。表明气阴两虚与脾肾阳虚及热毒炽盛型间的SLE-DAI积分、风湿内扰型与脾肾阳虚型的SLE-DAI积分差异有统计学意义。其它各证型之间的SLE-DAI积分无明显差异。
(7)临床表现：通过频数分析结果显示120例狼疮性肾炎患者中,肾外脏器的表现以血液系统损害最常见共92例(76.7%),发热36例(30%),关节炎49例(40.8%),面部红斑44例(36.7%),口腔溃疡25例(20.8%),光敏感25例(20.8%),浆膜炎18例(15%),神经系统损害6例(5.0%)；采用Spearman秩相关分析,结果提示中医证型与临床表现无相关性。
(8)理化指标：理化指标WBC、TG、LDL-C和HDL-C各证型组间对比无明显差异；热毒炽盛组LYM明显低于肝肾阴虚组和脾肾气虚组,脾肾阳虚组LYM低于肝肾阴虚组,其余各组间对比无明显差异；脾肾阳虚组RBC明显低于肝肾阴虚组和气阴两虚组,其它组间对比无明显差异；脾肾阳虚组HGB明显低于肝肾阴虚组,其它组间对比无明显差异；气阴两虚组PLT明显高于湿热组,其它组间对比无明显差异；血瘀组FIB水平明显高于其它各组；脾肾阳虚组ALB水平明显低于肝肾阴虚、脾肾气虚、风湿内扰和湿热组,血瘀组ALB水平明显低于肝肾阴虚,其它组间对比无明显差异；脾肾阳虚组BUN、CREA、UA和Cys水平明显高于其余各组,其它组间对比无明显差异；肝肾阴虚组24小时尿蛋白定量明显低于脾肾阳虚组和血瘀组,其它组间对比无明显差异；说明中医证型与LYM、RBC、HGB、PLT、FIB、BUN、CREA、UA和Cys水平可能有关。
2、实验研究
在蛋白质组学研究中,选取4例正常对照组、4例热毒炽盛型LN患者和4例脾肾阳虚型LN患者进行血清双向凝胶电泳和质谱分析。实验结果显示：LN患者热毒炽盛型与脾肾阳虚型比较表达的差异点有5个,热毒炽盛型高表达的蛋白质是免疫球蛋白亩链C和间a-胰蛋白酶抑制因子重链H4,脾肾阳虚型高表达的蛋白质是细胞角蛋白10；正常人与狼疮性肾炎患者的差异点有4个,狼疮性肾炎高表达的蛋白质是α1-抗胰蛋白酶和α1-抗糜蛋白酶,正常人高表达的是二硫键异构酶A6蛋白。
结论
1、临床研究
(1)狼疮性肾炎的中医证型与肾脏病理分型、临床分型、免疫指标及临床表现之间的相关性无统计学意义。
(2)狼疮性肾炎的中医证型与SLE-DAI存在相关性。
(3)狼疮性肾炎的中医证型与实验室指标LYM、RBC、HGB、PLT、FIB、BUN、CREA、 UA、CysC水平和24小时尿蛋白定量存在相关性；而与WBC、TG、LDL-C和HDL-C无相关性。
2、实验研究
正常人与狼疮性肾炎患者的血清蛋白质表达有差异,这些差异蛋白可能有助于揭示狼疮性肾炎的发病机制或作为其新的生物标记物,对狼疮性肾炎的诊断及治疗有重要意义；热毒炽盛型与脾肾阳虚型LN患者的血清蛋白质表达有差异,提示同一疾病不同中医证型间的蛋白质表达不同,利用蛋白质组学技术有望对中医证候的发生机制进行阐释。
Objectives
1. Clinical Study
The study established observation tables and databases of lupus nephritis with TCM syndrome elements、physicochemical index and pathological types etc through the collection of lupus nephritis patients in general、TCM syndromes、 physicochemical index and renal pathology, then analysed the relationship of TCM syndromes and physicochemical index、pathological types of lupus nephritis by using statistical analysis software, to provide evidence for microcosmic differentiation of TCM syndromes,which was beneficial to guide us for the diagnosis and treatment of LN with Integrated Traditional Chinese and Western Medicine.
2. Experimental Study
The study analysed serum proteomics of normal human and patients with lupus nephritis and lupus nephritis patients with excessive heat-toxic and yang deficiency of spleen and kidney, screening differential expression proteins, and try to found proteins that associated with lupus nephritis and important application perspective biomarkers, as well as to found proteins that related with TCM syndromes,then to explore the protein expression profiles and relationship between proteins and TCM types. To try to provide objective basis for syndrome differentiation and treatment of lupus nephritis.
Methods
1. Clinical studies
120cases of patients with lupus nephritis were divided into eight different TCM syndromes including (excessive heat-toxic、yin deficiency of liver and kidney、Qi deficiency of spleen and kidney、yang deficiency of spleen and kidney、deficiency of both Qi and Yin、wind-dampnes internal disturbance、 blood stasis and Damp-heat), and clinical physicochemical index and renal pathology collection, and then used SPSS statistical analysis software to analyse the relationship between TCM types and physicochemical index、renal pathological types of patients with lupus nephritis.
2. Experimental Study
To separate serum proteins by using two-dimensional gel electrophoresis for protein separation on lupus nephritis patients and normal persons and lupus nephritis with excessive heat-toxic and Yang deficiency of spleen and kidney, the gels were dyed with silver staining and scanned for gel pictures, to find out the differential proteins with software. The differential proteins were cut and digested with trypsin and identificated them with mass spectrographic analysis, and to obtain the finger print map of peptide, then to serch them through Protein Data Bank and identificate them.
Results
1. Clinical studies
The study selected120cases, according to the general condition of patients, symptoms, signs, physicochemical index and renal pathology were compared and analysed the relationship of TCM types and physicochemical index, renal pathology, as follows:
(1) General data analysis:120cases of patients with lupus nephritis,14cases were male, aged20years to38years, mean age was27.6±5.2years old, average disease course was28.3±6.5months;106cases were female, aged16years to58years, mean age was32.5±8.6years, average disease course was36.7±9.3months.
(2) The distribution of TCM types:The frequency analysis showed that Qi deficiency of spleen and kidney was the major type, there was35cases (29.2%); yin deficiency of liver and kidney、excessive heat-toxic、deficiency of both Qi and Yin、yang deficiency of spleen and kidney, respectively there was26cases (21.7%)、16cases (13.3%)、14cases (11.7%)、13cases (10.8%), wind-dampness internal distrubance was16cases (13.3%); blood stasis and damp-heat types, respectively was56cases (46.7%) and41cases (34.2%).
(3) Pathology types:The frequency analysis showed that type Ⅳ was the major type, there was87cases (72.5%), followed by the type Ⅴ+Ⅳ, type Ⅴ and type Ⅲ, there were10cases (8.3%)、8cases (6.7%)、8cases (6.7%); the type Ⅴ+Ⅲ was6cases (5%), type Ⅱ was5cases (4.2%), type Ⅰ and type Ⅵ each had4cases (3.3%) and2cases (1.7%). Using Spearman rank correlation analysis, results suggested that TCM types and renal pathology types without correlation.
(4)Clinical types:The frequency analysis showed that nephrotic syndrome was the major type,72cases (60%), chronic nephritis there was36cases (30%), latent nephritis and rapidly progressive glomerulonephritis respectively was6cases (5%) and4cases (3.3%). Using Spearman rank correlation analysis, results suggested that TCM types and clinical types without correlation.
(5) Immune index:The frequency analysis results showed that120cases of lupus nephritis patients with ANA positive (100%); Anti ds-DNA positive was101cases (84.2%), anti-Sm positive was41cases(34.2%), anti-ssA positive was55cases (45.8%), the anti-ssB positive was33cases(27.5%), serum C3decreased was104cases (86.7%), the ANCA positive only had4cases(3.3%). Using Spearman rank correlation analysis, results suggested no correlation between TCM types and immune index.
(6) Lupus disease activity level:The frequency analysis results showed that the lowest score of SLE-DAI integral was4points, the highest was28points, the average score was (16.28±5.46) points. Severe activity lupus (SLE-DAI>15)there was70cases (58.3%), followed by moderate activity was33cases (27.5%), mild activity was14cases (11.7%), no activity only had3cases (2.5%). Using Spearman rank correlation analysis, results suggested no correlation between TCM types and disease activity types.
Using single factor statistical analysis of variance on TCM types and SLE-DAI integrals, the results suggested:the SLE-DAI integral of deficiency of both Qi and Yin type was the lowest (12.69±5.06), significantly less than excessive heat-toxic and Yang deficiency of spleen and kidney (P<0.05). the SLE-DAI integral of wind-dampness internal distrubance less than Yang deficiency of spleen and kidney (P<0.05). The SLE-DAI integral between other TCM types had no statistical significance (P>0.05).
(7) Clinical manifestations:The frequency analysis results showed that the most common damage of the extra-renal organs was the blood system, there was92cases (76.7%), fever in36cases (30%),49cases of arthritis (40.8%), facial erythema was44cases (36.7%), oral ulcers was25cases (20.8%), light-sensitive was25cases (20.8%), serositis was18cases (15%), nervous system damage was6cases (5.0%). Using Spearmanrank correlation analysis, results suggested that TCM types and clinical manifestations without correlation.
(8)Physicochemical indicators:The results showed that physicochemical indexes of WBC, TG, LDL-C and HDL-C in groups of differential TCM types have no difference;the LYM of Excessive heat-toxic type was significantly lower than Yin deficiency of liver and kidney and Qi deficiency of spleen and kidney, LYM of yang deficiency of spleen and kidney group was significantly lower than Yin deficiency of liver and kidney, other groups showed no differences;the RBC of yang deficiency of spleen and kidney group was significantly lower than Yin deficiency of liver and kidney and deficiency of both Qi and Yin, other groups showed no significant difference; the HGB of yang deficiency of spleen and kidney group was significantly lower than Yin deficiency of liver and kidney, other groups showed no significant difference;the PLT of deficiency of both Qi and Yin group was significantly higher than damp-heat group, other groups had no significant difference; the FIB level of blood stasis type was significantly higher than the other groups;the ALB level of Yang deficiency of spleen and kidney group significantly lower than the types of yin deficiency of liver and kidney, Qi deficiency of spleen and kidney, wind-dampness internal disturbance and damp-heat,and the ALB level of blood stasis group significantly lower than yin deficiency of liver and kidney, the other groups had no obvious difference; the levels of BUN, CREA, UA and Cys C of Yang deficiency of spleen and kidney were significantly higher than those of other groups, the other groups there were not significantly different; the quantitates of24hour urine protein of Yin deficiency of liver and kidney group was significantly lower than that yang deficiency of spleen and kidney and blood stasis groups, other groups had no significant difference. These results may suggest that TCM types have relavent with LYM、RBC、HGB、PLT、FIB、BUN、CREA、UA、Cys C and the quanti tates of24hour urine protein.
2.Experimental Study
In the study we selected4cases of the normal persons,4cases of excessive heat-toxin type of LN patients and4cases of Yang deficiency of spleen and kidney type of LN patients, by using two-dimensional gel electrophoresis for protein separation. The experimental results showed:the excessive heat-toxin type of LN patients had5differential expression spots contrast to Yang deficiency of spleen and kidney of LN patients, the excessive heat-toxin type of LN patients high expressed of the proteins were Ig mu chain C and Inter-alpha-trypsin inhibitor heavy chain H4, yang deficiency of spleen and kidney type of LN patients high expressed of the protein was Keratin type1cytoskeletal10. the LN patients had4differential expression spots contrast to normal persons, LN patients high expressed of the proteins were Alpha-1-antitrypsin and Alpha-1-antichymotrypsin, normal persons high expressed of the protein was Protein disulfide-isomerase A6.
Conclusion
1. Clinical research
(1) The TCM types have no correlation with renal pathology, clinical classification, immune indicators and clinical manifestations in lupus nephritis patients.
(2)The SLE-DAI is correlated with the TCM types in Lupus Nephritis.
(3)The TCM types have relevant to physichemical indicators such as blood LYM、RBC、HGB、PLT、FIB、BUN、CREA、UA、Cys C and urinary protein; and have no significant correlation with blood WBC、TG、LDL-C and HDL-C.
2. Experimental Study
There are some difference of proteins expressing between normal persons and Lupus Nephritis patients, the different proteins may help to reveal the pathogenesis of lupus nephritis or as new biomarkers for lupus nephritis, it is important in the diagnosis and treatment of lupus nephritis;and also there are some difference of proteins expressing between excessive heat-toxic and yang deficiency of spleen and kidney in Lupus Nephritis patients, we may can use proteomics technology to reveal the mechanism of TCM.
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