重组人肿瘤坏死因子受体融合蛋白（rhTNFR：FC）对脓毒症大鼠心肌损伤的保护作用

发布时间：2018-05-02 10:56

  本文选题：重组人肿瘤坏死因子受体融合蛋白 + 脓毒症　； 参考：《安徽医科大学》2013年硕士论文

【摘要】：目的：本研究采用盲肠穿孔结扎法致大鼠脓毒症动物模型，观察脓毒症大鼠心肌损害情况，探讨应用人肿瘤坏死因子受体融合蛋白（rhTNFR：Fc）早期干预对脓毒症心肌损害的保护作用，并初步研究其作用机理。 方法：清洁级SD大鼠108只，全为雌性，随机分为3组：（1）假手术组（A组，n=36），脓毒症组（B组,n=36），脓毒症加rhTNFR：Fc组(C组,n=36)；（2）A组动物于称重后，麻醉，后行盲肠探查术后关闭腹腔，分别于术后3h,9h,24h,36h，48h和72h六时相点行腹主动脉采血，采用酶联免疫分析法检测肿瘤坏死因子（TNF-α）、白细胞介素-1（IL-1）、心肌肌钙蛋白I（cTnI）水平,留取心脏组织，采用蛋白质印迹实验（Western blot法）作NF-κB P65蛋白检测，在每一时相点取6只大鼠（3）B组动物称重后，麻醉，后行盲肠结扎穿孔术后关闭腹腔，分别于术后3h,9h,24h,36h，48h和72h六时相点行与假手术组相同的操作步骤，采用酶联免疫分析法检测肿瘤坏死因子（TNF-α）、白细胞介素-1（IL-1）、心肌肌钙蛋白I（cTnI）水平,留取心脏组织，作核转录因子-κB（NF-κB）P65蛋白检测，在每一时相点取6只大鼠；（4）C组动物行盲肠结扎穿孔（CLP）术后，立即从颈外静脉注入益赛普（rhTNFR：Fc）水溶液2mL／kg(1g／L)，分别于术后3h,9h,24h,36h，48h和72h六时相点行与假手术组相同的操作步骤，采用酶联免疫分析法检测肿瘤坏死因子（TNF-α）、白细胞介素-1（IL-1）、心肌肌钙蛋白I（cTnI）水平,留取心脏组织，作NF-κB P65蛋白检测，在每一时相点取6只大鼠。(5)所有数据以“均数±标准差”表示，采用SPSS13.0统计软件包进行分析，相关性分析采用Spearman等级相关分析。 结果： 1.血清TNF-α的变化 脓毒症加rhTNFR：Fc组与脓毒症组血清TNF-α水平在CLP术后均较假手术组均增高（p0.05），脓毒症加rhTNFR：Fc组在3h达峰值（257.619±5.558pg/ml）,随后两组均有下降趋势，（p0.05），从3h至72h脓毒症加rhTNFR：Fc组均明显低于脓毒症组（p0.05）。 2.血清IL-1的变化 与假手术组相比，脓毒症加rhTNFR：Fc组与脓毒症组血清IL-1水平在CLP术后均增高（p0.05），脓毒症加rhTNFR：Fc组在3h达峰值（179.23±2.506pg/ml）,随后两组均有下降趋势，（p0.05），，从3h至72h脓毒症加rhTNFR：Fc组均明显低于脓毒症组（p0.05）。 3.血清cTnI的变化 与假手术组相比，脓毒症加rhTNFR：Fc组与脓毒症组血清cTnI水平在CLP术后均增高（p0.05），脓毒症加rhTNFR：Fc组在3h达峰值（228.332±3.635pg/ml）,随后两组均有下降趋势（p0.05），从3h至72h均明显低于脓毒症组（p0.05）。 4.（NF-κB）P65蛋白水平表达情况 脓毒症加rhTNFR：Fc组与脓毒症组CLP术后3h （NF-κB）P65蛋白水平均高于假手术组（p0.05），随后均有下降趋势，脓毒症加rhTNFR：Fc组下降幅度较脓毒症组更快,在24h，36h，48h脓毒症加rhTNFR：Fc组（NF-κB）P65蛋白水平均低于脓毒症组，至72h两组无明显差异。 5.cTnI与TNF-α、IL-1之间的相关性分析 TNF-α与cTnI具有显著正相关,相关系数为0.393，P=0.164；IL-1与cTnI具有显著正相关,相关系数为0.980，P=0.000。 结论： 1.在脓毒症致急性心肌损伤的过程中,脓毒症大鼠血清cTnI早期升高，反映了脓毒症有心肌损伤，通过检测血清cTnI水平，可以反映急性心肌损伤的严重程度。 2.脓毒症发生发展过程中,脓毒症大鼠血清TNF-α、IL-1升高，与cTnI具有高度相关性，表明炎症因子过度表达参与了脓毒症心肌损伤，（NF-κB）P65蛋白表达增强，推测脓毒症可能通过NF-κB信号转导通路致细胞因子过度表达，从而参与脓毒症的心肌损伤的过程。 3.脓毒症大鼠早期应用rhTNFR：Fc干预后，cTnI下降，心肌损伤减轻，TNF-α、IL-1炎症因子水平下降，（NF-κB）P65蛋白表达下调，说明rhTNFR：Fc通过抑制TNF-α介导NF-κB信号转导阻断细胞因子过度表达，调节失控性炎症反应，对脓毒症心肌损伤起保护作用。
[Abstract]:Objective: in this study, the rat model of sepsis caused by cecal perforation and ligation was used to observe the myocardial damage in septic rats and to explore the protective effect of the early intervention of human tumor necrosis factor receptor fusion protein (rhTNFR:Fc) on the myocardial damage of sepsis, and the mechanism of its action was preliminarily studied.
Methods: 108 clean SD rats, all female, were randomly divided into 3 groups: (1) sham operation group (group A, n=36), sepsis group (group B, n=36), sepsis plus rhTNFR:Fc group (group C, n=36), and (2) group A, after weighing, anesthesia, and after cecum exploration, the abdominal aorta was closed after the operation of 3H, 9h, 24h, six, and six. The level of tumor necrosis factor (TNF- a), interleukin -1 (IL-1), cardiac troponin I (cTnI), cardiac tissue and NF- kappa B P65 protein were detected by enzyme linked immunosorbent assay (Western blot), and 6 rats (3), B group were weighed at each moment, anesthesia, and post cecal ligation and perforation. Closed abdominal cavity, the same operation steps as 3h, 9h, 24h, 36h, 48h and 72h six after the operation were the same as that of the sham operation group. The level of tumor necrosis factor (TNF- alpha), interleukin -1 (IL-1) and cardiac troponin I (cTnI) were detected by enzyme immunoassay, and the nuclear factor kappa B (nuclear factor kappa) protein was detected at each phase point. 6 rats were taken, and (4) after the C group was treated with cecal ligation and perforation (CLP), the Yasip (rhTNFR:Fc) aqueous solution 2mL / kg (1g / L) was injected immediately from the external jugular vein. The same operation steps as 3h, 9h, 24h, 36h, 48h and six were performed respectively after the operation, and the tumor necrosis factor (alpha) and leukocyte were detected by enzyme immunoassay. -1 (IL-1), cardiac troponin I (cTnI) level, cardiac tissue and NF- kappa B P65 protein were detected. 6 rats were taken at each phase point. (5) all data were expressed as "mean standard deviation", and SPSS13.0 statistical package was used for analysis. Correlation analysis used Spearman grade correlation analysis.
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