本文關(guān)鍵詞： 原發(fā)性高血壓惡性腎硬化 微血管 周細胞 巨噬細胞 生存分析　出處：《北京協(xié)和醫(yī)學(xué)院》2017年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景高血壓腎損害是慢性腎臟病的重要原因,其中惡性高血壓(Malignant Hypertension,MHT)腎臟病理表現(xiàn)為高血壓惡性腎硬化(Malignant Hypertensive Nephrosclerosis,MHN)靶器官受累多,病情進展快,預(yù)后差,發(fā)病率近年有上升趨勢。但目前文獻中缺乏有腎臟病理診斷的大樣本臨床研究,影響預(yù)后的危險因素存在爭議,相關(guān)機制的研究較少。腎臟具有兩套毛細血管網(wǎng),分別為腎小球毛細血管和腎小管周毛細血管(Peritubular capillary,PTC)。在多種合并高血壓的慢性腎小球腎炎、包括IgA腎病患者中均可觀察到毛細血管顯著減少,并與腎功能、尿蛋白水平、腎小球硬化和間質(zhì)纖維化相關(guān),目前在MHT患者中并無評價。血管內(nèi)皮穩(wěn)定性喪失是導(dǎo)致PTC損傷的重要途徑,而血管內(nèi)皮穩(wěn)定性則依賴于血管周細胞結(jié)構(gòu)和功能的完整性,炎癥在其中發(fā)揮重要作用。血管周成纖維細胞和周細胞,可識別細胞損傷刺激,分泌趨化因子,募集單核巨噬細胞。巨噬細胞向M1型分化有促炎作用,向M2方向轉(zhuǎn)化可促進纖維化。目前巨噬細胞不同的表型和來源在惡性腎硬化患者血管微環(huán)境中是否發(fā)揮作用尚無研究。因此,本研究將在以病理診斷為基礎(chǔ)的較大樣本惡性腎硬化隊列中,分析臨床病理特點、應(yīng)用虛擬切片技術(shù)自動化病理定量評分,分析影響患者長期預(yù)后的危險因素;探討腎小球和小管周微血管損傷對腎功能和預(yù)后的預(yù)測價值,并初步探討局部微環(huán)境中PDGFRβ(+)細胞和巨噬細胞對血管微環(huán)境損傷可能的機制。研究目的1.回顧性觀察原發(fā)性高血壓惡性腎硬化(MHN)的臨床和病理特點,分析影響預(yù)后的因素;2.觀察MHN病例腎小球微血管和小管周毛細血管缺失的情況,分析其和臨床指標(biāo)及預(yù)后的關(guān)聯(lián)性及其與局部微環(huán)境PDGFRβ陽性細胞的關(guān)系;3.初步觀察MHN病例腎臟局部巨噬細胞分型和外周血單核細胞分型、初步探討其對血管微環(huán)境的可能作用。研究方法2003年1月至2016年12月間在北京協(xié)和醫(yī)院住院的100例經(jīng)腎活檢確診的、臨床資料完整的原發(fā)性惡性高血壓腎損害病例,收集其臨床、病理及隨訪資料(大于6月),終點事件為長期透析(≥3個月)、腎移植或死亡。腎臟病理:腎小球、腎小管萎縮和間質(zhì)纖維化半定量分析,應(yīng)用虛擬切片方法分析腎小球密度、腎小球體積;對腎組織切片分別進行CD34、PDGFRp、TGFp免疫組化染色,PDGFRβ/CD34,PDGFRβ/CCL2免疫熒光共染,分析其與臨床、病理和預(yù)后關(guān)系;免疫熒光雙染D68/CD86,CD68/TLR4,CD68/CD163,CD68/CD206,CD68/TGFβ 研究腎臟巨噬細胞分型和功能,用流式細胞學(xué)方法檢測惡性腎硬化患者和健康對照組外周血單核細胞分型比例。統(tǒng)計方法:連續(xù)變量以均值±標(biāo)準(zhǔn)差的形式表示,計數(shù)資料以構(gòu)成比表示。統(tǒng)計軟件為SPSS 19.0軟件(IBM,USA)。正態(tài)分布連續(xù)變量比較用t檢驗、方差;非正態(tài)分布連續(xù)變量應(yīng)用秩和檢驗,構(gòu)成比應(yīng)用卡方分析;單因素相關(guān)分析正態(tài)分布變量應(yīng)用Pearson相關(guān)分析,非正態(tài)分布變量用spearman相關(guān)分析,評價臨床與病理資料的相關(guān)性,采用單因素或多重線性回歸分析MHT發(fā)生和腎功能損害的相關(guān)因素,應(yīng)用Kaplan-Meier方法和COXX比例風(fēng)險模型評價影響預(yù)后的獨立危險因素。研究結(jié)果1.原發(fā)性高血壓惡性腎硬化臨床病理特點及預(yù)后100例MHN患者以男性為主(男/女7.3:1),平均年齡34.7±8.9歲,就診時最高收縮壓(SBP)225.2±26.4mmHg,最高舒張壓(DBP)152.0±25.0mmH,血肌酐(Scr)465.3±333.9μmol/L,eGFR 22.13± 15.17 ml/min/1.73m2,24h 尿蛋白為1.64±1.22g/24h,貧血(48.5%)、高尿酸血癥(73.5%)和高甘油三脂血癥(67%)突出,同時存在全身炎癥反應(yīng)活化,表現(xiàn)為血沉增快(65.2%)、CRP升高(42.1%)。病理半定量分析提示腎小球硬化(硬化指數(shù):1.56±0.50)、腎小管萎縮(比例:62.8±19.1%)和腎間質(zhì)纖維化(比例65.0±17.8%)重,并與腎功能和24h尿蛋白密切相關(guān)。腎小球密度與腎功能相關(guān)性好,非硬化腎小球密度與小球硬化指數(shù)負相關(guān)。炎癥指標(biāo)(ESR、淋巴細胞絕對值、血清補體C3和血IgM水平)與血肌酐及腎小管萎縮比例密切相關(guān)。多因素回歸分析腎小球硬化指數(shù)、小管萎縮比例、出院舒張壓水平和淋巴細胞計數(shù)(度R=0.603)為腎功能(Scr)的獨立預(yù)測因素;小管萎縮比例與24h尿蛋白量獨立相關(guān)�；颊呷朐汉舐�(lián)合使用的多種降壓藥物,出院血壓、血肌酐和24h尿蛋白較其入院顯著降低(P0.001),平均隨訪時間56.8±37.1月,第1年、3年、5年和10年的累積腎臟生存率分別為93%、80%、66%和27%,COX回歸分析顯示蛋白尿水平、腎活檢時eGFR水平和小管萎縮比例是終末期生病的獨立危險因素。2.原發(fā)MHN患者腎小球及腎小管周毛細血管損傷與預(yù)后分析MHN組腎小球微血管比例和腎小管周毛細血管(PTC)比例均顯著低于良性腎硬化組和輕微病變組,且與臨床指標(biāo)(血肌酐、24h尿蛋白和血紅蛋白)和腎臟病理(腎小球硬化指數(shù)、小管萎縮比例、間質(zhì)纖維化程度)相關(guān)性較好,二者減少均是獨立于血肌酐、24小時尿蛋白和血壓是否達標(biāo)的影響長期預(yù)后的危險因素。腎小球PDGFRβ表達下降,與腎小球CD34陽性面積比例正相關(guān),小管間質(zhì)PDGFRβ(周細胞表面標(biāo)志)高于良性腎硬化與腎小球輕微病變,且與微血管分離。3.單核巨噬細胞參與MHT血管微環(huán)境損傷的初步觀察MHN患者腎臟間質(zhì)可觀察到PDGFRβ(周細胞標(biāo)志之一)與CCL2(巨噬細胞募集信號)陽性細胞,同時伴有M2a型(CD206/CD68共染陽性)為主的巨噬細胞浸潤,部分巨噬細胞可表達TGFβ1。流式細胞學(xué)方法檢測到MHN患者外周血M2型單核細胞在經(jīng)典型單核細胞(75.14 ±3.66%,39.26 ±6.50%,P=0.002)和中間型單核細胞中(80.84± 5.72%,31.51 ±7.89%,P=0.003)均高于正常對照。結(jié)論本研究條件下觀察:1.MHN患者表現(xiàn)為嚴(yán)重腎功能損害伴代謝異常和炎癥激活,出院舒張壓、淋巴細胞計數(shù)、腎小球硬化指數(shù)和小管萎縮比例為腎功能的獨立相關(guān)因素;24h尿蛋白與小管萎縮比例獨立相關(guān)。蛋白尿、最高eGFR和小管萎縮比例是影響患者腎臟長期生存的獨立危險因素;2.MHN患者腎小球和小管間質(zhì)均存在微血管損傷,并與腎功能顯著相關(guān),是獨立于傳統(tǒng)腎小管間質(zhì)病變,預(yù)測腎臟結(jié)局的危險因素。腎小球PDGFRβ陽性細胞表達下降,與腎小球CD34陽性面積比例正相關(guān),小管間質(zhì)PDGFRβ表達高于良性腎硬化與腎小球輕微病變,且與微血管分離。3.MHN患者外周血經(jīng)典型和中間型單核細胞中M2比例升高,腎臟間質(zhì)PDGFRβ陽性細胞可分泌CCL2募集M2a型為主的巨噬細胞,后者可分泌TGFβ1調(diào)節(jié)血管微環(huán)境。
[Abstract]:The research background of hypertensive renal injury is an important cause of chronic kidney disease, malignant hypertension (Malignant Hypertension MHT) renal pathology showed malignant hypertensive nephrosclerosis (Malignant Hypertensive Nephrosclerosis, MHN) of target organ involvement, rapid progression, poor prognosis, the incidence rate is rising in recent years. But lacking in the current literature have kidney sample clinical study of pathological diagnosis, prognostic factors of the risk is controversial, mechanism research is less. The kidney has two sets of capillary network, respectively, the glomerular capillaries and peritubular capillaries (Peritubular, capillary, PTC). In a variety of hypertensive patients with chronic glomerulonephritis, including patients with IgA nephropathy were observed in the capillary was significantly reduced, and the renal function, urinary protein level, glomerular sclerosis and interstitial fibrosis related, at present in the patients with MHT no Evaluation of vascular endothelial. Loss of stability is an important way to cause PTC damage, integrity and stability is dependent on the vascular endothelial cell structure and function of the week, which played an important role in inflammation. Perivascular fibroblasts and pericytes, can stimulate the identification of cell damage, chemokines secretion, monocyte macrophage recruitment. To M1 type differentiation proinflammatory effects, conversion to M2 direction can promote fibrosis. Currently macrophages with different phenotypes and sources in blood vessels in patients with malignant nephrosclerosis in the micro environment can play a role there is no research. Therefore, this study will be used in pathological diagnosis based on large sample of malignant nephrosclerosis cohort, analysis of clinical and pathological features. The application of virtual slice technology of automatic quantitative pathological score, analysis of the impact of risk factors on the prognosis of patients; glomerular and peritubular capillary injury on renal function and The predictive value of prognosis, and to investigate the microenvironment of PDGFR beta (+) cells and macrophages on vascular microenvironment injury mechanisms. Objective: 1. retrospective observation of primary hypertension and malignant nephrosclerosis (MHN) the clinical and pathological features and prognostic factors of 2. cases of glomerular MHN observation; vascular and peritubular capillary loss, and to analyze the relationship between clinical parameters and prognosis and its relevance to the microenvironment of PDGFR beta positive cells; 3. cases of renal local preliminary observation of MHN type macrophages and peripheral blood mononuclear cell types, and to explore the possible role of vascular microenvironment. Research methods from January 2003 to December 2016 in 100 cases hospitalized in Peking Union Medical College Hospital diagnosed by renal biopsy, the clinical data of primary malignant renal damage of hypertension patients, the clinical, pathological and follow-up data (more than June), end point events for long-term dialysis (over 3 months), renal transplantation or death. Renal pathology: semi quantitative analysis of glomerular, tubular atrophy and interstitial fibrosis, the application of virtual slice analysis method of glomerular density, glomerular volume; the renal tissue sections were CD34, PDGFRp, TGFp, immunohistochemistry PDGFR staining, beta /CD34, PDGFR beta /CCL2 immunofluorescence staining, analyzed the relationship with the clinical pathology and prognosis; double immunofluorescent staining of D68/CD86, CD68/TLR4, CD68/CD163, CD68/CD206, CD68/TGF beta of kidney type and macrophage function by flow cytometry method to detect malignant kidney cirrhosis patients and healthy control group peripheral blood nuclear cell type ratio. Statistical method: continuous variables expressed as mean standard deviation form, count data to proportion. Statistical software SPSS 19 software (IBM, USA). The normal distribution of continuous variables were compared with t test, variance; The non normal distribution of continuous variables using the rank sum test, constituent ratio using chi square analysis; single factor correlation analysis of normal distribution variables using Pearson correlation analysis, non normal distribution variables by Spearman correlation analysis, to evaluate the correlation between clinical and pathological data, using single factor or multiple linear regression analysis of factors related to the occurrence of MHT and kidney function damage, independent risk factors for prognosis by Kaplan-Meier method and COXX proportional risk evaluation model. The results of clinical and pathological features and prognosis of 1. primary hypertension and malignant renal sclerosis in 100 cases MHN patients were predominantly male (male / female 7.3:1), mean age 34.7 + 8.9 years old, the highest systolic blood pressure (SBP) treatment 225.2 + 26.4mmHg, the maximum diastolic blood pressure (DBP) 152 + 25.0mmH, serum creatinine (Scr) 465.3 + 333.9 mol/L, eGFR 22.13 + 15.17 ml/min/1.73m2,24h urine protein was 1.64 + 1.22g/24h (48.5%), anemia, high blood uric acid In (73.5%) and high glycerin three hyperlipidemia (67%) highlight the activation of systemic inflammatory response, and, as the ESR (65.2%), CRP (42.1%) increased. Semi quantitative pathological analysis showed glomerular sclerosis (hardening index: 1.56 + 0.50), tubular atrophy (ratio: 62.8 + 19.1%) and renal interstitial fibrosis (65 + 17.8%), and is closely related to renal function and 24h urinary protein and renal glomerular density. Good correlation, non glomerular sclerosis density and glomerular sclerosis index negatively correlated inflammation index (ESR, absolute value of lymphocyte, serum C3 and blood IgM level) is closely related with serum creatinine and the proportion of renal tubular atrophy. Multivariate regression analysis of glomerular sclerosis index, tubular atrophy ratio, discharge diastolic blood pressure level and lymphocyte count (R=0.603) for renal function (Scr) of the independent predictors; independent tubular atrophy and the proportion of 24h urinary protein of patients admitted to hospital. A variety of antihypertensive drugs, after the combined use of the discharge of blood pressure, serum creatinine and 24h urinary protein decreased significantly compared with those in the hospital (P0.001), the mean follow-up time was 56.8 + 37.1 months, first years, 3 years, 5 years and 10 years of cumulative renal survival rates were 93%, 80%, 66% and 27%, COX regression analysis. In the level of proteinuria, renal biopsy at the eGFR level and the proportion of tubular atrophy is an independent risk factor of.2. end-stage illness primary MHN patients with glomerular and peritubular capillary injury and prognosis of MHN group glomerular microvascular ratio and peritubular capillary (PTC) ratio were significantly lower than the benign nephrosclerosis group and mild lesion group. And with the clinical indicators (serum creatinine, 24h urine protein and hemoglobin) and renal pathology (glomerular sclerosis index, tubular atrophy ratio, interstitial fibrosis) good correlation, two decrease was independent of serum creatinine, urine protein and blood pressure is 24 hours Risk factors for long-term prognosis effect whether the target. The decreased expression of glomerular PDGFR beta, correlated with glomerular CD34 positive area ratio, tubulointerstitial PDGFR (beta cell surface marker week) was higher than that of benign nephrosclerosis and minor glomerular lesions, and microvessel.3. mononuclear macrophage MHT cells participate in vascular microenvironment damage preliminarily. MHN in kidneys of patients with interstitial separation can be observed in PDGFR (one of the beta cell marker (CCL2 weeks) and macrophage recruitment signal) positive cells, accompanied by a M2 (CD206/CD68 staining) infiltration of macrophages, part of macrophage cells can express TGF beta 1. flow cytometry detected MHN in peripheral blood of patients with type M2 mononuclear cells in classical monocytes (75.14 + 3.66%, 39.26 + 6.50%, P=0.002) and intermediate type mononuclear cells (80.84 + 5.72%, 31.51 + 7.89%, P=0.003) were higher than that of normal control. Observation under the conditions of this study 1.MHN patients showed severe renal impairment associated with metabolic disorders and inflammatory activation, diastolic pressure, lymphocyte count discharge, independent factors associated with glomerular sclerosis index and the ratio of renal tubular atrophy; 24h is independently related to urine protein and urine protein. The proportion of tubular atrophy, tubular atrophy and high eGFR proportion are independent risk factors for long-term survival the effect of kidney in patients with microvascular injury; there were 2.MHN patients with glomerular and tubular interstitial, and significantly correlated with renal function, is independent of traditional tubulointerstitial lesions and risk factors in predicting renal outcome. Glomerular PDGFR beta positive cells decreased and the expression of CD34 positive correlated with glomerular area ratio, tubulointerstitial PDGFR beta the expression is higher than that of benign nephrosclerosis and minor glomerular lesions, and the ratio of M2.3.MHN in peripheral blood of patients with the classic type and middle type mononuclear cells increased and renal interstitial microvascular separation. PDGFR positive beta cells can secrete CCL2 raise M2a type macrophages, which can secrete TGF beta 1 regulates vascular microenvironment.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R544.11;R692

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