狼疮性肾炎中医证型及血浆外泌体microRNA表达谱的生物信息研究

发布时间：2018-04-22 20:37

本文选题：狼疮性肾炎 + 脾肾阳虚证　；参考：《广州中医药大学》2016年博士论文

【摘要】：目的：1.临床研究探讨狼疮性肾炎患者年龄及性别、临床分型、病理类型及活动性指数等的分布情况,比较其中医主证虚、实证之间的上述指标及临床理化相关指标、免疫相关指标、炎症相关指标、尿液相关指标等的差异,以此来分析二者与疾病活动的相关性,为临床虚、实证的判定及分期论治提供客观依据。2.实验研究研究脾肾阳虚型狼疮性肾炎血浆外泌体microRNA表达谱与健康志愿者的差异,对差异表达的分子行靶基因预测及生物通路和基因功能富集分析,找出靶基因的富集功能及调控的信号通路,以探索狼疮性肾炎可能的发生发展机制；筛选出差异表达的microRNA分子,以期能发现具有良好应用前景的可用于辅助诊断的生物标志物；探讨差异表达的microRNA分子与狼疮性肾炎脾肾阳虚证型可能存在的相关性,为中医辨证提供分子生物学方面的客观依据。方法：1.临床研究回顾性调查2010年1月至2015年12月于广州中医药大学第一附属医院肾内科住院的符合诊断标准、纳入标准及排除标准的狼疮性肾炎患者,按照首次肾活检时记录的症状体征,将其分为热毒炽盛证、肝肾阴虚证、气阴两虚证、脾肾气虚证、脾肾阳虚证五型,其中热毒炽盛证纳入实证组,其余四种证型纳入虚证组。收集患者年龄及性别等一般情况、临床分型、病理分型、活动性指数、免疫相关指标及临床相关理化指标等资料,应用SPSS软件进行分析,并比较虚、实证两组患者上述指标的差异。2.实验研究选取脾肾阳虚型狼疮性肾炎患者及健康志愿者各3名,应用Exoquick试剂提取血浆外泌体,采用高通量测序方法测定两组外泌体的microRNA表达谱,筛选出差异表达的microRNA分子行靶基因预测,进一步对预测的靶基因行KEGG Pathway生物通路分析及GO基因功能富集分析,找出靶基因的富集功能及其调控的信号通路。结果：1.临床研究本研究共纳入患者134例,女性患者117例,男性患者仅17例,男女比例约1：6.9。中医主证以脾肾气虚证患者最多,共41例,所占比例约30.6%；其次为热毒炽盛证及脾肾阳虚证,二者均有33例患者,各占24.63%；之后依次为肝肾阴虚证患者14例(10.45%),气阴两虚证患者13例(9.70%)。实证组患者33例,虚证组患者101例。(1)性别、年龄等一般情况：实证组中男性2例,女性31例：年龄最小12岁,最大45岁,平均年龄(27.76±10.80)岁。虚证组中男性15例,女性86例；年龄最小10岁,最大60岁,平均年龄(30.68±12.07)岁。两组患者的年龄、性别比较,差异均无统计学意义。说明狼疮性肾炎患者虚、实证的发病与性别及年龄均无明显相关性。(2)临床分型：临床分型中以肾病综合征为最多见,共59例(44.03%),其次为慢性肾炎型48例(35.82%),之后依次为隐匿性肾炎型12例(8.96%),慢性肾衰竭型11例(8.21%),急进性肾炎型4例(2.98%)。虚、实证两组患者的临床分型分布比较,差异无统计学意义。但实证组患者以慢性肾炎型为最多,而虚证组患者以肾病综合征为最多。急进性肾炎均见于虚证组,且主要为脾肾阳虚证。(3)病理相关指标：病理类型中以Ⅳ型为最多见,共50例(37.31%),其次为V型23例(17.16%),之后依次为Ⅴ+Ⅳ型20例(14.93%),Ⅱ型17例(12.69%),Ⅰ型12例(8.96%),Ⅲ型7例(5.22%),Ⅴ+Ⅲ型3例(2.24%),Ⅵ型2例(1.49%)。虚、实证两组患者的病理类型分布及AI比较,差异无统计学意义；虚证组的CI明显高于实证组,差异有统计学意义。(4)临床理化指标：两组患者的血常规相关指标比较,WBC、LYM、RBC及HGB的差异均有统计学意义。虚证组患者WBC及LYM计数均显著高于实证组,RBC及HGB则显著低于实证组；两组患者的肾功能相关指标比较,BUN、UA及CysC差异均有统计学意义,而Cr水平比较无统计学差异。虚证组患者的BUN、UA及CysC水平均显著高于实证组患者；两组患者的肝功能相关指标比较,AST的差异有统计学意义,而ALT、ALB及G的差异均无统计学意义。虚证组患者的AST水平显著高于实证组。(5)免疫相关指标：所有患者ANA全部为阳性,而ds-DNA阳性患者113例(84.33%)。抗ENA抗体谱中,以抗SSA抗体阳性者为最多,共79例(58.96%)。其次是抗Sm抗体阳性者77例(57.46%),其后为抗核小体抗体(AnuA)阳性者73例(54.48%),之后依次为抗组蛋白抗体(AHA)阳性者58例(43.28%),抗nRNP抗体阳性者42例(31.34%),抗rRNP抗体阳性患者38例(28.36%),抗SSB抗体阳性者34例(25.37%),抗SCL-70抗体阳性者5例(3.73%),抗JO-1抗体阳性者仅1例(0.75%),未发现抗着丝点抗体阳性患者。虚、实证两组患者的各抗体阳性率比较,差异无统计学意义；两组患者自免五项相关指标比较,包括ANA滴度、dsDNA滴度、CIC、C3及C4比较,差异均无统计学意义。(6)炎症指标：部分患者未行CRP及ESR的检测,故实际纳入统计的实证组共20例,虚证组共64例。两组的CRP及ESR比较,差异无统计学意义。(7) SLEDAI积分：在收集的134例患者中,SLEDAI积分最高的为40分,最低的为2分,平均(13.39±0.48)分。病情处于重度活动期的患者62例(46.27%),中度活动期的患者36例(26.86%),轻度活动期患者28例(20.9%),非活动期患者8例(5.97%)。虚、实证两组患者中,各自也均以重度活动期患者为最多见,其次为中度活动期患者,之后依次为轻度活动期、非活动期患者。两组患者的SLEDAI积分及其分期比较,差异均无统计学意义。(8)尿液相关指标：在收集的134例患者中,尿蛋白阳性患者共128例,占95.52%,其中以尿蛋白3+为最多见,共95例(70.9%),尿蛋白阴性患者仅6例；而尿隐血阳性患者共115例,占85.82%,也是以尿隐血3+为最多,共77例患者(57.46%),尿隐血阴性患者仅19例。两组患者的尿蛋白定性、尿隐血及尿蛋白定量比较,差异均无统计学意义。2.实验研究(1)脾肾阳虚型狼疮性肾炎患者与健康志愿者血浆外泌体的microRNA表达谱分析比较,共筛选出148个差异表达的microRNA分子,其中上调表达的131个,而下调表达的仅17个。具有高显著差异的共2个,分别为hsa-miR-127-3p及hsa-miR-4497;一般显著差异的共6个,分别为hsa-miR-129-5p、hsa-miR-383-5p、hsa-miR-423-5p、 hsa-miR-629-3p、hsa-miR-3158-3p及hsa-miR-3184-3p;其余140个均为一般差异。(2)在表达差异显著的8个microRNA分子中,共预测出1664个靶基因,其中hsa-miR-629-3p预测到的靶基因最多,共439个,而hsa-miR-127-3p预测的数量最少,仅8个。Hsa-miR-3158-3p为表达下调的miRNA分子,其预测到的靶基因共205个。(3)靶基因经KEGG生物通路分析,得到具有统计学意义的信号及疾病通路共19个。其中以FoxO信号通路及TGF-β信号通路意义最为显著。(4)靶基因经GO基因功能富集分析,得到具有统计学意义的GO功能共108个。其功能主要体现在细胞自噬及调控基因表达、生物合成、转录因子活性等方面。结论：1.临床研究(1)狼疮性肾炎患者中医主证以脾肾两虚为最多见,包括脾肾气虚证及脾肾阳虚证;临床分型中以。肾病综合征为最多见,其次为慢性肾炎型；病理类型则以Ⅳ型为主,其次为Ⅴ型及Ⅴ+Ⅳ型;病情以重度活动期为主。(2)狼疮性肾炎患者虚、实证的发病与性别及年龄均无明显相关性。(3)实证组与虚证组患者临床分型、病理类型、自免五项、抗ENA抗体谱、SLEDAI积分、炎症指标及尿液相关指标比较,均无明显差异。实证、虚证与疾病活动性并无明显相关。(4)虚证组的CI、WBC、LYM、BUN、UA、CysC及AST等指标显著高于实证组,而RBC及HGB则显著低于实证组。说明虚证组肾肾功能损伤程度及贫血程度均重于实证组,肾脏的慢性损害程度也重于实证组。2.实验研究(1)Hsa-miR-127-3p及hsa-miR-4497两个分子在脾肾阳虚型狼疮性肾炎中表达显著升高,可能对辅助其诊断及辨证具有一定的意义,但需进一步的研究来验证。(2) FoxO信号通路及TGF-β信号通路可能在LN的发病中起着重要作用,细胞自噬及调控基因表达、生物合成、转录因子活性功能等异常可能是LN重要的发病机制,均需进一步的研究证实。
[Abstract]:Objective: 1. to investigate the distribution of age and sex, clinical typing, pathological type and activity index of patients with lupus nephritis, compare the deficiency of TCM syndrome, the above index and clinical physicochemical index, immunological related index, inflammation related index and urine related index, so as to analyze the two cases. In relation to disease activity, the difference between microRNA expression profiles of plasma exosbody of spleen and kidney yang deficiency type lupus nephritis and healthy volunteers was provided by.2. experimental study for clinical deficiency, empirical determination and staging, and the target gene was predicted and the target gene pathway and gene function enrichment analysis were used to find the target base. In order to explore the possible development mechanism of lupus nephritis, the differentially expressed microRNA molecules are screened for the discovery of biomarkers that can be used for auxiliary diagnosis, and the possibility of differentially expressed microRNA molecules and the spleen and kidney deficiency syndrome of lupus nephritis may be explored. The existing correlation provides an objective basis for the molecular biology of TCM syndrome differentiation. Methods: 1. clinical studies reviewed a retrospective study of patients with lupus nephritis that were hospitalized in the First Affiliated Hospital of Guangzhou University of Chinese Medicine from January 2010 to December 2015 in the nephrology department of the First Affiliated Hospital of Guangzhou University of Chinese Medicine. The symptoms and signs of the record were divided into heat and kidney yin deficiency syndrome, Qi Yin deficiency syndrome, spleen kidney qi deficiency syndrome, spleen kidney qi deficiency syndrome, spleen kidney yang deficiency syndrome five. Among them, the heat toxin flaming syndrome was included in the empirical group, and the other four types of syndrome types were included in the deficiency syndrome group. The general situation, clinical classification, pathological classification, activity index, immune related indexes and clinical symptoms were collected and four kinds of syndrome types were included in the group of deficiency syndrome. The data of bed related physical and chemical indexes were analyzed with SPSS software, and the difference between the two groups of the above indexes was analyzed. The.2. experimental study of the patients with spleen kidney yang deficiency type lupus nephritis and 3 healthy volunteers were selected. The Exoquick reagent was used to extract the plasma exote, and the high throughput sequencing method was used to determine the microRNA table of the two exosecreting groups. The target gene was predicted by microRNA, and the target gene was analyzed by KEGG Pathway biological pathway analysis and GO gene function enrichment analysis to find the target gene enrichment function and the signal transduction pathway. Results: 1. clinical studies included 134 patients, 117 female patients, and male patients. Only 17 cases, male and female proportion of about 1:6.9. TCM syndrome of spleen and kidney qi deficiency syndrome most, a total of 41 cases, the proportion of about 30.6%; the second is the heat poison and spleen and kidney yang deficiency syndrome, two of the 33 patients, each 24.63%; after the liver kidney yin deficiency syndrome in 14 cases (10.45%), 13 cases (9.70%), Qi Yin two deficiency syndrome patients 33, 33 cases, There were 101 cases in the asthenia syndrome group. (1) gender, age and other general conditions: 2 cases in the positive group and 31 cases in women: the age of 12 years, the maximum 45 years, and the average age (27.76 + 10.80) years. There are 15 males and 86 women in the deficiency syndrome group, the youngest 10 years old, the maximum age of 60, and the average age (30.68 + 5) years. Statistical significance. It showed that the asthenia of lupus nephritis had no significant correlation with sex and age. (2) clinical classification: the most common type of nephrotic syndrome was nephrotic syndrome in clinical typing, 59 cases (44.03%), followed by chronic nephritis in 48 cases (35.82%), followed by 12 cases of occult nephritis (8.96%), and 11 cases of chronic renal failure (8.21%). There were 4 cases of acute glomerulonephritis (2.98%). The clinical classification distribution of two groups of patients was not statistically significant, but the patients in the empirical group were most with chronic nephritis, and the patients in the deficiency syndrome group were the most in the nephrotic syndrome. The acute glomerulonephritis was found in the deficiency syndrome group and mainly for the spleen and kidney yang deficiency syndrome. (3) pathological type: pathological type. The most common cases were type IV, 50 (37.31%), followed by type V 23 (17.16%), followed by 20 (14.93%), 17 (12.69%), 12 (8.96%), 7 (5.22%), V + III 3 cases (8.96%), III type 7 (5.22%), V + III, VI). The distribution of pathological type and AI comparison of the patients in the empirical group were not statistically significant; the CI of the asthenia syndrome group Significantly higher than the empirical group, the difference was statistically significant. (4) clinical physical and chemical indicators: two groups of patients with blood routine related indicators, WBC, LYM, RBC and HGB differences were statistically significant. The number of WBC and LYM in the asthenia syndrome group were significantly higher than the positive group, RBC and HGB were significantly lower than the positive group; the renal function related indexes of the two groups were compared, BUN The differences in UA and CysC were statistically significant, but there was no statistical difference in the level of Cr. The levels of BUN, UA and CysC in the patients with deficiency syndrome were significantly higher than those in the positive group; the difference of AST in the two groups was statistically significant, but the difference in ALT, ALB and G was not statistically significant. The AST level in the asthenia syndrome group was significantly higher. In the positive group. (5) immune related indicators: all patients were positive for ANA, and 113 cases (84.33%) of ds-DNA positive patients. The anti ENA antibody was the most anti SSA antibody, 79 cases (58.96%), followed by anti Sm antibody positive 77 cases (57.46%), then 73 cases (54.48%) of anti nucleosome antibody (AnuA) positive, followed by anti group egg. White antibody (AHA) positive in 58 (43.28%), anti nRNP antibody positive in 42 cases (31.34%), anti rRNP antibody positive in 38 cases (28.36%), anti SSB antibody positive in 34 cases (25.37%), anti SCL-70 antibody positive in 5 cases (3.73%), only 1 cases (0.75%) with anti JO-1 antibody positive, no anti seropositive antibody positive patients. Deficiency, positive two group of patients antibody Yang There was no statistical difference in sex ratio, and there was no significant difference between the two groups of five related indexes, including ANA titer, dsDNA titer, CIC, C3 and C4. (6) some patients were not detected by CRP and ESR, so 20 cases were included in the actual statistics group, 64 cases in the deficiency syndrome group. The two groups of CRP and ESR were compared. The difference was not statistically significant. (7) SLEDAI score: among the 134 patients collected, the highest SLEDAI score was 40, the lowest was 2, the average (13.39 + 0.48). 62 patients were in the severe active period (46.27%), 36 (26.86%) in moderate active period, 28 (20.9%) in mild active period, 8 cases (5.97%) in inactive patients. In the two groups of patients, they were also the most common patients with severe active stage, followed by moderate active period, followed by mild active period and inactive stage. The difference between the two groups was not statistically significant. (8) urine related indexes: in the 134 patients, urine protein positive patients were collected. A total of 128 cases, accounting for 95.52%, of which urinary protein 3+ was the most common, 95 cases (70.9%), only 6 cases of urinary protein negative, 115 cases of urinary occult blood positive, 85.82%, and urinary occult blood 3+ as the most, 77 patients (57.46%), and only 19 cases of urinary occult blood negative patients. The qualitative of urinary protein, urinary occult blood and urine protein were compared, and the difference between urine protein and urine protein was different. No statistical significance.2. experimental study (1) the microRNA expression profiles of the plasma exosbodies of the spleen kidney yang deficiency type lupus nephritis patients and healthy volunteers were compared, and 148 microRNA molecules with different expression were screened, in which 131 were up and only 17 were down regulated. There were 2 highly significant differences, respectively, hsa-miR-127-3p And hsa-miR-4497; a total of 6 significant differences were hsa-miR-129-5p, hsa-miR-383-5p, hsa-miR-423-5p, hsa-miR-629-3p, hsa-miR-3158-3p and hsa-miR-3184-3p, and the other 140 were all general differences. (2) 1664 target genes were detected in 8 microRNA molecules with significant differences in expression, of which the target gene predicted by hsa-miR-629-3p was predicted. The maximum number was 439, and the number of hsa-miR-127-3p predicted was the least, only 8.Hsa-miR-3158-3p were the miRNA molecules expressed down. The target genes were predicted by 205. (3) the target gene was analyzed by KEGG biological pathway, and the statistical signalling and the disease pathway were 19. Among them, the FoxO signal pathway and the TGF- beta signal pathway were the most important. (4) the target gene was enriched and analyzed by GO gene function, and 108 GO functions with statistical significance were obtained. The function was mainly manifested in the autophagy and regulation gene expression, biosynthesis, and the activity of transcription factors. Conclusion: 1. clinical study (1) the main syndromes of Chinese lupus nephritis with spleen and kidney two deficiency are the most common, including spleen and kidney qi. Deficiency syndrome and spleen kidney yang deficiency syndrome; in clinical classification, nephrotic syndrome was the most common type, followed by chronic nephritis type; pathological type was mainly type IV type, followed by type V and V + IV; severe active stage was the main disease. (2) patients with lupus nephritis had no significant correlation with sex and age. (3) positivist group and deficiency syndrome group There were no significant differences in clinical typing, pathological type, five self exemption, anti ENA antibody spectrum, SLEDAI score, inflammatory index and urine related index. (4) the indexes of CI, WBC, LYM, BUN, UA, CysC and AST in the asthenia syndrome group were significantly higher than those in the positive group, while RBC and HGB were significantly lower than the positive group. The degree of renal function injury and the degree of anemia in the group of Ming deficiency were all heavier than that in the empirical group. The degree of chronic renal damage was also heavier than the.2. experimental study in the empirical group (1) the expression of two molecules in the spleen and kidney yang deficiency type lupus nephritis was significantly elevated, which may be of certain significance in assisting the diagnosis and differentiation of the syndrome, but it needs to be further studied. It is verified that (2) FoxO signaling pathway and TGF- beta signaling pathway may play an important role in the pathogenesis of LN. Autophagy and regulatory gene expression, biosynthesis, transcription factor activity and other abnormalities may be important pathogenesis of LN, and all of them need further research.

【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R259

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