【作者】 王一梅；

【导师】 丁小强； 滕杰； 邹建洲；

【作者基本信息】 复旦大学 ， 内科学， 2008， 博士

【摘要】 研究目的1.研究不同剂量血液滤过对内毒素休克猪生存率、血流动力学、氧合作用和炎症的影响,探讨相应的机制,包括对血浆细胞因子（TNF-α、IL-6、IL-10和IL-18）水平、单核细胞功能和中性粒细胞凋亡和NK-κB的作用;2.研究连续性血液滤过治疗sepsis的合适剂量。研究方法1.动物模型的建立采用静脉注射内毒素（E.coli O111:B4,15.7μg/kg）的方法诱导内毒素休克猪模型,当平均动脉压（MAP）下降30%时,标志内毒素休克猪模型建立。2.建立体外循环行左侧股静脉和左侧颈外静脉穿刺术,分别置入8F单腔导管,作为CVVH时的动脉端以及静脉回血端,连接百特BM 25床旁CRRT机,采用Gambro Filtral12聚丙烯腈膜滤器（AN69,1.2 m²）,血流量为150 ml/min。3.分组将18只内毒素休克猪（28.1±5.1kg）随机分成三组:对照组（n=6）、CVVH组（n=6,置换液速度为55 ml/kg·h,前稀释法,相当于后稀释法的45 ml/kg·h）和高容量血液滤过（HVHF）组（n=6,置换液速度为100 ml/kg·h,前稀释法,相当于后稀释法的70 ml/kg·h）,治疗24h后处死动物。4.观察指标1)血流动力学指标:每小时监测内毒素休克猪心率（HR）、平均动脉压（MAP）、体温（T）、呼吸（P）、脉搏氧饱和度（SpO₂）、平均肺动脉压（Mean Pulmonary Arterial Pressure,mPAP）、肺动脉楔压（Pulmonary Artery Wedge Pressure,PAWP）、中心静脉压（Central Venous Pressure,CVP）、心排出量（Cardial Output,CO）、心脏指数（CardialIndex,CI）、每搏量（Stroke Volume,SV）、每搏指数（Stroke Index,SI）、体循环阻力指数（Systemic Vascular Resistance Index,SVRI）、肺循环阻力指数（PulmonaryVascular Resistance Index,PVRI）和左室每搏作功指数（Left Ventricular Stroke WorkIndex,LVSWI）。2)氧合作用相关指标:每2小时送检血气,测定动脉血pH值、动脉氧分压（PaO₂）、动脉二氧化碳分压（PaCO₂）、动脉血氧饱和度（SaO₂）、碱剩余（BE）、HCO₃^-和氧合指数（oxygenation index,OI=PaO₂/FIO₂）。3)生存时间、血常规和肾功能:记录内毒素休克猪的生存时间和尿量,测定造模前（Baseline）、成模时（TO）、成模后6h（T6）、12h（T12）、24h（T24）血常规和肾功能。4)细胞因子:ELISA法测定Baseline、T1、T0、T6、T12、T24各时点血浆细胞因子（TNF-α、IL-6、IL-10、IL-18）的水平。5)单核细胞功能和中性粒细胞凋亡:流式细胞术测定Baseline、T0、T6、T12、T24各时点单核细胞MHCⅡ的表达和中性粒细胞凋亡的水平,予LPS（10μg/ml）刺激各时点单核细胞24h,ELISA法测定上清中TNF-α的含量。6)肺和肾组织病理:肺和肾组织行HE染色,免疫组化法观察p65和细胞间粘附分子-1（intercellular adhesion molecule-1,ICAM-1）在肺和肾的表达,ELISA法测定肺和肾组织的NF-κB活性。结果1.第一部分内毒素休克猪成模时HR、mPAP、PAWP和PVRI升高,MAP、SVRI、SV、SI、LVSWI、OI和BE下降（P＜0.05）,其余指标无明显变化。治疗24h后,CVVH组和HVHF组的LVSWI和OI较对照组明显升高（P＜0.05）,但两组之间无显著性差异。2.第二部分对照组动物平均生存时间为（15.4±5.2）h,CVVH组为（21.4±4.1）h,HVHF组为（22±6.7）h,CVVH组和HVHF组的生存时间显著高于对照组（P＜0.05）。对照组Scr和BUN分别从成模后6h和12h开始升高,后进行性恶化。HVHF组和CVVH组的BUN分别于成模后12h和24h开始低于对照组（P＜0.05）,两组的Scr分别于成模后6h和12h开始低于对照组（P＜0.05）,三组动物成模后血小板进行性下降,各组之间的红细胞、血红蛋白、红细胞压积、血小板数量均无显著性差异（P＞0.05）。成模后各组尿量均下降,治疗后12h HVHF组尿量高于另外两组（P＜0.05）,治疗后18h、24h,CVVH组和HVHF组尿量高于对照组,两组之间无显著差异。3.第三部分对照组TNF-α和IL-6水平在T1达最高峰,IL-10水平在T0达最高峰,随后不断下降,IL-18水平在成模后上升,后无明显变化。CVVH组血浆IL-10（T6、T12、T24）水平低于对照组（P＜0.05）,HVHF组TNF-α（T6）和IL-10（T6、T12、T24）水平低于对照组和CVVH组（P＜0.05）,三组的IL-6和IL-18水平无显著性差异（P＞0.05）,IL-6（T6、T12）水平与动物生存时间呈负相关（P＜0.05）。4.第四部分成模时三组单核细胞分泌的TNF-α水平较成模前无明显变化（P＞0.05）,成模后6h TNF-α水平最低,后逐步回升,三组之间无显著差异（P＞0.05）。成模时三组MHCⅡ表达较成模前无明显变化（P＞0.05）,成模后MHCⅡ表达呈进行性下降,三组之间无显著性差异（P＞0.05）。成模时各组中性粒细胞（PMN）凋亡较成模前减少（P＜0.05）,治疗后24h HVHF组PMN凋亡高于对照组和CVVH组,差异有统计学意义（P＜0.05）。成模时,各组PMN数量均下降,但百分比无明显变化,成模后各时点PMN数量和百分比均大于成模前（P＜0.05）,治疗12h、24h后HVHF组PMN数量和百分比较对照组和CVVH组显著下降（P＜0.05）。5.第五部分CVVH组和HVHF组肺组织的水肿、间质浸润、透明膜和肺不张病变较对照组减轻（P＜0.05）,CVVH组和HVHF组肾小球和肾小管病变较对照组显著减轻（P＜0.05）,两组之间肺和肾的改变均无显著性差异。p65和ICAM-1在内毒素休克猪肺、肾组织中的表达均增加,CVVH组与HVHF组肺、肾组织中NF-κB活性、p65表达和ICAM-1表达较对照组均显著下降（P＜0.05）,两组之间无显著性差异（P＞0.05）。结论1.CVVH和HVHF治疗均能一定程度地改善内毒素休克猪的心脏泵血功能和氧合作用,增加置换液剂量对血流动力学和氧合作用无额外的作用。2.CVVH和HVHF治疗能显著延长内毒素休克猪的生存时间。内毒素休克猪出现急性肾损伤（acute kidney injury,AKI）,且肾功能进行性恶化,连续性血液滤过治疗能有效改善肾功能,增加实验动物的尿量。3.CVVH治疗能有效清除IL-10,HVHF治疗能清除TNF-α和IL-10,但CVVH和HVHF对IL-6和IL-18水平无明显影响。IL-6水平是预测内毒素休克预后的独立指标。4.内毒素休克猪成模后出现免疫抑制,表现为单核细胞MHCⅡ表达下降、分泌TNF-α水平的下降和中性粒细胞凋亡的减少。CVVH和HVHF治疗对单核细胞抗原递呈功能和分泌功能均无影响,但HVHF能抑制PMN凋亡的减少,减少外周PMN的数量和百分比。5.CVVH和HVHF通过抑制NF-κB的机制来减轻肾和肺组织损伤和减少ICAM-1的表达。更多还原

【Abstract】 Objective1.To study the impact of continuous veno-venous hemofiltration（CVVH） in different ultrafiltmtion rate on survival time,hemodynamics,arterial oxygenation and inflammation in porcine endotoxemic shock;To investigate the mechanism, including the impact of CVVH and high volume hemofiltration（HVHF） on plasma cytokines,monocyte function,neutrophil apoptosis and nuclear factor-κB （NF-κB）.2.To study the adequate dose of CVVH in sepsis.Methods1.Animal preparation and hemofiltration treatmentEighteen anesthetized mechanically ventilated pigs weighing（28.1±5.1） kg received a 15.7μg/kg endotoxin（E.coli O111:B4） infusion over 60 min.When mean arterial pressure（MAP） dropped 30%,they were randomized into three groups. Control group（n = 6） received no further intervention,CVVH group（n = 6） and HVHF group（n = 6） received pre-dilution continuous veno-venous hemofiltration for 24 hours with an ultrafiltration rate of 55 ml/kg·h and an ultrafiltration rate of 100 ml/kg·h respectively,the blood flow was 150 ml/min.A 1.2-m² polyacrylonitrile membrane（AN69） and a Baxter BM 25 hemofiltration device were used.2.Measurements1) Monitoring heart rate（HR）,mean arterial pressure（MAP）,body temperature,pulse, Mean Pulmonary Arterial Pressure（mPAP）,Pulmonary Artery Wedge Pressure （PAWP）,Central Venous Pressure（CVP）,Cardial Output（CO）,Cardial Index（CI）, Stroke Volume（SV）,Stroke Index（SI）,Systemic Vascular Resistance Index（SVRI）, Pulmonary Vascular Resistance Index（PVRI） and Left Ventricular Stroke Work Index （LVSWI）. 2) Monitoring PaO₂,SaO₂,FiO₂,oxygenation index（OI= PaO₂/FiO₂）,BE,PaCO₂ and HCO₃^-.3) Monitoring survival time and urine output.Renal function and the complete blood count were measured before endotoxin infusion（Baseline） and at 0h（T0）,6h（T6）, 12h（T12）,24h（T24） after the establishment of endotoxic shock model.4) Blood were taken before endotoxin infusion（Baseline） and at 0h（T0）,1h（T1）, 6h（T6）,12h（T12）,24h（T24） after the establishment of endotoxic shock model.The plasma levels of TNF-α,IL-6,IL-10 and IL-18 were tested by ELISA.5) At T0,T6,T12,T24 during CVVH,peripheral monocytes were isolated and stimulated with LPS（10μg/ml ）to detect the ability of production of TNF-α.MHCⅡexpression on rnonocytes was assessed to evaluate the antigen presenting function of monocytes.Apoptotic changes in PMNs were measured by flow cytometry.6) Wedge-shaped sections from kidneys and lungs were stained with H&E.The expressions of intercellular adhesion molecule-1（ICAM-1） and p65 were assessed by immunohistochemistry.Moreover,NF-κB/DNA binding activity was analyzed by ELISA.Results1.Part OneThe natural course of endotoxemic shock in our study was characterized by increases of HR,mPAP,PAWP,SVRI and PVRI,and decreases of MAP,SVRI,SV,SI, LVSWI,OI and BE.LVSWI and OI were improved significantly in HVHF group and CVVH group at T24（P＜0.05 ）.No significant differences between HVHF group and CVVH group were found.2.Part TwoThe survival time in control group was（15.4±5.2） h,CVVH group was（21.4±4.1） h,HVHF group was（22±6.7） h.The survival time in CVVH and HVHF group were significantly longer than that of control group（P＜0.05 ）.The urine output in CVVH and HVHF group was obviously higher than that in control group at T18 and T24（P＜0.05）.Platelet decreased progressively after the infusion of LPS. 3.Part ThreePlasma TNF-αand IL-6 peaked at T1,while IL-10 peaked at T0,then they declined gradually.While IL-18 increased and did not decline after T0.A significant decrease of plasma IL-10 levels was observed at T6,T12 and T24 in CVVH group compared with control groups（P＜0.05）.HVHF group accomplished a greater decrease in plasma TNF-α（T6） and IL-10（T6、T12、T24） levels compared with control group and CVVH group（P＜0.05 ）.The level of IL-6 and IL-18 showed no significant difference among three groups（P＞0.05 ）.There was a significant negative correlation between IL-6 and survival time（P＜0.05）.4.Part FourMHCⅡexpression of monocyte and the level of TNF-αwere markedly decreased in porcine endotoxemic shock（P＜0.05）.CVVH and HVHF had no effect on them.The infusion of endotoxin was followed by decrease ofPMN apoptosis. HVHF increased PMN apoptosis and decreased the number of peripheral PMN（P＜0.05）.5.Part FiveCVVH and HVHF resulted in significantly lower scores for hyaline membranes, interstitial infiltration,edema and atelectasis in lung compared with control group（P＜0.05）.CVVH and HVHF mitigated significantly severe tubular damage and glomerular damage compared with control group（P＜0.05）.The expression of ICAM-1 and p65,the degree of NF-κB/DNA binding activity in lung and kidney were reduced in CVVH group and HVHF group（P＜0.05）,there was no significant difference between them（P＞0.05 ）.Conclusions1.HVHF and CVVH can improve blood pumping and arterial oxygenation of porcine endotoxemic shock in some degree.2.HVHF and CVVH can prolong survival time and improve urine output compared with control group.3.IL-10 can be removed effectively with CVVH and HVHF in porcine endotoxemic shock and HVHF can also remove TNF-αeffectively.IL-6 was a powerful independent predictive factor for survival time in porcine endotoxemic shock.4.HVHF can alleviate the decrease of PMN apoptosis which may result from removal of TNF-αand IL-10.CVVH and HVHF can not improve monocyte function.5.CVVH and HVHF can offer both renal and lung protection,which may be related to suppression of inflammation and NF-κB.更多还原