| 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). |
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| 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 |
