本文選題：狼瘡性腎炎 + 脾腎陽(yáng)虛證�。� 參考：《廣州中醫(yī)藥大學(xué)》2016年博士論文

【摘要】：目的：1.臨床研究探討狼瘡性腎炎患者年齡及性別、臨床分型、病理類(lèi)型及活動(dòng)性指數(shù)等的分布情況,比較其中醫(yī)主證虛、實(shí)證之間的上述指標(biāo)及臨床理化相關(guān)指標(biāo)、免疫相關(guān)指標(biāo)、炎癥相關(guān)指標(biāo)、尿液相關(guān)指標(biāo)等的差異,以此來(lái)分析二者與疾病活動(dòng)的相關(guān)性,為臨床虛、實(shí)證的判定及分期論治提供客觀(guān)依據(jù)。2.實(shí)驗(yàn)研究研究脾腎陽(yáng)虛型狼瘡性腎炎血漿外泌體microRNA表達(dá)譜與健康志愿者的差異,對(duì)差異表達(dá)的分子行靶基因預(yù)測(cè)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調(diào)控的信號(hào)通路,以探索狼瘡性腎炎可能的發(fā)生發(fā)展機(jī)制；篩選出差異表達(dá)的microRNA分子,以期能發(fā)現(xiàn)具有良好應(yīng)用前景的可用于輔助診斷的生物標(biāo)志物；探討差異表達(dá)的microRNA分子與狼瘡性腎炎脾腎陽(yáng)虛證型可能存在的相關(guān)性,為中醫(yī)辨證提供分子生物學(xué)方面的客觀(guān)依據(jù)。方法：1.臨床研究回顧性調(diào)查2010年1月至2015年12月于廣州中醫(yī)藥大學(xué)第一附屬醫(yī)院腎內(nèi)科住院的符合診斷標(biāo)準(zhǔn)、納入標(biāo)準(zhǔn)及排除標(biāo)準(zhǔn)的狼瘡性腎炎患者,按照首次腎活檢時(shí)記錄的癥狀體征,將其分為熱毒熾盛證、肝腎陰虛證、氣陰兩虛證、脾腎氣虛證、脾腎陽(yáng)虛證五型,其中熱毒熾盛證納入實(shí)證組,其余四種證型納入虛證組。收集患者年齡及性別等一般情況、臨床分型、病理分型、活動(dòng)性指數(shù)、免疫相關(guān)指標(biāo)及臨床相關(guān)理化指標(biāo)等資料,應(yīng)用SPSS軟件進(jìn)行分析,并比較虛、實(shí)證兩組患者上述指標(biāo)的差異。2.實(shí)驗(yàn)研究選取脾腎陽(yáng)虛型狼瘡性腎炎患者及健康志愿者各3名,應(yīng)用Exoquick試劑提取血漿外泌體,采用高通量測(cè)序方法測(cè)定兩組外泌體的microRNA表達(dá)譜,篩選出差異表達(dá)的microRNA分子行靶基因預(yù)測(cè),進(jìn)一步對(duì)預(yù)測(cè)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調(diào)控的信號(hào)通路。結(jié)果：1.臨床研究本研究共納入患者134例,女性患者117例,男性患者僅17例,男女比例約1：6.9。中醫(yī)主證以脾腎氣虛證患者最多,共41例,所占比例約30.6%；其次為熱毒熾盛證及脾腎陽(yáng)虛證,二者均有33例患者,各占24.63%；之后依次為肝腎陰虛證患者14例(10.45%),氣陰兩虛證患者13例(9.70%)。實(shí)證組患者33例,虛證組患者101例。(1)性別、年齡等一般情況：實(shí)證組中男性2例,女性31例：年齡最小12歲,最大45歲,平均年齡(27.76±10.80)歲。虛證組中男性15例,女性86例；年齡最小10歲,最大60歲,平均年齡(30.68±12.07)歲。兩組患者的年齡、性別比較,差異均無(wú)統(tǒng)計(jì)學(xué)意義。說(shuō)明狼瘡性腎炎患者虛、實(shí)證的發(fā)病與性別及年齡均無(wú)明顯相關(guān)性。(2)臨床分型：臨床分型中以腎病綜合征為最多見(jiàn),共59例(44.03%),其次為慢性腎炎型48例(35.82%),之后依次為隱匿性腎炎型12例(8.96%),慢性腎衰竭型11例(8.21%),急進(jìn)性腎炎型4例(2.98%)。虛、實(shí)證兩組患者的臨床分型分布比較,差異無(wú)統(tǒng)計(jì)學(xué)意義。但實(shí)證組患者以慢性腎炎型為最多,而虛證組患者以腎病綜合征為最多。急進(jìn)性腎炎均見(jiàn)于虛證組,且主要為脾腎陽(yáng)虛證。(3)病理相關(guān)指標(biāo)：病理類(lèi)型中以Ⅳ型為最多見(jiàn),共50例(37.31%),其次為V型23例(17.16%),之后依次為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%)。虛、實(shí)證兩組患者的病理類(lèi)型分布及AI比較,差異無(wú)統(tǒng)計(jì)學(xué)意義；虛證組的CI明顯高于實(shí)證組,差異有統(tǒng)計(jì)學(xué)意義。(4)臨床理化指標(biāo)：兩組患者的血常規(guī)相關(guān)指標(biāo)比較,WBC、LYM、RBC及HGB的差異均有統(tǒng)計(jì)學(xué)意義。虛證組患者WBC及LYM計(jì)數(shù)均顯著高于實(shí)證組,RBC及HGB則顯著低于實(shí)證組；兩組患者的腎功能相關(guān)指標(biāo)比較,BUN、UA及CysC差異均有統(tǒng)計(jì)學(xué)意義,而Cr水平比較無(wú)統(tǒng)計(jì)學(xué)差異。虛證組患者的BUN、UA及CysC水平均顯著高于實(shí)證組患者；兩組患者的肝功能相關(guān)指標(biāo)比較,AST的差異有統(tǒng)計(jì)學(xué)意義,而ALT、ALB及G的差異均無(wú)統(tǒng)計(jì)學(xué)意義。虛證組患者的AST水平顯著高于實(shí)證組。(5)免疫相關(guān)指標(biāo)：所有患者ANA全部為陽(yáng)性,而ds-DNA陽(yáng)性患者113例(84.33%)�？笶NA抗體譜中,以抗SSA抗體陽(yáng)性者為最多,共79例(58.96%)。其次是抗Sm抗體陽(yáng)性者77例(57.46%),其后為抗核小體抗體(AnuA)陽(yáng)性者73例(54.48%),之后依次為抗組蛋白抗體(AHA)陽(yáng)性者58例(43.28%),抗nRNP抗體陽(yáng)性者42例(31.34%),抗rRNP抗體陽(yáng)性患者38例(28.36%),抗SSB抗體陽(yáng)性者34例(25.37%),抗SCL-70抗體陽(yáng)性者5例(3.73%),抗JO-1抗體陽(yáng)性者僅1例(0.75%),未發(fā)現(xiàn)抗著絲點(diǎn)抗體陽(yáng)性患者。虛、實(shí)證兩組患者的各抗體陽(yáng)性率比較,差異無(wú)統(tǒng)計(jì)學(xué)意義；兩組患者自免五項(xiàng)相關(guān)指標(biāo)比較,包括ANA滴度、dsDNA滴度、CIC、C3及C4比較,差異均無(wú)統(tǒng)計(jì)學(xué)意義。(6)炎癥指標(biāo)：部分患者未行CRP及ESR的檢測(cè),故實(shí)際納入統(tǒng)計(jì)的實(shí)證組共20例,虛證組共64例。兩組的CRP及ESR比較,差異無(wú)統(tǒng)計(jì)學(xué)意義。(7) SLEDAI積分：在收集的134例患者中,SLEDAI積分最高的為40分,最低的為2分,平均(13.39±0.48)分。病情處于重度活動(dòng)期的患者62例(46.27%),中度活動(dòng)期的患者36例(26.86%),輕度活動(dòng)期患者28例(20.9%),非活動(dòng)期患者8例(5.97%)。虛、實(shí)證兩組患者中,各自也均以重度活動(dòng)期患者為最多見(jiàn),其次為中度活動(dòng)期患者,之后依次為輕度活動(dòng)期、非活動(dòng)期患者。兩組患者的SLEDAI積分及其分期比較,差異均無(wú)統(tǒng)計(jì)學(xué)意義。(8)尿液相關(guān)指標(biāo)：在收集的134例患者中,尿蛋白陽(yáng)性患者共128例,占95.52%,其中以尿蛋白3+為最多見(jiàn),共95例(70.9%),尿蛋白陰性患者僅6例；而尿隱血陽(yáng)性患者共115例,占85.82%,也是以尿隱血3+為最多,共77例患者(57.46%),尿隱血陰性患者僅19例。兩組患者的尿蛋白定性、尿隱血及尿蛋白定量比較,差異均無(wú)統(tǒng)計(jì)學(xué)意義。2.實(shí)驗(yàn)研究(1)脾腎陽(yáng)虛型狼瘡性腎炎患者與健康志愿者血漿外泌體的microRNA表達(dá)譜分析比較,共篩選出148個(gè)差異表達(dá)的microRNA分子,其中上調(diào)表達(dá)的131個(gè),而下調(diào)表達(dá)的僅17個(gè)。具有高顯著差異的共2個(gè),分別為hsa-miR-127-3p及hsa-miR-4497;一般顯著差異的共6個(gè),分別為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個(gè)均為一般差異。(2)在表達(dá)差異顯著的8個(gè)microRNA分子中,共預(yù)測(cè)出1664個(gè)靶基因,其中hsa-miR-629-3p預(yù)測(cè)到的靶基因最多,共439個(gè),而hsa-miR-127-3p預(yù)測(cè)的數(shù)量最少,僅8個(gè)。Hsa-miR-3158-3p為表達(dá)下調(diào)的miRNA分子,其預(yù)測(cè)到的靶基因共205個(gè)。(3)靶基因經(jīng)KEGG生物通路分析,得到具有統(tǒng)計(jì)學(xué)意義的信號(hào)及疾病通路共19個(gè)。其中以FoxO信號(hào)通路及TGF-β信號(hào)通路意義最為顯著。(4)靶基因經(jīng)GO基因功能富集分析,得到具有統(tǒng)計(jì)學(xué)意義的GO功能共108個(gè)。其功能主要體現(xiàn)在細(xì)胞自噬及調(diào)控基因表達(dá)、生物合成、轉(zhuǎn)錄因子活性等方面。結(jié)論：1.臨床研究(1)狼瘡性腎炎患者中醫(yī)主證以脾腎兩虛為最多見(jiàn),包括脾腎氣虛證及脾腎陽(yáng)虛證;臨床分型中以。腎病綜合征為最多見(jiàn),其次為慢性腎炎型；病理類(lèi)型則以Ⅳ型為主,其次為Ⅴ型及Ⅴ+Ⅳ型;病情以重度活動(dòng)期為主。(2)狼瘡性腎炎患者虛、實(shí)證的發(fā)病與性別及年齡均無(wú)明顯相關(guān)性。(3)實(shí)證組與虛證組患者臨床分型、病理類(lèi)型、自免五項(xiàng)、抗ENA抗體譜、SLEDAI積分、炎癥指標(biāo)及尿液相關(guān)指標(biāo)比較,均無(wú)明顯差異。實(shí)證、虛證與疾病活動(dòng)性并無(wú)明顯相關(guān)。(4)虛證組的CI、WBC、LYM、BUN、UA、CysC及AST等指標(biāo)顯著高于實(shí)證組,而RBC及HGB則顯著低于實(shí)證組。說(shuō)明虛證組腎腎功能損傷程度及貧血程度均重于實(shí)證組,腎臟的慢性損害程度也重于實(shí)證組。2.實(shí)驗(yàn)研究(1)Hsa-miR-127-3p及hsa-miR-4497兩個(gè)分子在脾腎陽(yáng)虛型狼瘡性腎炎中表達(dá)顯著升高,可能對(duì)輔助其診斷及辨證具有一定的意義,但需進(jìn)一步的研究來(lái)驗(yàn)證。(2) FoxO信號(hào)通路及TGF-β信號(hào)通路可能在LN的發(fā)病中起著重要作用,細(xì)胞自噬及調(diào)控基因表達(dá)、生物合成、轉(zhuǎn)錄因子活性功能等異�？赡苁荓N重要的發(fā)病機(jī)制,均需進(jìn)一步的研究證實(shí)。
[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.

【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R259

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