【作者】 许煊；

【导师】 封志纯；

【作者基本信息】 南方医科大学 ， 儿童重病， 2011， 博士

【摘要】 研究背景儿童脓毒症是危重患儿死亡的第一位原因,是当今儿童危重病医学所面临的棘手难题。持续血液净化技术(CBP)在脓毒症和严重脓毒症患儿治疗中广泛应用和深入研究,但目前对CBP的治疗机制缺乏足够的理解,有必要对这一治疗过程中的相应机制进行研究。目前尚未见到CBP治疗脓毒症患儿前后典型蛋白标志物变化情况研究,本研究主要对CBP治疗脓毒症患儿前后临床疗效做比较,探讨CBP治疗脓毒症指证及时机,并对典型蛋白标志物软骨糖蛋白39(YKL-40)及C反应蛋白(CRP)动态变化做深入研究,探讨CBP治疗脓毒症机制。目的本课题研究CBP治疗脓毒症患儿前后临床疗效,CBP治疗脓毒症指证及时机,分析CBP治疗脓毒症前后病情危重程度、炎性因子及YKL-40和CRP动态变化情况,并分析比较YKL-40、CRP与IL-6、TNF-α和脓毒症患儿病情危重程度做相关性分析,探讨脓毒症患儿疾病严重程度、预后与典型蛋白标志物的关系,为临床评价CBP治疗脓毒症提供治疗依据。第一部分方法根据2005年国际脓毒症会议发布小儿脓毒症诊断标准,收集北京军区总医院附属医院八一儿童重病中心2008年8月至2010年8月收治的符合严重脓毒症诊断标准的25例患儿,以同期门诊健康体检儿童25例为对照组。25例患儿在行CBP治疗前均进行了常规综合治疗、原发病的治疗、抗生素、营养对症支持治疗,维持水电解质酸碱平衡；25例患儿均需机械通气辅助呼吸,15例患儿需应用多巴胺及去甲肾上腺素维持血压。(1)在持续血液净化治疗儿童脓毒性休克患儿开始治疗即0小时、治疗开始12小时、24小时、48小时从桡动脉端抽血5mL和2mL,5mL血样低温离心后取上层血清冻存；2mL血样做血常规生化检查肾功能、肝功能、血电解质、血乳酸,并测定4个时间点的动脉血气,对以上结果做统计分析。根据1995年5月中华医学会儿科学会急救学组讨论通过的《小儿危重病例评分法(草案)》(评分法)对CBP治疗前后脓毒症患儿评分,反映患儿危重病情程度和预测死亡风险。(2)采用酶联免疫法测定4个时间点的IL-6、TNF-α水平值并做统计分析。结果(1)经CBP治疗,25例脓毒症患儿经临床好转18例,恶化死亡5例,放弃治疗2例,18例患儿经后续治疗后治愈出院,治愈率72%。①在CBP治疗脓毒症患儿开始后,Oh、12h、24h和48h平均动脉压结果分别为42.84±8.84 mmHg,54.80±12.67mmHg,55.96±12.48mrnHg,58.27±13.00 mmHg,平均动脉压不断上升,治疗后平均动脉压与治疗前比较,有显著性差异(F=54.031,P<0.001); 0h、12h、24h和48h多巴胺使用量为17.61±4.07μg/kg min、14.24±2.36μg/kg·min、8.51±2.39μg/kg·min、4.31±1.47μg/kg·min,治疗后多巴胺使用量与治疗前比较,有显著性差异(F=159.681,P<0.001),治疗后12h(P<0.001)、24h(P<0.001)和48h(P<0.001)多巴胺使用量与治疗前比较,均有显著性差异；0h、12h、24h和48h肾上腺素使用量为1.41±0.34μg/kg·min、0.63±0.21μg/kg·min、0.08±0.03μg/kg·min、0.03±0.02μg/kg·min,治疗后肾上腺素使用量与治疗前比较,有显著性差异(F=277.775,P<0.001),治疗后12h(P<0.001)、24h (P<0.001)和48h(P<0.001)肾上腺素使用量与治疗前比较,均有显著性差异；Oh、12h、24h和48h尿量分别为0.86±0.58 mL/kg. h,0.88±0.42 mL/kg·h,0.90±0.45 mL/kg. h,1.36±0.61 mL/kg. h,治疗后尿量与治疗前比较,有显著性差异(F=27.752,P<0.001),治疗后48h尿量与开始CBP治疗前治疗前比较,有显著性差异(P<0.001)。②在CBP治疗脓毒症患儿开始后,Oh、12h、24h和48h血肌酐水平分别为363.02±83.73μmol/L、230.20±58.39μmol/L、191.20±51.17μmol/L 103.71±30.89μmol/L,治疗后血肌酐水平与治疗前比较,有显著性差异(F146.343=,P<0.001),治疗后12h(P<0.001)、24h(P<0.001)和48h(P<0.001)血肌酐水平与治疗前比较,均有显著性差异；0h、12h、24h和48h尿素氮水平分别为39.83±8.72 mmol/L、23.55±6.44mmol/L、14.64±4.68mmol/L、7.71±1.61mmol/L,治疗后尿素氮水平与治疗前比较,有显著性差异(F=179.995=,P<0.001),治疗后12h(P<0.001)、24h(P<0.001)和48h(P<0.001)尿素氮水平与治疗前比较,均有显著性差异。③在CBP治疗脓毒症患儿开始后,0h、12h、24h和48h血气PH分别为7.14±0.43、7.33±0.48、7.36±0.44、7.31±0.40,治疗后血气PH与治疗前比较,无显著性差异；0h、12h、24h和48h血气BE分别为19.72±4.71、8.32±2.33、4.61±1.62、0.61±0.17,治疗后血气BE与治疗前比较,有显著性差异(F=264.709,P<0.001),治疗后12h(P<0.001)、24h (P<0.001)和48h(P<0.001)血气BE与开始CBP治疗前治疗前比较,均有显著性差异；0h、12h、24h和48h血气PO2/FiO2分别为187.49±44.28、247.01±70.07、300.97±55.27、351.53±80.59,治疗后PO2/FiO2水平与治疗前比较,有显著性差异(F=70.274,P<0.001),治疗后12h (P<0.001)、24h(P<0.001)和48h(P<0.001)PO2/FiO2水平与治疗前比较,均有显著性差异。④经CBP治疗后48h,患儿危重评分与治疗前比较,有显著性差异(t=-6.096,P<0.001)。(2)在CBP治疗脓毒症患儿开始后,0h、12h、24h和48h IL-6水平分别为706.90±275.95 ng/L.325.18±126.94 ng/L.212.07±82.79 ng/L. 162.59±63.47ng/L,治疗后IL-6水平与治疗前比较,有显著性差异(F=164.057,P<0.001),治疗后12h(P<0.001).24h(P<0.001)和48h(P<0.001) IL-6水平与治疗前比较,均有显著性差异；Oh、12h、24h和48h TNF-α水平分别为989.67±386.33ng/L.872.27±209.28 ng/L.541.25±207.28ng/L.439.08±159.37 ng/L,治疗后TNF-α水平与治疗前比较,有显著性差异(F=106.836,P<0.001),治疗后12h(P=0.033).24h(P<0.001)和48h(P<0.001)TNF-α水平与治疗前比较,均有显著性差异。治疗后IL-6、TNF-α水平呈现下降趋势,经CBP治疗脓毒症患儿48h后,患儿血清中炎性因子IL-6(t=4.653,P<0.001)和TNF-α水平(t=9.008,P<0.001)与正常患儿比较,有显著性差异。(3)8例存在肝功能衰竭患儿,CBP治疗前,患儿内环境严重紊乱,凝血机制差,总胆红素(258.98±117.38)U/L、血氨(189.31:±46.23)μg/dL. GPT(1281.19±363.18)U/L.GOT(1018.71±72.24)U/L.PTA(28.22±6.76)%. CBP治疗12h后症状减轻,内环境、出血指标显著改善,总胆红素(93.51±28.34)U/L、血氨(56.26±10.16)μg/dL.GPT(390.58±169.12)U/L.GOT(300.77±141.46)U/L.PTA(61.46±13.75)%.总胆红素(t=3.797,P=0.007)、血氨(t=9.391, P<0.001)、GPT(t=7.017,P<0.001)、GOT(t=13.897,P<0.001)、PTA (t=.12.553,P<0.001)治疗前后均有统计学意义。(4)掌握好脓毒症患儿CBP治疗指证、时机及实施方法,可明显提高脓毒症患儿治愈率。第二部分方法(1)采用酶联免疫法测定待测血清中YKL-40浓度,采用免疫比浊法测定血浆中CRP水平,根据标准品浓度与吸光度之间关系绘制标准曲线,算出回归方程,根据所测血清标本吸光度算出样本中YKL-40和CRP浓度。(2)制备脓毒症患儿全血总RNA,以RT-PCR法测定全血中YKL-40 mRNA相对表达含量。(3)获取脓毒症患儿外周血单核细胞,收集培养的细胞,加入PBS离心洗涤1遍,吸出上清,加入cell staining buffer,充分混匀,加抗人FITC-CD14抗体或FITC-CD16抗体,室温避光孵育,用cell staining buffer重悬后行FCM检测,每次重复3次,取平均值CXP软件分析；用细胞免疫化学法检测脓毒症患儿外周血单核细胞中YKL-40的定位表达。结果(1)在CBP治疗脓毒症患儿开始后,患儿外周血中0h、12h、24h和48h YKL-40蛋白浓度分别为177.93±58.74μg/L、100.57±37.37μg/L、80.16±28.46μg/L、59.75±21.03μg/L,治疗后YKL-40与治疗前比较,有显著性差异(F=172.466,P<0.001)；患儿外周血中0h、12h、24h和48h CRP蛋白浓度分别为664.49±259.40μg/L、619.01±206.81μg/L、201.13±75.42μg/L、120.17±46.91μg/L,治疗后CRP水平与治疗前比较,有显著性差异(F=172.262,P<0.001),治疗后12h (P=0.026).24h(P<0.001)和48h(P<0.001)CRP水平与治疗前比较,均有显著性差异。CBP治疗48h后脓毒症患儿外周血YKL-40浓度与健康儿童比较,无显著性差异。(2)在CBP治疗后YKL-40mRNA△Ct表达含量与治疗前比较,无显著性差异；患儿外周血中0h、12h、24h和48h YKL-40 mRNA相对表达含量(2-△△CT)分别为1(0.6-1.7)、0.5(0.4-0.6)、0.5(0.3-0.7)、0.4(0.2-0.6)。(3)YKL-40阳性外周血中CD14呈低表达,CD16呈阳性表达,且在CBP治疗脓毒症患儿开始后,患儿外周血中0h、12h、24h和48h CD16表达率分别为35.23±8.15%、26.42±6.11%、27.86±6.57%、23.67±4.85%,呈下降趋势,治疗前后有显著性差异(F=153.901,P<0.001);CBP治疗脓毒症患儿48h后,外周血中CD16表达率接近健康儿童；经细胞免疫化学法检测,外周血细胞单核细胞中YKL-40阳性表达。(4)经CBP治疗恶化死亡5例脓毒症患儿外周血中YKL-40.CRP水平和CD16表达率较生存组患儿显著升高,与生存组患儿比较,有显著性差异(P<0.001)。第三部分方法收集第一部分、第二部分CBP治疗脓毒症患儿YKL-40、CRP、IL-6、TNF-α、IL-6浓度及危重评分数据,经SPSS13.0统计分析,采用Pearson相关分析分别做YKL-40、CRP对TNF-α、IL-6和YKL-40、CRP对CBP治疗脓毒症患儿前后危重评分做相关性分析。结果经CBP治疗48小时后, YKL-40浓度与IL-6浓度相关具显著性,且呈密切正相关(r=0.905, P<0.001), YKL-40浓度与TNF-α浓度相关具显著性,且呈密切正相关(r=0.890, P<0.001); CRP浓度与IL-6浓度相关具显著性,且呈密切正相关(r=1.000, P<0.001), CRP浓度与TNF-α浓度相关具显著性,且呈密切正相关(r=0.987,P<0.001)。经CBP治疗48小时后, YKL-40浓度与危重评分相关具显著性,且呈中度正相关(r=0.662, P<0.001); CRP浓度与危重评分相关具显著性,且呈密切正相关(r=0.772,P<0.001)。结论1持续性血液净化技术治疗能明显提高脓毒症患儿氧合能力、纠正酸中毒、改善组织灌注、清除小分子物质、稳定血压并减少升压药用量,改善预后并降低死亡率。2掌握好上CBP的指证及时机和实施方法是提高脓毒症患儿治愈率的重要一环。3持续性血液净化技术是脓毒症患儿有效治疗手段,其清除体内炎性细胞因子是其治疗脓毒症的可能机制。合并急性肝功能衰竭的患儿,肝性脑病ⅡⅡ级以前及时行CBP是较好的时间点。4 YKL-40和CRP均有可能成为评价持续性血液净化技术治疗脓毒症疗效的蛋白指标,CBP治疗后,外周血中YKL-40、CRP水平高及持续CD16高表达提示脓毒症患儿预后较差。更多还原

【Abstract】 BackgroudsPediatric sepsis, a thornic problem in critically ill children, is the primary cause of death among children. Continuous blood purification technology (CBP) is much effective in treatment of pediatric sepsis and generally applied in pediatric sepsis. By far, there is not enough comprehension in treatment mechanism of CBP treatment in pediatric sepsis. It is essential to investigate the mechanism. The change of typical biomarkers in treatment mechanism of CBP treatment in pediatric sepsis has not been investigated by scientifically controlled studies. In the research, we investigated the curative effect, the therapeutic indications and juncture of CBP treatment in pediatric sepsis, the feasibility of 39 human cartilage glycoprotein-39 (YKL-40) and C-reactive protein (CRP) as typical biomarkers in treatment mechanism of CBP applied in pediatric sepsis.ObjectivesIn the study, we report the curative effect, the therapeutic indications and juncture of CBP treatment in pediatric sepsis, the disease severity of ill children, the changes of inflammatory factors, YKL-40 and CRP concentration in peripheral blood when treated pediatric sepsis with CBP. Then made correlation analysis between YKL-40 concentration to IL-6, TNF-a concentration and disease severity scores, CRP concentration to IL-6, TNF-a concentration and disease severity scores. By the study, we investigated the relationship with prognosis and YKL-40 and CRP as typical biomarkers in treatment mechanism of CBP treatment in pediatric sepsis, offered treatment basis for clinical evaluation of CBP.Part oneMethods25 cases of pediatric sepsis children were collected from Byi Children’s Hospital, Beijing Command Hospital People Liberation Army from August 2008 to August 2010, all ill children were selected in line with the 2005 Meeting of the International Sepsis published diagnostic criteria for severe sepsis in children, healthy children to the same period illness children as control group. All illness children received routine comprehensive treatment with primary disease treatment, antibiotic treatment, nutrition treatment, support treatment and liquid, electrolyte acid-alkali balancing before CBP treatment. All 25 cases of illness children required assist respiration with mechanical ventilation,15 cases needed maintain with dopamine and noradrenaline.(1)Blood samples were collected from radial artery at four points of CBP treatment 0 hours,12 hours,24 hours,48 hours respectively. Each volume 5 mL blood samples were made blood serum with centrifugating in low temperature, then were cryopreserved. Each volume 2 mL blood samples were taken for conventional biochemical tests of the renal function, liver function, blood electrolyte and blood lactate at different time points. At the same time, arterial blood gas analysis was done at different time points. Score changes, in cases of pediatric critical illness before and after CBP, was calculated according with the pediatric critical illness score published by the subspecialty group of emergency medicine, society of pediatrics, Chinese medical association, was used to predict the ill children death risk.(2)A euzymelinked immunosorbent assay was used to determine inflammation mediators of IL-6 and TNF-a concentration at different time points.Results(1) Evaluation of clinical outcome was done in 25 cases of illness children with 48 hours of CBP treatment. Clinical symptoms improved in 18 cases, deteriorated and resulted in death in 5 cases, treatment were given up in two case,72% cure rate.①The mean arterial blood pressure in the four time points (the beginning if treatment,12 hour,24 hour and 48 hour) were 42±12.6 mmHg,54±9.6 mmHg, 55±7.9 mmHg,58±5.9 mmHg. The mean arterial blood pressure increased significantly (P<0.05) before and after CBP treatment. The dosages of dopamine in the four time points were 17.05±3.26μg/kg·min,14.19±4.95μg/kg·min, 8.60±6.51μg/kg·min,4.25±3.12μg/kg·min. The dosages of dopamine decreased significantly (P<0.05) before and after CBP treatment 12 hour. The dosages of noradrenaline were 1.41±0.39μg/kg·min,0.62±0.30μg/kg.min, 0.08±0.03μg/kg·min,0.03±0.02μg/kg·min. The dosages of noradrenaline decreased significantly (P<0.05) before and after CBP treatment 12 hour. The urine amount for illness children in the four time points were 0.85±0.54 mL/kg·h,0.86±0.55 mL/kg·h, 0.89±0.56 mL/kg. h,1.35±0.686 mL/kg. h. The difference was significant (P<0.05) compared at the beginning of treatment and 48 hour after treatment.②The level of blood serum creatinine in the four time points were 361.95±70.26μmol/L,279.69±61.56μmol/L,189.64±50.36μmol/L,103.21±33.45μmol /L. The difference was significant (P<0.05) compared at the beginning of treatment and 12 hour after CBP treatment. The difference was very significant (P<0.01) compared at the beginning of treatment and 24hour and 48hour after CBP treatment. The level of urea nitrogen in the four time points were 39.21±10.24 mmol/L, 23.2±7.7mmol/L,14.76±2.12mmol/L,7.78±1.63mmol/L. The difference was significant (P<0.05) compared at the beginning of treatment and 12 hour after CBP treatment. The difference was very significant (P<0.01) compared at the beginning of treatment and 24hour and 48hour after CBP treatment.③The blood gas PH level in the four time points were 7.14±0.22,7.33±0.12, 7.4±0.16,7.33±0.11. PH level increased significantly (P<0.05). The BE blood gas value in the four time points were-19.58±6.45,-8.21±2.73,-4.56±1.78,-0.61±1.75. BE value increased very significantly (P<0.01) compared at the beginning of treatment and CBP treatment. The oxygenation index (PO2/FiO2) in the four time points were 188.76±85.61,245.32±82.56,301.24±78.35,352.42±81.56. The difference was significant (P<0.05) compared at the beginning of treatment and 12 hour after CBP treatment. The difference was very significant (P<0.01) compared at the beginning of treatment and 24hour and 48hour after CBP treatment.④Severity scores of illness cases increased significantly (P<0.05) before and after CBP treatment 48 hour.(2) IL-6 concentration in the four time points were 705.21±87.62 ng/L, 324.31±86.34 ng/L,213.64±62.35 ng/L,164.35±51.23ng/L. IL-6 concentration decreased significantly (P<0.05) before and after CBP treatment 12 hour. IL-6 concentration was significant (P<0.05) compared at the beginning of treatment and CBP treatment 24 hour and 48 hour. TNF-a concentration in the four time points were 987.52±91.25ng/L,871.36±97.56 ng/L,541.23±111.25 ng/L,437.69±107.19 ng/L. TNF-a concentration was significant (P<0.05) compared at the beginning of treatment and CBP treatment 12 hour. TNF-a concentration was very significant (P<0.01) compared at the beginning of treatment and CBP treatment 24 hour and 48 hour.(3) There were 8 cases liver failure in pediatric sepsis children. The total bilirubin concentration was 258.98±117.38 U/L and 93.51±28.34 U/L at the beginning of treatment and CBP treatment 12 hour. Blood ammonia concentration was 189.31±46.23μg/dL and 56.26±10.16μg/dL, glutamate-pyruvate transaminase (GPT) was 1281.19±363.18 U/L and 390.58±169.12 U/L, glutamic oxaloacetic transaminase(GOT) was 1018.71±72.24 U/L and 300.77±141.46 U/L, prothrombin activity(PTA) was 28.22±6.76% and 61.46±13.75%.There were all have very significant (P<0.01) compared at the beginning of treatment and CBP treatment12 hour.(4) The cure rate of CBP treatment in pediatric sepsis maybe obiously increasing when we made best CBP treatment therapeutic indications, juncture and method.Part twoMethods(1) A euzymelinked immunosorbent assay was used to determine YKL-40 concentration blood serum and immunoturbidimetry assay to determine CRP concentration. The blood serum of YKL-40 and CRP concentration were calculated by regression equation made by standard curve of YKL-40 and CRP concentration. (2) YKL-40 mRNA expression in pediatric sepsis illness blood was determined by RT-PCR.(3) Peripheral blood monouclear cells (PBMC) in pediatric sepsis illness blood was obtained and then cultivated. The flow cytometry (FCM) was used to detect CD 14 and CD 16. The YKL-40 allocated expression in pediatric sepsis illness blood was detected by immunohistochemistry (IHC).Results(1) YKL-40 concentration of pediatric sepsis illness blood in the four time points were180.56±23.25μg/L,100.21±21.32μg/L,80.35±35.26μg/L, 60.53±26.21μg/L. YKL-40 concentration decreased significantly (P<0.05) before and after CBP treatment 12 hour. IL-6 concentration was very significant (P<0.01) compared at the beginning of treatment and CBP treatment. CRP concentration of pediatric sepsis illness blood in the four time points were 662.23±69.56μg/L, 620.21±58.32μg/L,200.32±42.36μg/L,120.56±29.21μg/L. CRP concentration was significant (P<0.05) compared at the beginning of treatment and CBP treatment 12 hour, was very significant (P<0.01) compared at the beginning of treatment and CBP treatment 24 hour and 48 hour.(2) The YKL-40 mRNA of pediatric sepsis illness blood relative expression in the four time points were 1 (0.6-1.7),0.5 (0.4-0.6),0.5 (0.3-0.7),0.4 (0.2-0.6) CRP concentration was significant (P<0.05) compared at the beginning of treatment and CBP treatment 12 hour, was very significant (P<0.01) compared at the beginning of treatment and CBP treatment 24 hour and 48 hour. The YKL-40 mRNA relative expression was significant (P<0.05) compared at the beginning of treatment and CBP treatment 12 hour and 24 hour, was very significant (P<0.01) compared at the beginning of treatment and CBP treatment 48 hour.(3) The YKL-40 protein was expressed in CD16+ monocytes with a dim expression of CD14. The FCM showed that the CD16 expression in the four time points were 35.2±5.930%、26.3±5.21%、27.8±6.92%、23.8±5.12%, the expression was decreased. The YKL-40 expression in histoleucocyte was positive by IHC. (4) The YKL-40 concentration, CRP concentration and CD 16 expression of 5 pediatric sepsis death cases blood in the four time points were higher the live pediatric sepsis illness, there were very significant (P<0.01)Part threeMethodsTo make correlation analysis with Bivariate Correlation between YKL-40 concentration with IL-6, TNF-a concentration and disease severity scores, CRP concentration with IL-6, TNF-a concentration and disease severity scores.ResultsThere were positive correlation between YKL-40 concentration with IL-6, TNF-a concentration and disease severity scores, CRP concentration with IL-6, TNF-a concentration and disease severity scores.Conclusions1 CBP can improve the treatment of pediatric sepsis, reduce the death rate of the treatment, was effective in the treatment by improving oxygenation improvement, correcting metabolic acidosis, increasing the tissue perfusion, stabilizing blood pressure and educing boosting pressure drugs usage.2 The cure rate of CBP treatment in pediatric sepsis maybe obiously increasing when we made best CBP treatment therapeutic indications, juncture and method.3 CBP is an effective treatment method for pediatric sepsis. It effectively removes blood cytokines which may be the possible mechanisms for the CBP treatment of pediatric sepsis.4 YKL-40 and CRP may be as biomarkers to evaluate CBP treatment for pediatric sepsis. There are bad prognosis if the YKL-40,CRP concentration and CD16 expression keep in higher level.更多还原