中国图书资料分类号R576

摘要

目的观察静脉内滴注谷氨酰胺(Gln)对急性重症胰腺炎(ASP)后肠源性细菌/内毒素易位的保护作用.

方法选健康长白种猪27头，体重17kg～22kg，雌雄不限，随机分为5组. 组Ⅰ：假手术对照组(n=5)；组Ⅱ：ASP对照组(n=5)；组Ⅲ：ASP+甘氨酸(Gly)组(n=5)；组Ⅳ：ASP+Gln组(n=6)；组Ⅴ：ASP+Gln+盲肠造口、结肠灌洗组(n=6). 在麻醉状态下，进腹向胰总管内注入1mL/kg 50g/L牛磺胆酸钠混合液〔内含胰蛋白酶(8～10)×10⁶ BAEEU/L，pH 7.6〕诱导ASP. 以9g/L NaCl磷酸盐缓冲液取代50g/L牛磺胆酸钠混合液即为假手术对照组. 采集腔静脉血作内毒素测定(鲎试剂法). ASP后72h，采集门、腔静脉血作系列细菌定量培养和细菌鉴定. 然后推注100g/L氯化钾20mL处死后立即进胸腹取大小肠系膜淋巴结、肺组织、肺门淋巴结、胰腺组织，称重后研磨成组织匀浆，并作细菌定量培养和细菌鉴定.

结果静脉内直接输注Gln后可使血浆中Gln浓度较组Ⅰ、组Ⅱ明显升高(P均＜0.01). 静脉内Gln支持或Gln+盲肠造口、结肠灌洗均可明显降低血浆内毒素水平，且使血液和组织器官中易位的细菌数量均出现非常显著的下降.

结论静脉内Gln支持可显著地升高血浆Gln水平. 同时可显著地减轻ASP后肠源性细菌/内毒素易位.

Influence of glutamine and

caecostomy/colonic irrigation on

gut bacteria/endotoxin translocation

in acute severe pancreatitis in pigs

TU Wei-Feng^1,2， LI Jie-Sou¹, ZHU Wei-Ming¹, LI Zheng-Da¹, LIU Fang-Nan¹, CHEN Yong-Ming¹, XU Jian-Guo¹, SHAO Hai-Feng¹, XIAO Guang-Xia² and LI Ao²

Subject headingspancreatitis; glutamine; endotoxins; bacterial translocation

Abstract

AIMTo observe the protection of intravenous glutamine (Gln) on gut-originated bacteria/endotoxin translocation following acute severe pancreatitis (ASP) induced by sodium taurocholate.

METHODSTwenty seven pigs weighing 17kg-22kg, were divided into five groups. Group Ⅰ (n=5): sham-control; Group Ⅱ (n=5): ASP-control; Group Ⅲ (n=5): ASP+glycine (Gly); Group Ⅳ (n=6): ASP+Gln; Group Ⅴ (n=6): ASP+Gln+caecostomy/colonic irrigation. Anesthesized pigs were infected with 1mL/kg of combined solution of 50g/L sodium taurocholate and (8-10)×10⁶ BAEE units trypsin/L by injection via pancreatic duct. Plasma glutamine were measured by the high performance liquid-phase technique (the product of Waters, U.S.). Systemic plasma endotoxin levels was quantitated by the chromogenic limulus amebocyte lysate (LAL) technique. Both portal and systemic blood samples were obtained before and 72hours following ASP and cultured for aerobic as well as anaerobic bacterial growth. Subsequently, All pigs were sacrificed by injecting 20mL of 100g/L KCl intravenously. Tissue specimens from mesenteriolum and mesocolon lymph nodes, lung, pulmonary portal lymph nodes and pancreas were removed, weighed and homogenized in grinding tubes. Aliquots of the homogenate were cultured as mentioned above. Positive specimens were subcultured and the bacteria identified by standard procedure.

RESULTSIntravenous glutamine could effectively prevent the decrease of plasma glutamine levels following acute severe pancreatitis. Both intravenous glutamine and intravenous glutamine + caecostomy/colonic irrigation reduced significantly the levels of plasma endotoxin and the magnitude of bacteria translocation to the portal and systemic blood and remote organs.

CONCLUSIONIntravenous glutamine support can enhance plasma glutamine concentrations. Both intravenous glutamine and intravenous glutamine + caecostomy/colonic irrigation can effectively decrease gut-originated bacteria/endotoxin translocation.

0引言

肠道细菌和内毒素易位是导致急性重症胰腺炎(ASP)并发感染和脓毒症致死亡率居高不下的重要原因^［1-3］. 肠道内外给予谷氨酰胺(Gln)具有增加肠粘膜厚度和蛋白质、增强粘膜屏障功能、降低肠道内细菌/内毒素易位的作用，从而明显降低和减轻大面积烧伤、重症胰腺炎、重症监护患者继发感染的发生率和严重程度^［4-7］. 但由于Gln极不稳定，遇热易降解并释放聚谷氨酸等毒性物质，难以常规配制在TPN制剂中供临床使用. 我们使用日本进口的高纯度(99.0%) Gln粉制备成粉剂供猪试用，以观察Gln对ASP后肠源性细菌/内毒素易位的保护作用.

1材料和方法

1.1动物分组①健康长白种猪27头(由南京军区动物实验中心提供)，体重17kg～22kg，雌雄不限，随机分为5组：组Ⅰ：假手术对照组(n=5)，组Ⅱ：胰腺炎组(n=5)；组Ⅲ：胰腺炎+甘氨酸(Gly)组(n=5)，组Ⅳ：胰腺炎+Gln组(n=6)，组Ⅴ：胰腺炎+Gln+盲肠造口/结肠灌洗组(n=6). ②Gln粉剂的制备、鉴定和投给方法：日本进口的高纯度(99.0%)Gln粉在无菌条件下分装成0.5g,1g,2g,2.5g，经⁶⁰Co辐照后备用. 随机取5份⁶⁰Co辐照后的Gln经系列真菌、细菌培养和致热原试验证实均为阴性，可供静脉内使用(此项工作由我院药剂制剂科和微生物科协助完成). Gln用量按总氮量0.56g·kg^-1.d^-1的25%提供，即0.74g·kg^-1.d^-1. Gln或Gly在成功诱导ASP后6h开始使用. Gln在临用前15min以9g/L NaCl溶解并配成20g/L浓度，经上腔静脉均匀输入(约需1h左右，1次/8h). 以等氮量Gly作为Gln对照组.

1.2方法①急性重症胰腺炎的诱导：无菌条件下，正中腹直肌切口进腹，暴露胰十二指肠，在胰管开口对侧切一纵型小切口(约1.5cm～ 2.5cm). 仔细暴露胰管开口处，并放置直径为1mm的聚氯乙烯导管，深约3cm～5cm，并暂时结扎胰管，逆行在4kPa压力下缓慢地注入50g/L牛磺胆酸钠混合液〔内含胰蛋白酶(8～10)×10⁶ BAEE U/L，pH 7.6,牛磺胆酸钠和胰蛋白酶均为Sigma产品〕. 10min后拔除胰管内导管. 以9g/L缓冲生理盐水(pH 7.6)取代50g/L牛磺胆酸钠混合液即为对照组. 然后，依次缝合胰十二指肠和腹壁. 液体在手术期间及手术后8h以内按250mL/kg输入，以后按(125±25)mL/kg，交替使用100g/L葡萄糖注射液和复方乳酸林格氏液. ②Gln+盲肠造口/结肠灌洗组：先在诱导ASP前，取右下腹斜切口，进腹后取出盲肠，周围用盐水纱布保护，用1号线在盲肠前结肠带处做两个同心荷包缝合，彼此相距1cm，在荷包缝合中央切一小口，并插入一根蕈状导管，结扎第1荷包缝线，剪去线尾，作第2荷包缝合时，使盲肠壁内翻. 再将线尾穿过腹膜后打结，使盲壁固定于腹膜上，造口管从腹壁切口引出，逐层缝合腹壁切口，并将导管固定于皮肤上. 在诱导ASP后6h静脉内输注Gln同时，开始进行结肠灌洗，逆向自盲肠造口导管灌入(重力法)生理盐水2000mL，1次/2h，直至结肠流出液澄清无残渣后，改为1次/8h，每次500mL～1000mL，以洗去自小肠流入结肠的肠内容物，来确保结肠灌洗的效果. ③观察指标和方法：每日注意猪的全身情况，包括血压、脉搏、呼吸、体温、动静脉血气分析、白细胞计数，以及厌食、嗜睡或烦燥和留置导尿管记24h尿量等. 分别于ASP诱导前30min(T₀)，ASP诱导后、6h(T₁)，24h(T₂)，48h(T₃)，72h(T₄)采集腔静脉血样本以测定血浆内毒素(采用上海医化所内毒素定量测定试剂盒)和Gln水平(采用美国Waters公司生产的高效液相色谱仪测定). 72h时点同时采集门静脉血作细菌定量培养，并按常规作细菌学鉴定. ASP后72h，存活的猪用100g/L KCl处死(0.1～0.2)g/kg，在无菌条件下剖胸腹取肺组织、肺门淋巴结、大小肠系膜淋巴结、胰腺组织作细菌学定量培养和常规细菌学鉴定. 在上述采样过程中所涉及到的任何器具均经去热原处理和高压灭菌消毒.

统计学处理所列计量资料均采用均数±标准差表示，应用统计软件包stat 5.0作方差分析、Newman-Keuls'检验和t检验.

2结果

2.1血浆Gln组Ⅱ和组Ⅲ血浆Gln在诱导ASP后均出现较明显的下降，ASP后24，48和72h时点血浆Gln与正常对照组比较均有非常显著的统计学差别(P均＜0.01). 静脉内Gln支持后，在ASP后24，48，72h个时点血浆Gln均较组Ⅱ和组Ⅲ有非常显著的上升(表1和图1)，但组Ⅳ和组Ⅴ两组间无明显统计学差别.

2.2血浆内毒素的变化组Ⅲ血浆内<