终末期肾脏病的血小板凋亡及多囊肾病DNA损伤的临床与实验研究

发布时间：2018-07-24 20:13

【摘要】：第一部分终末期肾脏病血小板凋亡的临床与实验研究目的：探究终末期肾脏病（ESRD）患者临床出血风险、血小板参数的变化特点及其影响因素与相互关系；探讨尿毒症患者的血小板凋亡及其机制。方法：分临床研究与实验研究两部分。在临床研究中，通过回顾性分析278例ESRD患者的出血情况、治疗方法及血清肌酐（SCr）、甲状旁腺激素（PTH）、血小板（PLT）及其参数的改变，分别探究肾功能分期、原发疾病、替代治疗方法等因素对出血、血小板参数的影响。在实验研究中，首先对16例已进入ESRD阶段的尿毒症患者分血液透析组（HD）、腹膜透析组（PD）及非透析治疗组（ND）进行血小板凋亡和活化检测。制备患者富含血小板的血浆（PRP）,以血小板聚集仪检测血小板聚集功能，流式细胞仪检测血小板线粒体跨膜电位（ΔΨm）的去极化、磷脂酰丝氨酸（PS）的外翻表达、血小板活化指标P-选择素（P-Selectin）的表达；在此基础上以尿毒症患者少血小板血浆（PPP）与正常人洗涤血小板孵育后，用流式细胞仪检测ΔΨm、PS及P-Selectin，用Western Blot检测血小板Caspase-3及抗凋亡蛋白Bcl-2家族的表达变化。最后，分别以尿毒症毒素肌酐、尿素与甲状旁腺激素的不同浓度梯度刺激正常血小板并检测血小板凋亡。结果：临床研究发现ESRD患者出血发生率为25%，其中狼疮性肾炎、高血压肾病、糖尿病肾病及慢性肾小球肾炎的患者占出血患者总数92.86%，，各原发病种间Hb、PLT、MPV、HCT有显著性差异。慢性肾脏病（CKD）4期及CKD5期出血率高（26.47%、28.85%），CKD3期-5期Hb、PLT、PDW、HCT组间有显著性差异，PLT与SCr呈负相关关系。三种不同的治疗方案间比较，出血率分别为HD32.61%、PD30.95%、ND15.09%，HD及PD组PLT明显低于ND组。实验研究发现16例尿毒症患者的血小板聚集功能明显降低；与正常对照组相比，尿毒症患者血小板ΔΨm降低（43.48±9.58,52.76±15.36, P=0.005)，PS外翻表达增加（1.36±0.51,0.99±0.27, P0.001)，P-Selectin表达无差异（P=0.14）；三组间上述检测指标无差异。尿毒症PPP与正常血小板孵育后检测发现，血小板ΔΨm明显降低（P=0.0001），PS外翻显著增加（P=0.002），P-Selectin无明显增加（P=0.14）；Western-Blot检测到Caspase-3活化裂解的17Kd片段，抗凋亡蛋白Bcl-2、Bcl-xL低表达，促凋亡蛋白Bax高表达。毒素刺激实验发现随着浓度升高，肌酐、尿素、甲状旁腺激素均未能导致血小板ΔΨm去极化。结论：终末期肾脏病患者具有较高出血倾向，出血风险与原发疾病有关并随着肾功能的进展而增加，患者血小板数量随着肾功能进展而减少。血液透析与腹膜透析患者较高的出血风险与血小板减低有关。尿毒症患者存在增强的血小板凋亡，这种凋亡趋势是多种毒素长期综合作用的结果，不能被透析纠正且与透析方式无关。血小板凋亡可能导致尿毒症血小板减少及血小板功能障碍。第二部分多囊肾病DNA损伤的实验研究目的：研究常染色体显性遗传多囊肾病（ADPKD）患者及其家系成员外周血淋巴细胞DNA的损伤及其基因稳定性。方法：采用单细胞凝胶电泳技术（SCGE）研究10例ADPKD患者（A组）、1个ADPKD家系中3例无症状者（B组）、20例健康对照者（C组）外周血淋巴细胞的DNA损伤及在0.5Gy照射后的DNA损伤情况。采用彗星分析软件（CASP）进行图像分析，以尾DNA含量（TDNA%）评价淋巴细胞的DNA损伤程度。结果： A组照射前、后TDNA%（8.85%±0.14%，14.84±0.77%）及照射后TDNA%的增加值（5.99%±0.77%）均显著高于C组（7.50%±0.37%，12.46%±0.26%，4.96%±0.44%）；B组均无临床症状，但其中2例照射前、后TDNA%均与C组相似，1例照射前、后TDNA%均值均接近A组。结论：ADPKD患者外周血淋巴细胞具有基因不稳定性，在环境因素刺激下，有可能通过基因突变启动多脏器囊肿形成。SCGE为进一步阐明ADPKD发病机制及其预后判断提供了一种新的方法和思路。
[Abstract]:Part one clinical and experimental study of platelet apoptosis in patients with end-stage renal disease
Objective: To explore the risk of clinical bleeding in patients with end-stage renal disease (ESRD), the characteristics of changes in platelet parameters and their relationship, and to explore the apoptosis and mechanism of platelet in patients with uremia.
Methods: two parts were divided into clinical and experimental studies. In the clinical study, 278 cases of ESRD patients' bleeding, treatment methods and serum creatinine (SCr), parathyroid hormone (PTH), platelet (PLT) and their parameters were analyzed, and the renal function staging, primary disease, replacement therapy and other factors on bleeding and blood were small. In the experimental study, 16 cases of uremia who had entered ESRD stage were divided into hemodialysis group (HD), peritoneal dialysis group (PD) and non dialysis treatment group (ND) for platelet apoptosis and activation. The platelet rich plasma (PRP) was prepared, platelet aggregation was detected by platelet aggregation apparatus, flow cytometry was used. The depolarization of the platelet mitochondrial transmembrane potential (delta m), the expression of phosphatidylcholine (PS) and the expression of P- selectin (P-Selectin) of platelet activation index (P-Selectin) were detected. On this basis, after incubation of platelets in uremic patients with less platelet plasma (PPP) and normal human washed platelets, a flow cytometry was used to detect delta m, PS and P-Selectin, using Western. The expression changes of platelet Caspase-3 and anti apoptotic protein Bcl-2 family were detected by Blot. Finally, the normal platelets were stimulated by the different concentrations of uremic toxin creatinine, urea and parathyroid hormone, and the apoptosis of platelets was detected.
Results: the clinical study found that the incidence of bleeding in ESRD patients was 25%, including lupus nephritis, hypertensive nephropathy, diabetic nephropathy and chronic glomerulonephritis, which accounted for 92.86% of the total bleeding. There were significant differences in Hb, PLT, MPV and HCT among the primary diseases. The 4 and CKD5 bleeding rates of chronic renal disease (CKD) were high (26.47%, 28.85%), CKD3 stage -5 H. There were significant differences in B, PLT, PDW, and HCT groups, and PLT and SCr were negatively correlated. The rate of bleeding was significantly lower in HD32.61%, PD30.95%, ND15.09%, HD and PD group than in the group of HD32.61%, PD30.95%, ND15.09%, HD and PD, respectively. The experimental study found that the platelet aggregation work of 16 patients with uremia was significantly reduced; compared with the normal control group, the blood of uremia patients was compared. The expression of M decreased (43.48 + 9.58,52.76 + 15.36, P=0.005), PS expression increased (1.36 + 0.51,0.99 + 0.27, P0.001), P-Selectin expression was no difference (P=0.14), and there was no difference between the three groups. After incubation of the uremia PPP and normal platelets, it was found that the delta M of the platelets decreased significantly (P=0.0001), and PS ectropion increased significantly. There was no significant increase in Selectin (P=0.14); Western-Blot detected the 17Kd fragment of Caspase-3 activation cracking, the anti apoptotic protein Bcl-2, the low expression of Bcl-xL and the high expression of apoptotic protein Bax. The toxin stimulation experiment found that the creatinine, urea and parathyroid hormone did not cause the platelet delta m depolarization with the increase of concentration.
Conclusion: Patients with end-stage renal disease have a higher bleeding tendency. The risk of bleeding is associated with the primary disease and increases with the progression of renal function. The number of platelets in patients decreases with the progression of renal function. The higher risk of bleeding in hemodialysis and peritoneal dialysis patients is associated with thrombocytopenia. Apoptosis, which is the result of a long-term comprehensive effect of multiple toxins, can not be corrected by dialysis and is not related to the mode of dialysis. Platelet apoptosis may lead to thrombocytopenia and platelet dysfunction in uremia.
Experimental study of DNA damage in the second part of polycystic kidney disease
Objective: To study DNA damage and its gene stability in peripheral blood lymphocytes of patients with autosomal dominant polycystic kidney disease (ADPKD) and their family members.
Methods: single cell gel electrophoresis (SCGE) was used to study 10 cases of ADPKD (group A), 3 asymptomatic patients in 1 ADPKD families (group B), 20 healthy controls (group C), DNA damage and DNA damage after 0.5Gy irradiation. The comet analysis software (CASP) was used to analyze the lymph nodes, and the tail DNA content (TDNA%) was used to evaluate the lymph nodes. The degree of DNA damage in cells.
Results: before A group, the value of TDNA% (8.85% + 0.14%, 14.84 + 0.77%) and TDNA% (5.99% + 0.77%) after irradiation were significantly higher than those in group C (7.50% + 0.37%, 12.46% + 0.26%, 4.96% + 0.44%), and B group had no clinical symptoms, but before exposure to 2 cases, TDNA% was similar to that in group C, and the mean value of TDNA% was close to the A group before irradiation.
Conclusion: the peripheral blood lymphocytes of ADPKD patients have genetic instability. Under the environmental stimulus, it is possible to start multiple organ cysts by gene mutation to form.SCGE to further clarify the pathogenesis of ADPKD and to provide a new way of thinking for the prognosis.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692.5

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