大肠杆菌亚型致脓毒症模型比较研究

目的 探讨不同种类大肠杆菌诱导的脓毒症模型损伤程度。方法 将152只小鼠随机数字表法分为对照组、DH5α 组、44102 组和 25922 组，每组 38 只。DH5α 组、44102 组和 25922 组分别腹腔注射 300 μL 浓度为 1.0×109CFU/kg的大肠杆菌DH5α、44102和25922，以不同种类大肠杆菌诱导脓毒症模型，对照组腹腔注射等体积生理盐水。（1）造模8 h后，每组各取4只小鼠，进行外周血细菌培养，计算细菌菌落数。（2）造模12 h后，通过酶联免疫吸附试验（ELISA）的方法检测血清中肿瘤坏死因子（TNF）-α、白介素（IL）-6水平（每组10只）；采用蛋白免疫印迹法（Western blot）测定血清中晚期炎症因子高迁移率族蛋白1（HMGB1）水平（每组4只）；使用自动生化分析仪检测血清中丙氨酸 转氨酶（ALT）、天冬氨酸转氨酶（AST）、尿素氮（BUN）、血肌酐（CR）水平（每组10只）；取肺肝肾组织经甲醛固定后，进行苏木精-伊红（HE）染色（每组10只）。结果 DH5α组、44102组、25922组细菌菌落形成数量，炎症因子TNF-α、IL-6以及HMGB1蛋白，肝肾功能指标ALT、AST、CR、BUN均呈依次增高趋势（P＜0.01）。DH5α组、44102组、25922组的肺泡结构损伤程度、肝炎性细胞浸润程度及肾小球萎缩程度由重到轻依次为25922组、44102组、DH5α组，对照组肺、肝、肾组织无明显损伤。结论 大肠杆菌25922诱导脓毒症模型损伤严重，可用于研究脓毒症初期炎症风暴阶段的动物模型；大肠杆菌44102诱导脓毒症模型损伤适中，可用于无需限定脓毒症分期实验的动物模型；大肠杆菌 DH5α诱导脓毒症模型损伤较轻，可用于探索脓毒症免疫抑制期治疗方法的动物模型。

Comparative study on sepsis models induced by Escherichia coli subtypes

Objective To investigate the degrees of injury severity of sepsis models made by different kinds of Escherichia coli. Methods The 152 mice were randomly divided into control group, DH5α group, 44102 group, and 25922 group, with 38 rats in each group. DH5α group, 44102 group and 25922 group were intraperitoneally injected with 300 μL of Escherichia coli DH5α, 44102 and 25922 at the concentration of 1.0 × 109 CFU/kg to prepare sepsis models of different kinds of Escherichia coli. Control group was injected intraperitoneally with the same amount of normal saline. (1) After 8 h,four mice were taken from each group for peripheral blood bacterial culture . (2) After 12 h, ten mice in each group were used for measuring serum levels of TNF-α and IL-6 by enzyme-linked immunosorbent assay (ELISA). (3) Western blot assay was used to determine the serum levels of high-mobility group protein (HMGB1) in four mice of each group. (4) Ten mice in each group were used to measure serum levels of alanine transaminase (ALT), aspartate aminotransferase (AST), creatinine (CR) and blood urea nitrogen (BUN) by automatic biochemical analyzer. (5) After liver, lung and kidney tissues were fixed with formaldehyde, hematoxylin-eosin (HE) staining was performed (n=10 for each group). Results In DH5α group, 44102 group and 25922 group, bacteria, inflammatory cytokines TNF- α, IL-6 and HMGB1 protein, liver and kidney indicators ALT, AST, CR and BUN showed a sequential increasing trend (P＜0.01). The severe degrees of alveolar structure damage,hepatic cell infiltration and renal glomerular atrophy were DH5α group, 44102 group and 25922 group in turn. There were no obvious damages of lung, liver or kidney tissues in control group. Conclusion Escherichia coli 25922 induces severe sepsis injury and can be used to study the animal models of the initial inflammatory phase of sepsis. Escherichia coli 44102 induces moderate damage of sepsis and can be used in animal models that do not require definitive sepsis staging experiments. Escherichia coli DH5α induces less damage of sepsis and can be used to explore immunosuppressive therapy of the animal model of sepsis.