M2型巨噬细胞在原发性高血压恶性肾硬化微血管病变中的作用初探

发布时间：2018-02-10 00:20

本文关键词： 原发性高血压恶性肾硬化微血管周细胞巨噬细胞生存分析　出处：《北京协和医学院》2017年博士论文　论文类型：学位论文

【摘要】：研究背景高血压肾损害是慢性肾脏病的重要原因,其中恶性高血压(Malignant Hypertension,MHT)肾脏病理表现为高血压恶性肾硬化(Malignant Hypertensive Nephrosclerosis,MHN)靶器官受累多,病情进展快,预后差,发病率近年有上升趋势。但目前文献中缺乏有肾脏病理诊断的大样本临床研究,影响预后的危险因素存在争议,相关机制的研究较少。肾脏具有两套毛细血管网,分别为肾小球毛细血管和肾小管周毛细血管(Peritubular capillary,PTC)。在多种合并高血压的慢性肾小球肾炎、包括IgA肾病患者中均可观察到毛细血管显著减少,并与肾功能、尿蛋白水平、肾小球硬化和间质纤维化相关,目前在MHT患者中并无评价。血管内皮稳定性丧失是导致PTC损伤的重要途径,而血管内皮稳定性则依赖于血管周细胞结构和功能的完整性,炎症在其中发挥重要作用。血管周成纤维细胞和周细胞,可识别细胞损伤刺激,分泌趋化因子,募集单核巨噬细胞。巨噬细胞向M1型分化有促炎作用,向M2方向转化可促进纤维化。目前巨噬细胞不同的表型和来源在恶性肾硬化患者血管微环境中是否发挥作用尚无研究。因此,本研究将在以病理诊断为基础的较大样本恶性肾硬化队列中,分析临床病理特点、应用虚拟切片技术自动化病理定量评分,分析影响患者长期预后的危险因素;探讨肾小球和小管周微血管损伤对肾功能和预后的预测价值,并初步探讨局部微环境中PDGFRβ(+)细胞和巨噬细胞对血管微环境损伤可能的机制。研究目的1.回顾性观察原发性高血压恶性肾硬化(MHN)的临床和病理特点,分析影响预后的因素;2.观察MHN病例肾小球微血管和小管周毛细血管缺失的情况,分析其和临床指标及预后的关联性及其与局部微环境PDGFRβ阳性细胞的关系;3.初步观察MHN病例肾脏局部巨噬细胞分型和外周血单核细胞分型、初步探讨其对血管微环境的可能作用。研究方法2003年1月至2016年12月间在北京协和医院住院的100例经肾活检确诊的、临床资料完整的原发性恶性高血压肾损害病例,收集其临床、病理及随访资料(大于6月),终点事件为长期透析(≥3个月)、肾移植或死亡。肾脏病理:肾小球、肾小管萎缩和间质纤维化半定量分析,应用虚拟切片方法分析肾小球密度、肾小球体积;对肾组织切片分别进行CD34、PDGFRp、TGFp免疫组化染色,PDGFRβ/CD34,PDGFRβ/CCL2免疫荧光共染,分析其与临床、病理和预后关系;免疫荧光双染D68/CD86,CD68/TLR4,CD68/CD163,CD68/CD206,CD68/TGFβ 研究肾脏巨噬细胞分型和功能,用流式细胞学方法检测恶性肾硬化患者和健康对照组外周血单核细胞分型比例。统计方法:连续变量以均值±标准差的形式表示,计数资料以构成比表示。统计软件为SPSS 19.0软件(IBM,USA)。正态分布连续变量比较用t检验、方差;非正态分布连续变量应用秩和检验,构成比应用卡方分析;单因素相关分析正态分布变量应用Pearson相关分析,非正态分布变量用spearman相关分析,评价临床与病理资料的相关性,采用单因素或多重线性回归分析MHT发生和肾功能损害的相关因素,应用Kaplan-Meier方法和COXX比例风险模型评价影响预后的独立危险因素。研究结果1.原发性高血压恶性肾硬化临床病理特点及预后100例MHN患者以男性为主(男/女7.3:1),平均年龄34.7±8.9岁,就诊时最高收缩压(SBP)225.2±26.4mmHg,最高舒张压(DBP)152.0±25.0mmH,血肌酐(Scr)465.3±333.9μmol/L,eGFR 22.13± 15.17 ml/min/1.73m2,24h 尿蛋白为1.64±1.22g/24h,贫血(48.5%)、高尿酸血症(73.5%)和高甘油三脂血症(67%)突出,同时存在全身炎症反应活化,表现为血沉增快(65.2%)、CRP升高(42.1%)。病理半定量分析提示肾小球硬化(硬化指数:1.56±0.50)、肾小管萎缩(比例:62.8±19.1%)和肾间质纤维化(比例65.0±17.8%)重,并与肾功能和24h尿蛋白密切相关。肾小球密度与肾功能相关性好,非硬化肾小球密度与小球硬化指数负相关。炎症指标(ESR、淋巴细胞绝对值、血清补体C3和血IgM水平)与血肌酐及肾小管萎缩比例密切相关。多因素回归分析肾小球硬化指数、小管萎缩比例、出院舒张压水平和淋巴细胞计数(度R=0.603)为肾功能(Scr)的独立预测因素;小管萎缩比例与24h尿蛋白量独立相关。患者入院后联合使用的多种降压药物,出院血压、血肌酐和24h尿蛋白较其入院显著降低(P0.001),平均随访时间56.8±37.1月,第1年、3年、5年和10年的累积肾脏生存率分别为93%、80%、66%和27%,COX回归分析显示蛋白尿水平、肾活检时eGFR水平和小管萎缩比例是终末期生病的独立危险因素。2.原发MHN患者肾小球及肾小管周毛细血管损伤与预后分析MHN组肾小球微血管比例和肾小管周毛细血管(PTC)比例均显著低于良性肾硬化组和轻微病变组,且与临床指标(血肌酐、24h尿蛋白和血红蛋白)和肾脏病理(肾小球硬化指数、小管萎缩比例、间质纤维化程度)相关性较好,二者减少均是独立于血肌酐、24小时尿蛋白和血压是否达标的影响长期预后的危险因素。肾小球PDGFRβ表达下降,与肾小球CD34阳性面积比例正相关,小管间质PDGFRβ(周细胞表面标志)高于良性肾硬化与肾小球轻微病变,且与微血管分离。3.单核巨噬细胞参与MHT血管微环境损伤的初步观察MHN患者肾脏间质可观察到PDGFRβ(周细胞标志之一)与CCL2(巨噬细胞募集信号)阳性细胞,同时伴有M2a型(CD206/CD68共染阳性)为主的巨噬细胞浸润,部分巨噬细胞可表达TGFβ1。流式细胞学方法检测到MHN患者外周血M2型单核细胞在经典型单核细胞(75.14 ±3.66%,39.26 ±6.50%,P=0.002)和中间型单核细胞中(80.84± 5.72%,31.51 ±7.89%,P=0.003)均高于正常对照。结论本研究条件下观察:1.MHN患者表现为严重肾功能损害伴代谢异常和炎症激活,出院舒张压、淋巴细胞计数、肾小球硬化指数和小管萎缩比例为肾功能的独立相关因素;24h尿蛋白与小管萎缩比例独立相关。蛋白尿、最高eGFR和小管萎缩比例是影响患者肾脏长期生存的独立危险因素;2.MHN患者肾小球和小管间质均存在微血管损伤,并与肾功能显著相关,是独立于传统肾小管间质病变,预测肾脏结局的危险因素。肾小球PDGFRβ阳性细胞表达下降,与肾小球CD34阳性面积比例正相关,小管间质PDGFRβ表达高于良性肾硬化与肾小球轻微病变,且与微血管分离。3.MHN患者外周血经典型和中间型单核细胞中M2比例升高,肾脏间质PDGFRβ阳性细胞可分泌CCL2募集M2a型为主的巨噬细胞,后者可分泌TGFβ1调节血管微环境。
[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.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R544.11;R692

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