【作者】 陈志强；

【导师】 何杨；

【作者基本信息】 苏州大学 ， 临床检验诊断学（专业学位）， 2016， 硕士

【摘要】 第一部分终末期肾病患者血管生成相关因子的表达目的:检测终末期肾病(End stage renal disease,ESRD)患者外周血中血清转化生长因子-β1(Transforming growth factorβ1,TGF-β1)、血管内皮生长因子(Vascular endothelial growth factor-A,VEGF-A)、可溶性神经钙黏蛋白(Solubleneural cadherin,sN-cadherin)和可溶性血管内皮钙黏蛋白(Soluble vascular endothelial cadherin,sVE-cadherin)表达水平,探讨ESRD患者外周血中与血管生成相关因子的表达情况,为ESRD患者的诊疗效果提供新的指标。方法:根据改善全球肾脏病预后组织(Kidney Disease:Improving Global Outcomes,KDIGO)指南连续选取2014年8月-2015年2月于我院血液透析中心诊断为ESRD的患者72例,作为ESRD组,平均年龄(54±17)岁,其中男45例,女27例。同时选取我院体检中心健康体检者39名作为对照组,平均年龄(55±18)岁,其中男27例,女12例。血细胞分析仪检测ESRD组和对照组血红蛋白(Hb)、血细胞比容(Hct),全自动生化分析仪检测ESRD组和对照组血清肌酐(Cr)、尿素(UREA)、超敏C反应蛋白(hsCRP)、血钙(Ca)、血磷(P)、总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)和高密度脂蛋白(HDL)等生化指标,化学发光法检测ESRD组血清甲状旁腺素(PTH)、维生素D3(Vit D3)、铁蛋白(Fer)和β2-微球蛋白(β2-MG),酶联免疫吸附法检测ESRD组和对照组血清TGF-β1、VEGF-A、sN-cadherin和sVE-cadherin的表达情况,分析各组差异及关系。结果:(1)ESRD组和对照组在年龄、性别、体重指数、Ca、P、TC、TG、LDL和HDL等方面的差异无统计学意义(P>0.05),但ESRD组血清UREA、Cr水平显著高于对照组(P<0.01)。ESRD组血清TGF-β1、VEGF-A和sN-cadherin水平显著高于对照组(P<0.01);ESRD组血清sVE-cadherin和s VE-cadherin/sN-cadherin显著低于对照组(p<0.01)。(2)72例esrd患者,23.6%患者处于非炎症状态,76.4%患者处于微炎症状态。esrd患者非炎症组和微炎症组在血清tgf-β1、vegf-a、sn-cadherin和sve-cadherin/sn-cadherin水平之间的差异无统计学意义(p>0.05),但esrd患者非炎症组血清sve-cadherin水平显著低于微炎症组(p<0.01)。(3)72例esrd患者,16.6%患者vitd3缺乏,41.7%患者vitd3不足,41.7%患者vitd3适量。血清tgf-β1和sn-cadherin水平在vitd3缺乏组、vitd3不足组和vitd3适量组三组之间的差异无统计学意义(p>0.05);血清vegf-a、sve-cadherin和sve-cadherin/sn-cadherin水平在vitd3缺乏组、vitd3不足组和vitd3适量组三组之间的差异有统计学意义(p<0.01),sve-cadherin、sve-cadherin/sn-cadherin与vitd3水平正相关(r=0.324,p=0.006;r=0.28,p=0.017)。(4)单因素pearson相关分析示,esrd患者血清tgf-β1与pth、ldl-c水平正相关(r=0.328,p=0.0049;r=0.2319,p=0.0499);esrd患者血管生成相关因子间比较,血清tgf-β1与vegf-a、sn-cadherin正相关(r=0.258,p=0.028;r=0.306,p=0.009),而sn-cadherin与sve-cadherin负相关(r=-0.255,p=0.039);esrd患者sve-cadherin/sn-cadherin与透析年限、tgf-β1负相关(r=-0.435,p<0.001;r=-0.289,p=0.014)。(5)多因素线性回归分析示,透析年限与血清vitd3水平是esrd患者sve-cadherin/sn-cadherin比值的独立预测因子(r=-0.454,p<0.01;r=0.246,p=0.019)。结论:(1)esrd患者血清高表达tgf-β1、sn-cadherin,提示可能存在上皮间质转化或内皮间质转化,同时血清vegf-a水平升高,提示可能存在与肾脏血管新生相关。(2)esrd患者在微炎症状态下较非炎症状态外周血高表达sve-cadherin,提示血管内皮细胞膜表面上ve-cadherin的脱落与炎症相关。(3)esrd患者sve-cadherin水平及sve-cadherin/sn-cadherin与vitd3水平正相关,提示vitd3水平可通过改变vegf-a和sve-cadherin水平而可能参与血管内皮细胞损伤修复的病理生理过程。(4)esrd患者的透析年限和vitd3水平是sve-cadherin/sn-cadherin值的独立预测因子,透析年限越长,Vit D3水平越低,提示患者肾脏内皮功能障碍越严重。第二部分终末期肾病患者血液透析前后血清TGF-β1、VEGF-A、sN-cadherin和sVE-cadherin水平的变化目的:根据ESRD患者聚醚砜(polyethersulfone,PES)膜血液透析前、后血清TGF-β1、VEGF-A、sN-cadherin和sVE-cadherin水平的变化,探讨血液透析对ESRD患者血管新生的影响,为临床治疗提供新的综合评定标准。方法:根据KDIGO指南连续入选2014年8月-2015年2月在我院血液透析中心长期维持透析的ESRD患者66例,作为治疗组,平均年龄(52±19)岁,其中男43例,女23例。酶联免疫吸附法检测患者血液透析前、后血清TGF-β1、VEGF-A、sN-cadherin和sVE-cadherin水平,行配对样本t检验分析其差异。同时,与第一部分的39名健康体检者作对照,探讨ESRD患者血管生成因子的变化。结果:治疗组透析后VEGF-A水平升高(P<0.01),sN-cadherin水平降低(P<0.01),sVE-cadherin水平及sVE-cadherin/sN-cadherin比值升高(P<0.01),而血清TGF-β1水平无显著性改变(P>0.05)。结论:PES膜血液透析前后ESRD患者血清TGF-β1水平没有改变,血清VEGF-A水平增高,VE-cadherin和N-cadherin之间的平衡趋向正常化,提示PES膜血液透析可能改善ESRD患者血管内皮细胞功能,有促进有效血管新生的趋势。更多还原

【Abstract】 PART ONE Expression of Serum Angiogenic factors in Patients with End-stage Renal DiseaseObjective: To evaluate the effect of angiogenic factors in End stage renal disease(ESRD) patients based on analysis of circulating transforming growth factor β1(TGF-β1), vascular endothelial growth factor-A(VEGF-A), soluble neural cadherin(s N-cadherin) and soluble vascular endothelial cadherin(s VE-cadherin).Methods: 72 patients(45 male and 27 female, average age 54±17), diagnosed as ESRD according to KDIGO guideline between August 2014 and February 2015 from the blood purification center of the 1st Affiliated Hospital of Soochow University, were enrolled in this study. 39 healthy individuals(male 27, female 12, average age 55±18) served as controls. Hemoglobin and Hematocrit was carried out on an automated hematology analyzer. Biochemical parameters such as serum creatinine(Cr), urea, high sensitivity C-reactive protein(hs CRP), calcium(Ca), phosphorus(P), total cholesterol(TC), triglycerides(TG), low density lipoprotein cholesterol(LDL-C) and high density lipoprotein cholesterol(HDL-C) were estimated using an automated biochemical analyzer. Serum parathyroid hormone(PTH), vitamin D3(Vit D3), ferritin(Fer) and β2-microglobin(β2-MG) were determined using a Chemiluminescence Immunoassay system. Serum TGF-β1, VEGF-A, soluble N-cadherin and soluble VE-cadherin were measured by enzyme-linked immunosorbent assay(ELISA), respectively. The differences and relationships between each parameter were analyzed.Results:(1) Except serum Cr and urea(P<0.01), no significant differences between ESRD patients and healthy controls were found in age, gender, body mass index, Ca, P, TC, TG, LDL-c and HDL-c(P>0.05). Serum concentrations of TGF-β1, VEGF-A and s N-cadherin were significant higher in ESRD patients than in healthy controls(P<0.01). In contrast, serum s VE-cadherin level and the ratio of s VE-cadherin to s N-cadherin in ESRD patients were significant lower when comparing to healthy controls(P<0.01).(2) Of the ESRD patients, 23.6% of them were innon-inflammatory state, 76.4% were in a state of micro inflammation. Instead of TGF-β1, VEGF-A, s N-cadherinand s VE-cadherin/s N-cadherin(P>0.05), serum s VE-cadherin concentration was significant higher in micro-inflamed patients in comparison with non-inflammatory patients(P<0.01).(3) Of the ESRD cohort, 16.6% of patients were Vit D3 deficient, 41.7% had inadequate Vit D3 values whereas 41.7% had optimal Vit D3 values. Serum concentrations of VEGF-A, s VE-cadherin as well as s VE-cadherin/s N-cadherin(P<0.01) did differ significantly between the subgroups by Vit D3 category, whereas neither TGF-β1 nor s N-cadherin was found significant difference among each group(P>0.05). Furthermore, Serum concentration of s VE-cadherin as well as s VE-cadherin/s N-cadherin was positively correlated with Vit D3(r=0.324,P=0.006; r=0.28,P=0.017).(4) In univariate analysis of ESRD patients, significant positive correlations with TGF-β1 were found for PTH and LDL-c(r=0.328,P=0.0049;r=0.2319,P=0.0499). Of the angiogenic factors studied, serum TGF-β1 was positive associated with VEGF-A and s N-cadherin(r=0.258,P=0.028; r=0.306,P=0.009), while aninverse correlation was noted between s N-cadherin and s VE-cadherin(r=-0.255,P=0.039). The ratio of s VE-cadherin to s N-cadherin was significant negatively associated with dialysis therapy duration and Vit D3 state, r=-0.454, P<0.01, r=0.246, P=0.019, respectively.(5) In multiple regressions, dialysis therapy duration and Vit D3 were the independent explanation variables of s VE-cadherin/s N-cadherin(r=-0.454, P<0.01; r=0.246, P=0.019).Conclusions:(1) Elevated serum TGF-β1 and s N-cadherin indicated possible EMT or End MT in ESRD patients. In terms of high levels of VEGF-A, there was a possibility of angiogenesis in kidney also.(2) Increased concentration of serum s VE-cadherin in ESRD patients under micro-inflamed state suggested that the shedding of VE-cadherin from endothelial cell membrane was associated with inflammation.(3) The positive correlations with Vit D3 between s VE-cadherin and s VE-cadherin/s N-cadherin indicated Vit D3 was involved in the pathophysiological process of the repair of dysfunctional endothelial cells.(4) The dialysis therapy duration and serum Vit D3 levels were the independent predictors of the ratio of s VE-cadherin to s N-cadherin in ESRD patients. The longer dialysis therapy duration and lower levels of Vit D3, the more severe of renal endothelial cells dysfunction.PART TWO The Analysis of Serum TGF-β1, VEGF-A, s N-cadherin and s VE-cadherin in End-stage Renal Disease Patients upon HemodialysisObjective: To compare the angiogenic profile of ESRD patients before and after polyethersulfone(PES) hemodialysis based on the analysis of serum TGF-β1, VEGF-A, s N-cadherin and s VE-cadherin.Methods: Serum concentrations of TGF-β1, VEGF-A, s N-cadherin and s VE-cadherin were measured in 66 ESRD patients(male 43 and female 23, average age 52±19) pre- and post- PES dialysis and 39 healthy controls(male 27, female 12, average age 55±18).Results: After a duration of hemodialysis in ESRD patients, serum levels of VEGF-A, s VE-cadherin and the ratio of s VE-cadherin to s N-cadherin were significantly higher(P<0.01), whereas serum concentrations ofs N-cadherin were significantly lower(P<0.01). However, no significant change was observed in terms of serum TGF-β1 levels(P>0.05).Conclusion: Increased concentrations of serum TGF-β1 in ESRD patients could not be attenuated by PES hemodialysis therapy. However, after hemodialysis, elevated serum levels of VEGF-A may promote angiogenesis activity, and the balance between VE-cadherin and N-cadherin was tend to normalization, suggesting PES hemodialysis therapy may contribute to the improvement of endothelial function and thus facilitated effective angiogenesis.更多还原