【打印本页】 【下载PDF全文】 【HTML】 查看/发表评论下载PDF阅读器关闭

←前一篇|后一篇→

过刊浏览    高级检索

本文已被:浏览 2161次   下载 2824 本文二维码信息
码上扫一扫!
血液滤过联合灌流治疗对重症急性胰腺炎继发肺损伤患者血清TNF-α及血管内皮细胞RhoA磷酸化修饰的影响
郑倩1,杜晓刚1*,陈雪梅2,李正荣1,龚颖1,何俊伶1
0
(1.重庆医科大学附属第一医院肾内科,重庆 400016
4.第二军医大学长海医院超声科,上海 200433
*通信作者)
摘要:
目的 探讨血液滤过联合血液灌流(HF+HP)治疗对重症急性胰腺炎(SAP)继发肺损伤患者血清中TNF-α的清除作用及对内皮细胞中RhoA的188位丝氨酸磷酸化修饰(p-RhoA)的影响。方法 选取35例SAP患者纳入实验研究,其中SAP继发急性肺损伤(ALI)/急性呼吸窘迫综合征(ARDS) 28例,未继发ALI/ARDS 7例,同时设正常对照组20例。全部SAP患者收入中心ICU治疗,其中9例SAP继发ARDS患者给予连续性静脉-静脉血液滤过(CVVH)联合HP治疗(HF+HP组)。应用ELISA法检测不同程度肺损伤的SAP患者血清中TNF-α水平的变化,以及采用HF+HP治疗的9例SAP继发ARDS患者治疗不同时间点血清中TNF-α的浓度。体外实验采用HF+HP治疗不同时间点患者的血清分别刺激体外培养的人脐静脉内皮细胞(HUVECs),并以重组人TNF-α刺激细胞作为阳性对照,蛋白质印迹分析检测各组HUVECs中p-RhoA与总RhoA蛋白的表达,免疫荧光染色观察p-RhoA在细胞内的分布。结果 与未继发ALI/ARDS患者比较,SAP继发ALI/ARDS的患者血清中TNF-α水平明显升高(P<0.05),尤以继发ARDS的患者升高最为明显(约为正常对照组的7倍)。采用HF+HP治疗的9例SAP继发ARDS患者治疗6 h后,血清中TNF-α水平与HF+HP治疗前比较开始下降,至治疗20 h下降最为明显,接近正常组水平;HF+HP治疗后,SAP继发ARDS患者动脉血氧分压(PaO2)、HCO3-水平、氧合指数(PaO2/FiO2)均较治疗前上升(P<0.05)。蛋白质印迹分析结果表明,HF+HP治疗后SAP继发ARDS患者血清诱导的HUVECs中p-RhoA水平逐渐升高(P<0.05),但各组RhoA总蛋白表达水平差异无统计学意义(P>0.05)。免疫荧光结果显示,在HUVEsC中,p-RhoA主要分布在细胞质,各组变化趋势与蛋白质印迹分析结果一致。结论 SAP继发ALI/ARDS患者血循环中存在高水平的TNF-α。HF+HP治疗可有效地清除SAP继发ALI/ARDS患者血液中大量生成的TNF-α,并抑制RhoA的活化,从而起到降低内皮细胞高通透性的作用。
关键词:  血液滤过  血液灌流  重症急性胰腺炎;肺损伤;内皮细胞;RhoA;肿瘤坏死因子α
DOI:
投稿时间:2013-02-26修订日期:2013-06-08
基金项目:重庆市教委科研资助项目(KJ100318),重庆市科委资助项目(cstc2012jjA10136),重庆市卫生局重点资助项目(2011-1-016).
Effect of hemofiltration combined with hemoperfusion on serum TNF-α in severe acute pancreatitis patients with lung injury and on RhoA serine 188 phosphorylation in endothelial cells
ZHENG Qian1,DU Xiao-gang1*,CHEN Xue-mei2,LI Zheng-rong1,GONG Ying1,HE Jun-ling1
(1. Department of Nephrology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
2. Department of Emergency, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
*Corresponding author.)
Abstract:
Objective To study the effect of hemofiltration plus hemoperfusion (HF+HP) on removal of serum TNF-α in severe acute pancreatitis (SAP) patients with lung injury and on RhoA serine 188 phosphorylation (p-RhoA) in endothelial cells. Methods Totally 35 SAP patients, including 28 with acute lung injury (ALI)/ acute respiratory distress syndrome (ARDS), and 20 healthy subjects were involved in this study. All the SAP patients were treated in ICU, and the 9 combined with ARDS underwent continuous veno-venous hemofiltration (CVVH) and HP therapy (HF+HP group). The serum TNF-α in SAP patients with different degrees of lung injury and the serum TNF-α in SAP patients with ARDS was determined by ELISA at different time points during HF+HP. For in vitro study the human umbilical vein endothelial cells (HUVECs) were treated with sera from SAP patients with ARDS during HF+HP, and those treated with recombinant TNF-α were taken as a positive control. The changes of p-RhoA and total RhoA were observed by Western blotting analysis. Subcellular distribution of p-RhoA protein was observed by immunofluorescence staining. Results We found that, compared with SAP patients without ALI/ARDS, those with ALI/ARDS, especially those with ARDS (about 7 folds that of the normal control) had a significantly increased serum TNF-α level (P<0.05). The serum TNF-α level began to decrease in the 9 SAP patients with ARDS 6 h after HF+HP treatment, with the decrease reached maximal 20 h after treatment, closing to the level of the normal control group. Meanwhile, the arterial blood PaO2 , HCO3- and PaO2/FiO2 ratio were significantly elevated after HF+HP treatment in SAP patients with ARDS(P<0.05). Western blotting analysis showed that p-RhoA level was significantly increased in HUVECs treated with the serum of HF+HP-treated SAP patients with ARDS (P<0.05), and the total RhoA protein expressions were not significantly different among different groups (P>0.05). Immunofluorescence indicated that p-RhoA was largely distributed in the cytoplasm of HUVECs, and the changes were consistent with the data from Western blotting analysis. Conclusion SAP patients with ALI/ARDS have high circulation TNF-α. Treatment with HF+HP can effectively remove the excessive TNF-α in the blood of SAP patients with ALI/ARDS and inhibit activation of RhoA, subsequently decreasing the high permeability of endothelial cells.
Key words:  hemofiltration  hemoperfusion  severe acute pancreatitis  lung injuries  endothelial cells  RhoA  tumor necrosis factor-alpha