中华危重病急救医学
中華危重病急救醫學
중화위중병급구의학
Chinese Critical Care Medicine
2015年
10期
805-810
,共6页
陈颖颖%李慧贤%马帅%邓博%卢建新%丁峰
陳穎穎%李慧賢%馬帥%鄧博%盧建新%丁峰
진영영%리혜현%마수%산박%로건신%정봉
脓毒症%动物模型%“二次打击”%免疫麻痹
膿毒癥%動物模型%“二次打擊”%免疫痳痺
농독증%동물모형%“이차타격”%면역마비
Sepsis%Animal model%"Two-hit"%Immunoparalysis
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）高峰出现在术后1 d〔TNF-α为（18.03±2.88）ng/L，IL-6为（10.37±4.20）ng/L，IL-1β为（102.44±51.46）ng/L〕，高迁移率族蛋白B1（HMGB1）高峰出现在术后4 d〔（1.76±0.71）μg/L〕；抗炎因子转化生长因子-β1（TGF-β1）和可溶性肿瘤坏死因子受体-Ⅰ（sTNFR-Ⅰ）于脓毒症后期仍维持在较高水平〔术后7 d TGF-β1为（0.90±0.56）ng/L，sTNFR-Ⅰ为（1.56±0.39） ng/L〕。苏木素-伊红（HE）染色显示，单纯脓毒症组CLP术后脾脏组织病理改变随时间延长逐渐加重，术后4 d脾脏凋亡细胞数明显高于术后1 d（个/HP：52.99±20.79比16.05±3.28，P＜0.05）。③与单纯脓毒症组同期比较，脓毒症继发肺炎4 d组外周血菌落数明显增多（log cfu/mL：1.78±0.54比0.25±0.18，P＜0.05），而7 d组则差异无统计学意义（log cfu/mL：0.57±0.46比0.13±0.12，P＞0.05）。脓毒症继发肺炎4 d、7 d组各脾脏免疫细胞计数及生化指标变化趋势与单纯脓毒症组相似，虽较单纯脓毒症组同期有所减少，但差异均无统计学意义。与单纯脓毒症组同期比较，脓毒症继发肺炎4 d组HMGB1进一步减少（μg/L：1.17±0.74比1.76±0.71，P＜0.05），脓毒症继发肺炎7 d组IL-1β进一步升高（ng/L：105.73±25.06比61.04±31.29，P＜0.05），其余炎症因子水平则差异无统计学意义。脓毒症继发肺炎4 d组脾脏凋亡细胞较单纯脓毒症组同期明显增加（个/HP：74.48±22.47比52.99±20.79，P＜0.05），而7 d组则差异无统计学意义（个/HP：28.70±4.13比30.43±14.55， P＞0.05）。结论 CLP术后4 d继发肺炎“二次打击”模型较单纯CLP致脓毒症模型死亡大鼠明显增加，细菌清除力下降，免疫细胞数量减少且凋亡加重，这可能与病程中期机体存在的免疫麻痹有关。CLP术后7 d继发肺炎“二次打击”模型表现为宿主免疫功能重构，可能与免疫应答能力逐渐恢复有关。
目的：模擬臨床膿毒癥繼髮感染的髮病情況，構建“二次打擊”動物模型，評價“二次打擊”對機體損害及免疫功能的影響。方法81隻雄性SD大鼠按隨機數字錶法分組，其中45隻分為單純膿毒癥組及膿毒癥繼髮肺炎4 d、7 d組，每組15隻，用于觀察生存情況；另外36隻分為正常對照組，單純膿毒癥1、4、7 d組，膿毒癥繼髮肺炎4 d、7 d組，每組6隻，用于指標檢測。採用盲腸結扎穿孔術（CLP）建立膿毒癥模型；于CLP術後4 d或7 d分彆經鼻腔註射肺炎鏈毬菌（含菌量1×1010 cfu/mL）繼髮肺部感染構建“二次打擊”模型。分彆于相應時間點以及給菌後1 d處死大鼠，收集血液及脾髒組織，進行外週血菌落計數、脾髒細胞計數，檢測血清生化指標及相關細胞因子水平，觀察脾髒病理改變及細胞凋亡情況。結果①與單純膿毒癥組同期比較，膿毒癥繼髮肺炎4 d組大鼠存活數明顯減少（隻：4比11，χ2＝6.533，P＝0.011），而7 d組則差異無統計學意義（隻：9比11，χ2＝0.600，P＝0.439）。②單純膿毒癥組術後1 d外週血菌落計數、生化指標均明顯升高，隨後逐漸下降；術後4 d時脾髒免疫細胞計數已明顯低于1 d〔樹突細胞（DC）：（0.69±0.09）%比（0.87±0.31）%， CD4+ T細胞：（21.05±2.89）%比（24.84±4.59）%，CD8+ T細胞：（10.62±1.79）%比（13.40±1.31）%，均P＜0.05〕，而調節性T細胞（Treg）明顯高于正常對照組〔（3.14±0.74）%比（2.87±1.08）%，P＜0.05〕；術後7 d血生化指標丙氨痠轉氨酶（ALT）、天鼕氨痠轉氨酶（AST）、尿素氮（BUN）、血肌酐（SCr）較1 d明顯下降〔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〕；血清促炎因子腫瘤壞死因子-α（TNF-α）、白細胞介素（IL-6、IL-1β）高峰齣現在術後1 d〔TNF-α為（18.03±2.88）ng/L，IL-6為（10.37±4.20）ng/L，IL-1β為（102.44±51.46）ng/L〕，高遷移率族蛋白B1（HMGB1）高峰齣現在術後4 d〔（1.76±0.71）μg/L〕；抗炎因子轉化生長因子-β1（TGF-β1）和可溶性腫瘤壞死因子受體-Ⅰ（sTNFR-Ⅰ）于膿毒癥後期仍維持在較高水平〔術後7 d TGF-β1為（0.90±0.56）ng/L，sTNFR-Ⅰ為（1.56±0.39） ng/L〕。囌木素-伊紅（HE）染色顯示，單純膿毒癥組CLP術後脾髒組織病理改變隨時間延長逐漸加重，術後4 d脾髒凋亡細胞數明顯高于術後1 d（箇/HP：52.99±20.79比16.05±3.28，P＜0.05）。③與單純膿毒癥組同期比較，膿毒癥繼髮肺炎4 d組外週血菌落數明顯增多（log cfu/mL：1.78±0.54比0.25±0.18，P＜0.05），而7 d組則差異無統計學意義（log cfu/mL：0.57±0.46比0.13±0.12，P＞0.05）。膿毒癥繼髮肺炎4 d、7 d組各脾髒免疫細胞計數及生化指標變化趨勢與單純膿毒癥組相似，雖較單純膿毒癥組同期有所減少，但差異均無統計學意義。與單純膿毒癥組同期比較，膿毒癥繼髮肺炎4 d組HMGB1進一步減少（μg/L：1.17±0.74比1.76±0.71，P＜0.05），膿毒癥繼髮肺炎7 d組IL-1β進一步升高（ng/L：105.73±25.06比61.04±31.29，P＜0.05），其餘炎癥因子水平則差異無統計學意義。膿毒癥繼髮肺炎4 d組脾髒凋亡細胞較單純膿毒癥組同期明顯增加（箇/HP：74.48±22.47比52.99±20.79，P＜0.05），而7 d組則差異無統計學意義（箇/HP：28.70±4.13比30.43±14.55， P＞0.05）。結論 CLP術後4 d繼髮肺炎“二次打擊”模型較單純CLP緻膿毒癥模型死亡大鼠明顯增加，細菌清除力下降，免疫細胞數量減少且凋亡加重，這可能與病程中期機體存在的免疫痳痺有關。CLP術後7 d繼髮肺炎“二次打擊”模型錶現為宿主免疫功能重構，可能與免疫應答能力逐漸恢複有關。
목적：모의림상농독증계발감염적발병정황，구건“이차타격”동물모형，평개“이차타격”대궤체손해급면역공능적영향。방법81지웅성SD대서안수궤수자표법분조，기중45지분위단순농독증조급농독증계발폐염4 d、7 d조，매조15지，용우관찰생존정황；령외36지분위정상대조조，단순농독증1、4、7 d조，농독증계발폐염4 d、7 d조，매조6지，용우지표검측。채용맹장결찰천공술（CLP）건립농독증모형；우CLP술후4 d혹7 d분별경비강주사폐염련구균（함균량1×1010 cfu/mL）계발폐부감염구건“이차타격”모형。분별우상응시간점이급급균후1 d처사대서，수집혈액급비장조직，진행외주혈균락계수、비장세포계수，검측혈청생화지표급상관세포인자수평，관찰비장병리개변급세포조망정황。결과①여단순농독증조동기비교，농독증계발폐염4 d조대서존활수명현감소（지：4비11，χ2＝6.533，P＝0.011），이7 d조칙차이무통계학의의（지：9비11，χ2＝0.600，P＝0.439）。②단순농독증조술후1 d외주혈균락계수、생화지표균명현승고，수후축점하강；술후4 d시비장면역세포계수이명현저우1 d〔수돌세포（DC）：（0.69±0.09）%비（0.87±0.31）%， CD4+ T세포：（21.05±2.89）%비（24.84±4.59）%，CD8+ T세포：（10.62±1.79）%비（13.40±1.31）%，균P＜0.05〕，이조절성T세포（Treg）명현고우정상대조조〔（3.14±0.74）%비（2.87±1.08）%，P＜0.05〕；술후7 d혈생화지표병안산전안매（ALT）、천동안산전안매（AST）、뇨소담（BUN）、혈기항（SCr）교1 d명현하강〔ALT（U/L）：35.33±11.52비81.00±38.40，AST（U/L）：70.33±42.16비156.00±28.11，BUN（mmol/L）：5.30±2.27비9.13±4.04，SCr（μmol/L）：55.33±10.67비96.67±45.79，균P＜0.05〕；혈청촉염인자종류배사인자-α（TNF-α）、백세포개소（IL-6、IL-1β）고봉출현재술후1 d〔TNF-α위（18.03±2.88）ng/L，IL-6위（10.37±4.20）ng/L，IL-1β위（102.44±51.46）ng/L〕，고천이솔족단백B1（HMGB1）고봉출현재술후4 d〔（1.76±0.71）μg/L〕；항염인자전화생장인자-β1（TGF-β1）화가용성종류배사인자수체-Ⅰ（sTNFR-Ⅰ）우농독증후기잉유지재교고수평〔술후7 d TGF-β1위（0.90±0.56）ng/L，sTNFR-Ⅰ위（1.56±0.39） ng/L〕。소목소-이홍（HE）염색현시，단순농독증조CLP술후비장조직병리개변수시간연장축점가중，술후4 d비장조망세포수명현고우술후1 d（개/HP：52.99±20.79비16.05±3.28，P＜0.05）。③여단순농독증조동기비교，농독증계발폐염4 d조외주혈균락수명현증다（log cfu/mL：1.78±0.54비0.25±0.18，P＜0.05），이7 d조칙차이무통계학의의（log cfu/mL：0.57±0.46비0.13±0.12，P＞0.05）。농독증계발폐염4 d、7 d조각비장면역세포계수급생화지표변화추세여단순농독증조상사，수교단순농독증조동기유소감소，단차이균무통계학의의。여단순농독증조동기비교，농독증계발폐염4 d조HMGB1진일보감소（μg/L：1.17±0.74비1.76±0.71，P＜0.05），농독증계발폐염7 d조IL-1β진일보승고（ng/L：105.73±25.06비61.04±31.29，P＜0.05），기여염증인자수평칙차이무통계학의의。농독증계발폐염4 d조비장조망세포교단순농독증조동기명현증가（개/HP：74.48±22.47비52.99±20.79，P＜0.05），이7 d조칙차이무통계학의의（개/HP：28.70±4.13비30.43±14.55， P＞0.05）。결론 CLP술후4 d계발폐염“이차타격”모형교단순CLP치농독증모형사망대서명현증가，세균청제력하강，면역세포수량감소차조망가중，저가능여병정중기궤체존재적면역마비유관。CLP술후7 d계발폐염“이차타격”모형표현위숙주면역공능중구，가능여면역응답능력축점회복유관。
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 on the 4th day after CLP compared with sepsis-alone rats [(3.14±0.74 )% vs. (2.87±1.08)%,P< 0.05]. The biochemical indices, including alanine transaminase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and serum creatinine (SCr) were obviously lowered on 7 days post-CLP compared with 1 day after CLP [ALT (U/L): 35.33±11.52 vs. 81.00±38.40, AST (U/L): 70.33±42.16 vs. 156.00±28.11, BUN (mmol/L): 5.30±2.27 vs. 9.13±4.04, SCr (μmol/L): 55.33±10.67 vs. 96.67±45.79, allP< 0.05]. The serum levels of tumor necrosis factor-α (TNF-α) and interleukins (IL-6, IL-1β) peaked on the 1st day after CLP [TNF-α:(18.03±2.88) ng/L, IL-6: (10.37±4.20) ng/L, IL-1β: (102.44±51.46) ng/L], and high mobility group box-1 (HMGB1) peaked on the 4th day after CLP [(1.76±0.71)μg/L]. The levels of anti-inflammatory cytokines transforming growth factor-β1 (TGF-β1 ) and soluble tumor necrosis factor receptor-Ⅰ (sTNFR-Ⅰ) maintained at high levels [7 days post-CLP: TGF-β1 was (0.90±0.56) ng/L, sTNFR-Ⅰ was (1.56±0.39) ng/L]. The spleen pathology became more marked with the time in the group of sepsis-alone, meanwhile the number of apoptotic spleencytes increased 4 days post-CLP as compared with that of the 1st day post-CLP (cells/HP: 52.99±20.79 vs. 16.05±3.28,P< 0.05).③ Compared with the same period of sepsis-alone group, the rats with pneumonia 4 days post-CLP group showed a higher blood bacterial count (log cfu/mL: 1.78±0.54 vs. 0.25±0.18,P< 0.05), while no difference was found between 7-day of post-CLP pneumonia group and sepsis-alone group (log cfu/mL: 0.57±0.46 vs. 0.13±0.12,P> 0.05). The same trend of changes, with slight reduction in splenocytes and biochemical indices were found between the groups of sepsis followed by pneumonia and sepsis-alone, but no significant difference was found. The level of HMGB1 in the 4-day group of sepsis with complication of pneumonia was further decreased compared with sepsis-alone group (μg/L:1.17±0.74 vs. 1.76±0.71,P< 0.05), and IL-1β in the 7-day group of sepsis complicated pneumonia was further higher than those of sepsis-alone group in the same period (ng/L: 105.73±25.06 vs. 61.04±31.29,P< 0.05), while there were no differences in levels of other cytokines between "two-hit" group and sepsis-alone group. Apoptosis of spleencytes in the 4-day group of sepsis complicated pneumonia was more marked than that of sepsis-alone group at the same period (cells/HP: 74.48±22.47 vs. 52.99±20.79,P< 0.05), while no difference was found between the 7-day groups of sepsis complicated pneumonia and the sepsis-alone group (cells/HP: 28.70±4.13 vs. 30.43±14.55, P> 0.05).Conclusions The mortality of this "two-hit" model with complication of pneumonia 4 days after CLP was significantly higher than that of single sepsis model. The ability of bacteria clearance was decreased, and immunocyte apoptosis was exacerbated. These findings may be with the result of the occurrence of immunoparalysis in the mid stage of sepsis. The "two-hit" model reproduced on 7 days after CLP might suggest reconstruction of host immune function, and maybe associated with the recovery of immune response.
