“二次打击”脓毒症继发肺炎大鼠模型的构建与评价  

Reproduction of a model of ＂two-hit＂ sepsis model with complication of pneumonia in rat

作　　者：陈颖颖[1] 李慧贤[2] 马帅[2] 邓博[2] 卢建新[2] 丁峰[2] 

机构地区：[1]厦门大学附属中山医院肾内科 [2]上海交通大学医学院附属第九人民医院肾脏科,200011

出　　处：《中华危重病急救医学》2015年第10期805-810,共6页Chinese Critical Care Medicine

基　　金：国家自然科学基金（81270850,81470990）;国家科技支撑项目（2011BAI10B08）;上海市科委重点项目（11441901400）

摘　　要：目的：模拟临床脓毒症继发感染的发病情况，构建“二次打击”动物模型，评价“二次打击”对机体损害及免疫功能的影响。方法81只雄性SD大鼠按随机数字表法分组，其中45只分为单纯脓毒症组及脓毒症继发肺炎4 d、7 d组，每组15只，用于观察生存情况；另外36只分为正常对照组，单纯脓毒症1、4、7 d组，脓毒症继发肺炎4 d、7 d组，每组6只，用于指标检测。采用盲肠结扎穿孔术（CLP）建立脓毒症模型；于CLP术后4 d或7 d分别经鼻腔注射肺炎链球菌（含菌量1×1010 cfu/mL）继发肺部感染构建“二次打击”模型。分别于相应时间点以及给菌后1 d处死大鼠，收集血液及脾脏组织，进行外周血菌落计数、脾脏细胞计数，检测血清生化指标及相关细胞因子水平，观察脾脏病理改变及细胞凋亡情况。结果①与单纯脓毒症组同期比较，脓毒症继发肺炎4 d组大鼠存活数明显减少（只：4比11，χ2＝6.533，P＝0.011），而7 d组则差异无统计学意义（只：9比11，χ2＝0.600，P＝0.439）。②单纯脓毒症组术后1 d外周血菌落计数、生化指标均明显升高，随后逐渐下降；术后4 d时脾脏免疫细胞计数已明显低于1 d〔树突细胞（DC）：（0.69±0.09）%比（0.87±0.31）%， CD4＋ T细胞：（21.05±2.89）%比（24.84±4.59）%，CD8＋ T细胞：（10.62±1.79）%比（13.40±1.31）%，均P＜0.05〕，而调节性T细胞（Treg）明显高于正常对照组〔（3.14±0.74）%比（2.87±1.08）%，P＜0.05〕；术后7 d血生化指标丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）、尿素氮（BUN）、血肌酐（SCr）较1 d明显下降〔ALT（U/L）：35.33±11.52比81.00±38.40，AST（U/L）：70.33±42.16比156.00±28.11，BUN（mmol/L）：5.30±2.27比9.13±4.04，SCr（μmol/L）：55.33±10.67比96.67±45.79，均P＜0.05〕；血清促炎因子肿瘤坏死因子-α（TNF-α）、白细胞介素（IL-6、IL-1β）ObjectiveTo reproduce a clinically relevant ＂two-hit＂ model of sepsis complicated by pneumonia and to explore the correlation between ＂two-hit＂ and immune state.Methods Eighty-one male Sprague-Dawley （ SD ） rats were divided into groups according to the random number table. Forty-five male rats were assigned respectively to sepsis-alone group, pneumonia 4 days and 7 days after sepsis groups, respectively. Survival rate of each group was observed. Another group of 36 male rats were divided into normal control group, sepsis-alone for 1, 4 and 7 days groups, and sepsis complicated by pneumonia for 4 days and 7 days after sepsis groups, each group consisted of 6 rats. Cecal ligation and puncture （CLP） was done in rats, andStreptococcus pneumoniae suspension （bacteria count 1×1010 cfu/mL） was injected via the nose on the 4th day or 7th day after CLP. Rats were sacrificed at corresponding time points, and 1 day after challenge ofStreptococcus pneumoniae on the 4 days or 7 days post CLP for the collection of blood and tissue samples to make bacterial count of the blood, splenocyte count, biochemical indices, cytokines concentration, pathological changes in spleen and apoptotic cells.Results① Compared with the rats of sepsis-alone group, the rats in pneumonia 4 days after CLP group had poor survival rate （4 vs. 11,χ2 = 6.533,P = 0.011）, while no difference was found between pneumonia 7 days after CLP group and sepsis-alone group （9 vs. 11,χ2 = 0.600,P = 0.439）.② The blood bacterial count and all the biochemical indexes were sharply increased on 1 day post-CLP in the rats of sepsis-alone group, and then they gradually lowered. Compared with the rats of 1 day post-CLP, the proportion of splenocytes were decreased on the 4th day post-CLP [dendritic cells （DC）： （0.69±0.09）% vs. （0.87±0.31）%, CD4＋T cells： （21.05±2.89）% vs. （24.84±4.59）%, CD8＋ T cells： （10.62±1.79）% vs. （13.40±1.31）%, allP〈 0.05], but T-regulatory cell （Treg） count was higher

关 键 词：脓毒症 动物模型 “二次打击” 免疫麻痹 "Two-hit" 

分 类 号：R459.7[医药卫生—急诊医学] R-332[医药卫生—治疗学]