内毒素预处理对内毒素血症的作用及其机制探讨
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摘要
前言
感染性休克(septic shock)是指严重脓毒症病人在给予足量液体复苏仍无法纠正的持续性低血压，常伴有低灌流状态及器官功能障碍，是病毒、真菌和细菌脱落的蛋白碎片或免疫原作用于机体诱发多种细胞因子如TNF-α、IL-1、IL-6、IL-8、血小板活化因子(PAF)等以及炎症介质如前列环素、缓激肽、白三烯等释放所导致的全身性过度炎症反应产生破坏作用的结果。革兰氏阴性菌胞壁成分内毒素是引起感染性休克的最常见原因之一。
目前感染性休克的治疗远未达到满意的疗效，死亡率仍在50％左右。现在的防治原则是早期清除原发病灶；使用有效的抗生素控制感染；积极的多系统器官的综合支持疗法。除此之外，各种抗炎症介质、细胞因子的抗体，如LPS、TNF-α、IL-1的抗体以及NOS抑制剂等，在动物实验中似乎都有一定的作用。然而，临床试验不能证明任何一项临床有效。其中三项包括非选择性NOS抑制剂、TNF及抗内毒素单克隆抗体治疗严重感染性休克试验均因受试者病死率增加而中途停止。这些方法主要是从外源性角度来进行治疗，但总的效果并不令人满意。因此在目前感染性休克治疗无突破的情况下，能否利用刺激或激发机体细胞在漫长的进化过程中获得的内源性抗损伤能力来减轻致病因素对机体的损伤程度，在一些可预防性脓毒症高危病人开展有效预防措施，是降低脓毒症及感染性休克病人死亡率的另一思路。研究证明热休克反应可提高鼠对随后感染性休克的耐受力，但热休克反应实施起来有一定困难，而药物预处理具有方便、易行的特点。LPS耐受现象的存在，尤其是其减毒衍生物预处理为这种设想提供了可能。
迄今为止，虽然对内毒素耐受作了大量的实验研究，但大都着重于以离体的单核-巨噬系统细胞和内皮细胞为观察对象，缺乏在动物整体的、器官实质细胞的系统研究。因此，我们设计了以下的系列研究，从整体、器官、组
织以及好水平剧内毒素预处理对内毒素血症的作用及其机制，为进一
步研究内毒素耐受、内毒素减毒衍生物预处理的作用及应用提供实验基础。
实验材料
1．实验动物：昆明种小鼠120只，20－229，雌雄不拘；雄性Wistar大鼠
102 ／q，200－2809，由中国医科大学实验动物中心提供。
2．主要实验试剂与药品：内毒素（Escherichia colt LPS Oill：B4，美国
Sima公司八戊巴比妥（消化学试剂采购供应站八 TNF叱 ELISA试剂盒
（购自深圳晶美生物工程公司，进口分装八个羟基香豆素（美国 Siaa公
司hTUNEL检测试剂盒（购自武汉博士德公司）中主要包括：末端脱氧核
糖核酸转移酶（Termina de…ucleoti邮Trans咖se，TdT，美国Promega 公
司太地高辛标记的规Th（DIG－dUTP，德国 B．M．公司人生物素标记抗地
高辛抗体（Anh刀IGEiotin，美国 SIGMA公司）和 SABC（博士德公司人 Bcl－
20SP＊明心一抗及SP免疫组化试剂盒（购自北京中山公司人 TNF呕、
ICAM＊、iNOS一抗及免疫组化试剂盒（购自武汉博土德公司人 Tizol Rea－
gellt（Promega公司人逆转录试剂盒、酶及RT－PCR所需的其他试剂（Takara
公司厂其它均为国产分析纯试剂。
3．主要实验器材：RM6000多导生理检测仪（日本光电厂 Dupont ST－ZI
型低温离心机（美国h Eh 800型酶标仪（美国 BIO－TEK＊日立 F2000荧光
分光光度计（日本人 A200 DS型电子天平（美国人 JUNGC 型切片
机；Ol卿us显微镜（日本）； Obmpus AX 70显微摄像系统（日本）； MetaMo。
pVO…Us DPIWBXS二显微图像分析系统（日本人Mebm。ph1呷system
（美国UIC公司人LKB力型超薄切片机；透射电镜门ppON EM10C，美国＊
IIEIDOLPH DIAX 900型匀浆机（德国hPTC－100PCR扩增仪（美国人DYY－
Ill31A型电泳仪问京＊ KODAKID型凝胶成像分析系统（美国h GIS－
700D数码凝胶扫描分析系统（上海＊日立7170 AU全自动生化分析仪；
5500P紫外光、可见光连续分光光谱仪（法国人 L X J—11型离心沉淀机（上
海医疗器械工厂h SHH WZI Cr 600型电热恒温水浴箱问京长安科学仪器
厂＊KS—150超声波细胞粉碎机（宁波科生仪器厂厂722光栅分光光度计
（上海第三分析仪器厂厂 B碉rown微量泵（德国人 Hitachi 8080型原子吸
·2·
收分光光度计（美国人 Cell羽yn 1700型全自动血常规分析仪（美国厂 ACL
200型全自动血凝分析仪（美国＊WZZA型微量震荡器问京人
实验方法
一、动物模型制备、分组及取材
雄性Wistar大鼠102只，随机分为三组：生理盐水组门组，n＝18八
LPS对照组几组，n＝42＊ LPS预处理组u组，n＝42入J组：首次，经腹腔
注射 Lp 0·25mg／kg；24hr后再经腹腔注射 LPS 0．s略kg，其余两组在上述
时间均给予等量NS，预处理期间大鼠可自由进食水；第二次腹腔注射72hr
后，3％戊巴比妥钠门0mg／kg）腹腔注射麻醉，股静脉插管用于输液和给药，
股动脉插管持续监测动脉压，部分大鼠＊＝6）经右颈动脉插管至左心室人
组和 P组经股静脉插管一次注人脱 10mg／kgJ组给予等量
Sepsis has been redefined as " the systemic inflammatory response to infection" . Septic shock is that sepsis-induced hypotension persists despite adequate fluid resuscitation, along with the hypoperfusion and organ dysfunction, lactic acidosis, oliguria, or altered mental status are frequent manifestations of hypoperfusion. Septic shock is a highly complex pathophysiological state representing a major challenge to physicians. Despite aggressive treatment and increased understanding of the molecule aspects of septic shock, mortality in established septic shock is more than 50%. In the United States, septicemia is the 13th leading cause of death and causes more than 100 000 deaths per year. Experimental and clinical evidence have demonstrated that a variety of inflammatory response mediators, such as TNF-a, IL-1β, IL-6 and NO, are released by activated blood cells, vascular cells and different parenchyma! cells during sepsis. Regulation of the expression of these mediators is critical for normal homeostasis a
nd host defence, whereas overexpression may result in tissue injury, multiple organ dysfunction and death. Laboratory studies have shown protection against experimentally induced sepsis through the use of agents that inhibit specific mediators in the cascade, including endotoxin, TNF-a and IL-1β. However, despite promising results in animal trial, clinical trials applying such specific ther-
apies have been unable to duplicate the success seen in the laboratory, and have so far failed to show improved survival. Hence, these is a continuous search for new therapeutic options.
The induction of tolerance to the systemic effects of endotoxin is a potential form of immunomodulatory therapy. A transient refractoriness to the effects of LPS known as endotoxin tolerance was encountered almost a century ago by physicians who employed bacterial vaccines for fever therapy, and first studied by Beeson, who observed a decrease in febrile response when rabbits were injected repeatedly with this compound. It is well recognized that when a health human (or experimental animal) is repeatedly injected intravenously with nonlethal doses of LPS, increasing resistance developes to its pyrogenic and subjective toxic effects. It is also known - in different animal models - that tolerance can be induced to its metabolic and lethal effects. LPS tolerance was not a permanent phenomenon, which was found to last 2 weeks in the rat. Based on studies in the rabbit, Greisman et al. suggested that LPS tolerance was controlled at the cellular level. In fact, adoptive transfer experiments have shown that LPS tolerance operates at the level of monocytes/macrophages. Consistant with this concept, multiple studies have demonstrated ex vivo tolerance of these cells; When, for instance, rabbits were repeatedly injected with LPS and their peritoneal macro-phages were harvested and stimulated with LPS in vitro, their TNF response was strongly decreased as compared to cells from nontolerant animals. A few problems have to be considered with these in vitro studies:
Too short a primary culture may result in a hangover of response into the secondary culture.
A proper dose of LPS in the primary culture is important, as very low dose may even result in an enhanced response.
LPS contamination has to be excluded, since low amounts of contaminated LPS may induce tolerance already in the control cultures.
Especially for primary cells, explanted from blood or tissue, the effects of culture adaption and in vitro maturation must be considered.
Endotoxin tolerance can also be induced by using nontoxic derivatives of the
lipid A component of the lipoplysacchride molecule. A number of these derivatives , such as monophosphoryl lipid A, retain the immunoadjuvant properties of lipopolysacchride, but are significantily less toxic than the parent molecule.
Some experiments demonstrated that LPS pretreatment could attenuated en-dotoxemia, but the mechanism behind this effect is not well known. One possible hypothesis would be t
感染性休克(septic shock)是指严重脓毒症病人在给予足量液体复苏仍无法纠正的持续性低血压，常伴有低灌流状态及器官功能障碍，是病毒、真菌和细菌脱落的蛋白碎片或免疫原作用于机体诱发多种细胞因子如TNF-α、IL-1、IL-6、IL-8、血小板活化因子(PAF)等以及炎症介质如前列环素、缓激肽、白三烯等释放所导致的全身性过度炎症反应产生破坏作用的结果。革兰氏阴性菌胞壁成分内毒素是引起感染性休克的最常见原因之一。
目前感染性休克的治疗远未达到满意的疗效，死亡率仍在50％左右。现在的防治原则是早期清除原发病灶；使用有效的抗生素控制感染；积极的多系统器官的综合支持疗法。除此之外，各种抗炎症介质、细胞因子的抗体，如LPS、TNF-α、IL-1的抗体以及NOS抑制剂等，在动物实验中似乎都有一定的作用。然而，临床试验不能证明任何一项临床有效。其中三项包括非选择性NOS抑制剂、TNF及抗内毒素单克隆抗体治疗严重感染性休克试验均因受试者病死率增加而中途停止。这些方法主要是从外源性角度来进行治疗，但总的效果并不令人满意。因此在目前感染性休克治疗无突破的情况下，能否利用刺激或激发机体细胞在漫长的进化过程中获得的内源性抗损伤能力来减轻致病因素对机体的损伤程度，在一些可预防性脓毒症高危病人开展有效预防措施，是降低脓毒症及感染性休克病人死亡率的另一思路。研究证明热休克反应可提高鼠对随后感染性休克的耐受力，但热休克反应实施起来有一定困难，而药物预处理具有方便、易行的特点。LPS耐受现象的存在，尤其是其减毒衍生物预处理为这种设想提供了可能。
迄今为止，虽然对内毒素耐受作了大量的实验研究，但大都着重于以离体的单核-巨噬系统细胞和内皮细胞为观察对象，缺乏在动物整体的、器官实质细胞的系统研究。因此，我们设计了以下的系列研究，从整体、器官、组
织以及好水平剧内毒素预处理对内毒素血症的作用及其机制，为进一
步研究内毒素耐受、内毒素减毒衍生物预处理的作用及应用提供实验基础。
实验材料
1．实验动物：昆明种小鼠120只，20－229，雌雄不拘；雄性Wistar大鼠
102 ／q，200－2809，由中国医科大学实验动物中心提供。
2．主要实验试剂与药品：内毒素（Escherichia colt LPS Oill：B4，美国
Sima公司八戊巴比妥（消化学试剂采购供应站八 TNF叱 ELISA试剂盒
（购自深圳晶美生物工程公司，进口分装八个羟基香豆素（美国 Siaa公
司hTUNEL检测试剂盒（购自武汉博士德公司）中主要包括：末端脱氧核
糖核酸转移酶（Termina de…ucleoti邮Trans咖se，TdT，美国Promega 公
司太地高辛标记的规Th（DIG－dUTP，德国 B．M．公司人生物素标记抗地
高辛抗体（Anh刀IGEiotin，美国 SIGMA公司）和 SABC（博士德公司人 Bcl－
20SP＊明心一抗及SP免疫组化试剂盒（购自北京中山公司人 TNF呕、
ICAM＊、iNOS一抗及免疫组化试剂盒（购自武汉博土德公司人 Tizol Rea－
gellt（Promega公司人逆转录试剂盒、酶及RT－PCR所需的其他试剂（Takara
公司厂其它均为国产分析纯试剂。
3．主要实验器材：RM6000多导生理检测仪（日本光电厂 Dupont ST－ZI
型低温离心机（美国h Eh 800型酶标仪（美国 BIO－TEK＊日立 F2000荧光
分光光度计（日本人 A200 DS型电子天平（美国人 JUNGC 型切片
机；Ol卿us显微镜（日本）； Obmpus AX 70显微摄像系统（日本）； MetaMo。
pVO…Us DPIWBXS二显微图像分析系统（日本人Mebm。ph1呷system
（美国UIC公司人LKB力型超薄切片机；透射电镜门ppON EM10C，美国＊
IIEIDOLPH DIAX 900型匀浆机（德国hPTC－100PCR扩增仪（美国人DYY－
Ill31A型电泳仪问京＊ KODAKID型凝胶成像分析系统（美国h GIS－
700D数码凝胶扫描分析系统（上海＊日立7170 AU全自动生化分析仪；
5500P紫外光、可见光连续分光光谱仪（法国人 L X J—11型离心沉淀机（上
海医疗器械工厂h SHH WZI Cr 600型电热恒温水浴箱问京长安科学仪器
厂＊KS—150超声波细胞粉碎机（宁波科生仪器厂厂722光栅分光光度计
（上海第三分析仪器厂厂 B碉rown微量泵（德国人 Hitachi 8080型原子吸
·2·
收分光光度计（美国人 Cell羽yn 1700型全自动血常规分析仪（美国厂 ACL
200型全自动血凝分析仪（美国＊WZZA型微量震荡器问京人
实验方法
一、动物模型制备、分组及取材
雄性Wistar大鼠102只，随机分为三组：生理盐水组门组，n＝18八
LPS对照组几组，n＝42＊ LPS预处理组u组，n＝42入J组：首次，经腹腔
注射 Lp 0·25mg／kg；24hr后再经腹腔注射 LPS 0．s略kg，其余两组在上述
时间均给予等量NS，预处理期间大鼠可自由进食水；第二次腹腔注射72hr
后，3％戊巴比妥钠门0mg／kg）腹腔注射麻醉，股静脉插管用于输液和给药，
股动脉插管持续监测动脉压，部分大鼠＊＝6）经右颈动脉插管至左心室人
组和 P组经股静脉插管一次注人脱 10mg／kgJ组给予等量
Sepsis has been redefined as " the systemic inflammatory response to infection" . Septic shock is that sepsis-induced hypotension persists despite adequate fluid resuscitation, along with the hypoperfusion and organ dysfunction, lactic acidosis, oliguria, or altered mental status are frequent manifestations of hypoperfusion. Septic shock is a highly complex pathophysiological state representing a major challenge to physicians. Despite aggressive treatment and increased understanding of the molecule aspects of septic shock, mortality in established septic shock is more than 50%. In the United States, septicemia is the 13th leading cause of death and causes more than 100 000 deaths per year. Experimental and clinical evidence have demonstrated that a variety of inflammatory response mediators, such as TNF-a, IL-1β, IL-6 and NO, are released by activated blood cells, vascular cells and different parenchyma! cells during sepsis. Regulation of the expression of these mediators is critical for normal homeostasis a
nd host defence, whereas overexpression may result in tissue injury, multiple organ dysfunction and death. Laboratory studies have shown protection against experimentally induced sepsis through the use of agents that inhibit specific mediators in the cascade, including endotoxin, TNF-a and IL-1β. However, despite promising results in animal trial, clinical trials applying such specific ther-
apies have been unable to duplicate the success seen in the laboratory, and have so far failed to show improved survival. Hence, these is a continuous search for new therapeutic options.
The induction of tolerance to the systemic effects of endotoxin is a potential form of immunomodulatory therapy. A transient refractoriness to the effects of LPS known as endotoxin tolerance was encountered almost a century ago by physicians who employed bacterial vaccines for fever therapy, and first studied by Beeson, who observed a decrease in febrile response when rabbits were injected repeatedly with this compound. It is well recognized that when a health human (or experimental animal) is repeatedly injected intravenously with nonlethal doses of LPS, increasing resistance developes to its pyrogenic and subjective toxic effects. It is also known - in different animal models - that tolerance can be induced to its metabolic and lethal effects. LPS tolerance was not a permanent phenomenon, which was found to last 2 weeks in the rat. Based on studies in the rabbit, Greisman et al. suggested that LPS tolerance was controlled at the cellular level. In fact, adoptive transfer experiments have shown that LPS tolerance operates at the level of monocytes/macrophages. Consistant with this concept, multiple studies have demonstrated ex vivo tolerance of these cells; When, for instance, rabbits were repeatedly injected with LPS and their peritoneal macro-phages were harvested and stimulated with LPS in vitro, their TNF response was strongly decreased as compared to cells from nontolerant animals. A few problems have to be considered with these in vitro studies:
Too short a primary culture may result in a hangover of response into the secondary culture.
A proper dose of LPS in the primary culture is important, as very low dose may even result in an enhanced response.
LPS contamination has to be excluded, since low amounts of contaminated LPS may induce tolerance already in the control cultures.
Especially for primary cells, explanted from blood or tissue, the effects of culture adaption and in vitro maturation must be considered.
Endotoxin tolerance can also be induced by using nontoxic derivatives of the
lipid A component of the lipoplysacchride molecule. A number of these derivatives , such as monophosphoryl lipid A, retain the immunoadjuvant properties of lipopolysacchride, but are significantily less toxic than the parent molecule.
Some experiments demonstrated that LPS pretreatment could attenuated en-dotoxemia, but the mechanism behind this effect is not well known. One possible hypothesis would be t
引文
1. Beeson PB. Tolerance to bacterial pyrogens I. factors influencing its development. J Exp Med, 1947, 86: 29～44.
2. O'Mally WE, Achinstein B, Shear MJ. Action of bacterial polysaccharide on tumors. Ⅱ. damage of sarcoma 37 by serum of mice treated with Serratia Marcescens polysaccharide, and induced tolerance. J Natl Cancer Inst, 1962, 29, 1169～1175.
3. Sanchez-cantu L, Rode HN, Christou NV. Endotoxin tolerance is-associated with reduced secretion of tumor necrosis factor. Arch Surg, 1989, 124: 1432～1436.
4．王景华，杨世杰，杨贵贞．用荧光光度法测定组织和血液中一氧化氮．生物化学与生物物理进展，1997；24(4)：363～366．
5. Ghosh S, Latimer RD, Gray BM, et al. Endotoxin-induced organ injury. Crit Care Med, 1993, 21(2): S19～S24.
6. Sanchez-cantu L, Rode HN, Christou NV. Endotoxin tolerance is associated with reduced secretion of tumor necrosis factor. Arch Surg, 1989, 124: 1432～1436.
7. Billau A, Bone RC, Cheadle WG, et al. Cytokines and their interactions with other inflammatory mediators in the patl (?)genesisof sepsis and septic shock. Eur J Clin Invest, 1991, 21: 559-573.
8. Borrelli E, Roux Lombard P, Gran GE, et ax. Plasma concentrations of cytokines, their soluble receptors, and antioxident vitamins can predict the development of multiple organ failure in patients at risk. Crit Care Med, 1996, 24: 392～397.
9. Makhlouf M, Zingarelli B, Halushka PV, et al. Endotoxin tolerance Alters macrophage membrane regulatory G proteins. Prog Clin Biol Res, 1998, 397,217.
10. Medvedev AE, Kopydlowski KW, Vogel SN. Inhibition of LPS-induced signal transduction in endotoximia tolerized mouse macrophages dysregulation
of cytokine, chemokine and toll-like receptor 2 and 4 gene expression. J Immunol, 2000, 164(11): 5564.
11. Grisham MB, Jourdheuil D, Wink DA. Nitric oxide, Iphysiolgical chemistry of nitric oxide and its metabolities: implications in inflammation. Am J Physiol, 1999, 276(2pt1): G315～G321.
12. Liu S, Adcock IM, Old RW, et al. lipopolysaccharide treatment in vivo induces widespreade tissue expression of inducible nitric oxide synthase mRNA. Biochem Biophys Res Commun, 1993, 196(3): 1208～1213.
13. Sato K, Miyakawa K, Takeya M, et al. Immunohistochemical expression of inducible nitric oxide synthase (iNOS) in reversible endotoxemia studied by a novel monoclonal antibody against rat iNOS. J leukoc Biol, 1995, 57: 36.
14. Szabo C, Wu CC, Mitchell JA, et al. Platelet-activating factor contributes to the induction of nitric oxide synthase by bacterial lipopolysaccharide. Circ Res, 1993, 73: 991～999.
15. Haddad IY, Pataki G, Hu P, et al. Quantitation of nitrotyrosine levels in lung section of patients and animals with acute lung injury. J Clin Invest, 1994, 94: 2407.
16. Wizemann TM, Garndner CR, Laskin JD, et al. Production of nitric oxide and peroxynitrite in the lung during acute endotoxemia. J Leukoc Biol, 1994, 56(2): 759～768.
17. Freudenberg MA, Golanos C. induction of tolerance to lipoplysaccaride-dgalactosamine lethality by pretratement with LPS is mediated by macrophages. Infect Immun, 1988, 56: 1352～1357.
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