【作者】 张宏华；

【导师】 张玲；

【作者基本信息】 重庆医科大学 ， 内科学， 2009， 博士

【摘要】 第一部分维持性血液透析患者血浆钙浓度对体内炎症介质及脉搏波速的影响目的脉搏波速是能准确反映动脉粥样硬化程度的早期指标,能够综合反映各种危险因素对血管的损伤,探讨维持性血液透析(MHD)患者血浆钙浓度对体内炎症介质及脉搏波速的影响,进一步了解导致MHD患者心血管并发症的危险因素,防治并发症的发生发展。方法1 MHD患者63例分成两组(A组和B组),A组31例为血浆钙浓度正常范围的患者,B组32例为血浆钙浓度低于正常范围的患者,透析前空腹留血3ml ,乙二胺四乙酸(EDTA)抗凝,1500rpm离心10min,收集血浆,置-20℃冰箱保存待测。2由我院生化室检测肾功,肝功,血脂及血浆钙浓度;采用酶联免疫吸附实验(ELISA)检测C反应蛋白(CRP)、白介素6(IL-6)、总同型半胱氨酸(tHcy)、全段甲状旁腺素(iPTH)水平;3采用脉搏波传导速度(PWV)分析仪测定颈股脉搏波传导速度(CF-PWV);4应用SPSS13.0统计软件包处理数据,计量资料以( x±S)表示,分类变量间比较进行卡方检验,采用两均数比较的t检验,直线相关分析,多元线性回归分析,多元Logistic回归分析。P<0.05为统计学差异有显著性。结果1 A组CRP、IL-6、tHcy、iPTH、及CF-PWV明显低于B组(P<0.05);2 63例患者用直线相关分析表明MHD患者血浆钙浓度与CRP、IL-6、tHcy、iPTH及CF-PWV呈负相关关系;3分别以CRP、tHcy和IL-6为应变量,血钙、血肌酐、血清白蛋白、血胆固醇、甘油三酯及透析龄为自变量进行多元回归分析,血钙及透析龄进入CRP和tHcy的回归方程,说明血钙浓度高低及透析龄长短与CRP和tHcy水平相关,透析龄进入IL-6的回归方程,说明透析龄长短与IL-6水平相关;4以CF-PWV为应变量,透析龄、血钙、CRP、IL-6、tHcy、iPTH为自变量进行多元回归分析,透析龄、血钙、CRP、tHcy进入回归方程,说明透析龄、血钙浓度、CRP及tHcy水平与CF-PWV相关;5以CF-PWV增快(CF-PWV≧9.8m/s,中位数)将63例患者分为两组并以它为应变量,性别、年龄、吸烟、糖尿病、收缩压、舒张压、透析龄、血钙、CRP、IL-6、tHcy和iPTH为自变量进行多元逐步Logistic回归分析,透析龄,血钙及tHcy进入了回归方程,说明透析龄、血钙浓度及tHcy水平与CF-PWV增快有关。结论1 MHD患者血浆钙浓度降低与炎症介质CRP及tHcy水平升高密切相关;2除透析龄外,血钙及tHcy是影响颈股脉搏波传导速度的独立危险因素。第二部分不同浓度的氯化钙对MHD患者PBMC炎症介质及PKC,NF-κB的影响目的在临床观察结果基础上,进一步探讨不同的钙离子浓度对MHD患者外周血单个核细胞(PBMC)炎症介质及细胞内蛋白激酶C(PKC)、核转录因子-κB(NF-κB)的影响。方法1健康志愿者和MHD患者各6例,取外周静脉血分离获得外周血单个核细胞,单个核细胞以5×105个细胞/孔种入96孔细胞培养板,分5组,分别加入不同浓度的氯化钙。A组:健康志愿者PBMC;B组:MHD患者PBMC;C组:MHD患者PBMC+1.0mmol/LCaCL2;D组:MHD患者PBMC+1.25mmol/LCaCL2,E组:MHD患者PBMC+1.5mmol/LCaCL2,培养细胞12小时。2观察培养前后细胞形态变化;3收集各组细胞培养上清检测CRP及IL-6水平,收获细胞后分别检测胞内钙离子浓度、PKC及NF-κB。结果1各组细胞培养前后细胞形态、数量无明显变化;2各组细胞培养前后胞内钙离子浓度差异无显著性;3健康者与未加氯化钙培养的细胞,培养上清中CRP、IL-6水平及PKC、NF-κB含量无明显差异;4加入1.0mmol/LCaCL2组细胞培养12小时后CRP、IL-6水平及PKC、NF-κB含量明显低于1.5mmol/LCaCL2组。结论1不同浓度的氯化钙不影响MHD患者PBMC的形态和数量;2不同浓度的氯化钙对MHD患者PBMC胞内钙离子浓度无明显影响;3不同浓度的氯化钙不影响健康者和MHD患者PBMC培养上清CRP、IL-6的水平及胞内PKC、NF-κB的含量;4低浓度钙明显抑制MHD患者PBMC培养上清CRP及IL-6的生成,明显减少胞内PKC及NF-κB的含量。第三部分脂多糖和氯化钙对MHD患者PBMC炎症介质及PKC、NF-κB的影响目的MHD患者体内存在多种炎症介质,对患者的PBMC产生炎症因子有一定影响。本部分实验拟进一步探讨在炎症介质刺激下,不同浓度的氯化钙对MHD患者PBMC炎症因子的产生及细胞内PKC、NF-κB的影响。方法1健康志愿者和MHD患者各6例,取外周静脉血分离获得PBMC,PBMC以5×105个细胞/孔种入96孔细胞培养板,每孔均加入LPS,其终浓度为100ng/ml,分5组。A1组:正常人PBMC;B1组:患者PBMC;C1组:患者PBMC+1.0mmol/LCaCL2;D1组:患者PBMC+1.25mmol/LCaCL2;E1组:患者PBMC+1.5mmol/LCaCL2;培养12小时。2观察培养前后细胞形态变化;3收集各组细胞培养上清检测CRP及IL-6水平,收获细胞后分别检测胞内钙离子浓度、PKC及NF-κB。结果1各组细胞培养前后细胞形态、数量无明显变化;2各组细胞培养前后,细胞内钙离子浓度差异无显著性;3健康者与MHD患者未加氯化钙培养的PBMC,培养上清中CRP、IL-6水平及PKC、NF-κB含量无明显差异;4加入1.0mmol/LCaCL2组细胞培养12小时后,细胞产生CRP、IL-6及细胞内PKC、NF-κB含量明显高于1.5mmol/LCaCL2组。结论1在LPS的刺激下,不同浓度的氯化钙不影响培养的MHD患者PBMC形态和数量;2三种浓度的氯化钙对LPS刺激培养的MHD患者PBMC胞内钙离子浓度无明显影响(均P>0.05);3在LPS的刺激下,不同浓度的氯化钙不影响健康者和MHD患者PBMC培养上清CRP、IL-6的水平及胞内PKC、NF-κB的含量;4在LPS的刺激下,低浓度钙明显增加MHD患者PBMC培养上清CRP、IL-6的生成及胞内PKC及NF-κB的含量。更多还原

【Abstract】 Chapter 1EFFECTS OF THE BLOOD PLASM CALCIUM CONCENTRATION ON INFLAMMATION MEDIATORS AND PULSE WAVE VELOCITY IN MAINTENANCE HEMODIALYSIS PATIENTSObjectivePulse wave velocity is a earlier index of atherosclerosis level and can reflect damage of all kinds of risk factors on blood vessel. It conduced to understand risk factors of cardiovascular complication and prevent and cure these complications for investigating the effects of blood plasm calcium concentration on inflammation mediators and pulse wave velocity in maintenance hemodialysis patientsMethods1 Sixty three maintenance hemodialysis patients were divided into two groups(A group n=31 and B group n=32). The blood plasm calcium concentrations of A group were in normal range.The blood plasm of 3 mL venous blood,anticoagulation by ethylene diamine tetraacetic acid,centrifugation 10 minutes by 1500 rpm,were preserved in -20℃refrigerator for measuring.2 Results of hepatic function,renal function,blood fat and the blood plasm calcium density were from our biochemistry compartment,C reactive protein(CRP),interleukin-6(IL-6),total Homocysteine(tHcy),parathyroid hormone(iPTH) were measured by enzyme-linked immunosorbent assay.3 carotid-femoral pulse wave velocity(CF-PWV) were detected by PWV analysator.4 SPSS 13.0 statistical package were utilized for handling data,measurement data were expressed as arithmetic mean and standard deviation( x±s).The comparisons between the stydy groups were performed with chi square test,t test,linear correlation analysis,multiple linear regression analysis and multiple logistic regression analysis.Results1 The levels of CRP, IL-6,tHcy ,iPTH and CF-PWV in A group were lower than those of B group(p﹤0.05);2 There was a negative correlation among the blood calcium concentration and CRP and IL-6 and iPTH and tHcy and CF-PWV in 63 patients by linear correlation analysis; 3 By multiple linear regression analysis,levels of blood calcium concentration and dialysis time correlated with levels of CRP and tHcy , dialysis time correlated with levels of IL-6;4 The blood calcium concentration,dialysis time,levels of CRP and tHcy correlated with increase of CF-PWV by multiple linear regression analysis;5 Tthe blood plasm calcium concentration,dialysis time and levels of tHcy correlated with increase of CF-PWV by multiple logistic regression.Conclusions1 There is a significant negative correlation among the blood plasm calcium concentration and CRP and tHcy;2 In addition to dialysis time, blood plasm calcium concentration and tHcy are independent risk factors of increase of CF-PWV in maintenance hemodialysis.chapter 2EFFECT OF DIFFERENT CONCENTRATION CALCIUM CHLORIDE ON PERIPHERAL BLOOD MONONUCLEAR CELL INFLAMMATION MEDIATORS,PKC AND NF-κB IN MHD PATIENTS ObjectiveAccording to prophase clinical observe founds,continually investigating the effect of calcium chloride of different concentration on peripheral blood inflammation mediators,PKC and NF-κB in MHD patients .Methods1 Peripheral blood mononuclear cells,from 6 healthy volunteers and 6 MHD patients were obtained after centrifugalization,5×105PBMCs/well ,seeded in 96 wells cell culture plate were divided into 5 groups for cultivating 12 hours.A group:normal PBMC,B group:patient PBMC,C group: patient PBMC+1.0mmol/L Cacl2,D group: patient PBMC+1.25mmol/L Cacl2,E Group: patient PBMC+1.5mmol/L Cacl2;2 Cell morphologic changes before and after culture were observed; CRP and IL-6 of culture supernatant were detected by ELISA; calcium ion concentration within cells were measured by Fluo-3 AM;3 PKC and NF-κB within cells were detected by western blot.Results1 Cell appearance and amount before and after 12 hours culture didn,t show marked change in every groups;2 Discrepancies of calcium ion concentration within cells after cultivating 12 hours between every groups were not significant; 3 There were not obvious difference about levels of CRP and IL-6 after 12 hours culture between A and B groups,so were leves of PKC and NF-κB;4 The levels of CRP and IL-6 in C group after 12 hours culture were lower than E group;so were levels of PKC and NF-κB.Conclusions1 Calcium chloride of three kind concentration didn’t affect form and amount of PBMC of MHD patients after cultivating 12 hours ;2 Discrepancies were not significant about effects of Calcium chloride of three kind concentrations on calcium ion concentration within PBMCs of MHD patients after cultivating 12 hours ;3 There were not obvious difference about levels of CRP and IL-6 from PBMC between healthy volunteers and MHD patients ;levels of PKC and NF-κB within PBMC in healthy volunteers were not significant,compared with MHD patients after cultivating 12 hours ;4 Compared with 1.5mmol/L calcium chloride,1.0mmol/L calcium chloride significantly inhibited CRP and IL-6 production of PBMC,reduced levels of PKC and NF-κB within PBMC in MHD patients. Chapter 3SYNERGETIC EFFECT OF LIPOPOLYSACCHARIDE AND CALCIUM CHLORIDE ON PERIPHERAL BLOOD MONONUCLEAR CELL INFLAMMATION MEDIATORS ,PKC AND NF-κB IN MHD PATIENTSObjectiveConsidering the uremic medium of MHD patients,for instance,PBMC were stimulated by lipopolysaccharide ,advanced glycosylation ends and so on.for this reason,to explore the synergetic effect of lipopolysaccharide and calcium chloride on peripheral blood mononuclear cell inflammation mediators ,PKC and NF-κB in MHD patients progressively .Methods1 Peripheral blood mononuclear cells,from 6 healthy volunteers and 6 MHD patients were obtained after centrifugalization,5×105PBMCs/well seeded in 96 wells cell culture plate were divided into 5 groups for cultivating 12 hours.before cultivating,LPS were added in every well,adjusting LPS final concentration as 100ng/ml,A1 group:normal PBMC,B1 group:patient PBMC,C1 group: patient PBMC+1.0mmol/L CaCL2,D1 group: patient PBMC+1.25mmol/L CaCL2,E1 Group: patient PBMC+1.5mmol/L CaCL2.2 Cell morphologic changes before and after culture were observed,CRP and IL-6 of culture supernatant were detected by ELISA;calcium ion concentration within cells were measured by Fluo-3 AM;3 PKC and NF-κB within cells were detected by western blot.Results1 Cell appearance and amount before and after 12 hours culture didn,t show marked change in every groups;2 Discrepancies of calcium ion concentration within cells after 12 hours culture between every groups were not significant;3 There were not obvious difference about levels of CRP and IL-6 between A 1and B1 groups after 12 hours culture;so were levels of PKC and NF-κB;4 Compared with E1 group;the levels of CRP and IL-6 in C1 group were higher after 12 hours culture ;so were gray levelsof PKC and NF-κB.Conclusions1 By stimulus of LPS, Calcium chloride of three kind concentrations didn’t affect form and amount of PBMC of MHD patients after 12 hours culture;2 Discrepancies were not significant about effects of Calcium chloride of three kind concentrations on calcium ion concentration within PBMC of MHD patients after 12 hours culture under stimulus of LPS;3 Through stimulus of LPS,there were not obvious difference about levels of CRP and IL-6 from PBMC between healthy volunteers and MHD patients after 12 hours culture;so were levels of PKC and NF-κB;4 By stimulus of LPS,compared with 1.5mmol/L calcium chloride,1.0mmol/L calcium chloride significantly increased CRP and IL-6 production of PBMC,raised levels of PKC and NF-κB within PBMC in MHD patients after 12 hours culture.更多还原