慢性阻塞性肺疾病患者急性发作期血浆内毒素与IL-6、IL-8、TNF-α水平相关性研究
摘要
前言
慢性阻塞性肺疾病(COPD)是呼吸系统常见病、多发病，是一种以进行性、不可逆性气流阻塞为特征的慢性炎症性疾病。由于气道阻塞是不可逆的，最终多发展为肺心病。COPD的病因和发病机制尚未完全明了，国内外大量研究已证实：内毒素和细胞因子等多种炎性介质在COPD发生发展进程中起重要作用，COPD病人急性发作期血浆内毒素、IL-6、IL-8、TNF-α水平均明显升高，但其血浆内毒素水平与血浆IL-6、IL-8、TNF-α水平之间是否存在相关性，目前鲜有报道。本实验主要是通过检测COPD急性发作期病人血浆内毒素、IL-6、IL-8、TNF-α水平，探讨COPD急性发作期内毒素与IL-6、IL-8、TNF-α的相关性，进而探讨它们在COPD发病机制中的作用，为COPD急性发作期的合理治疗提供理论依据。
资料与方法
一、资料
1．COPD组：急性发作期患者32例，男15例，女17例，平均年龄(58.9±11.1)岁，诊断符合中华医学会呼吸病学会制定的《慢性阻塞性肺疾病(COPD)诊治规范(草案)》。
2．正常对照组：无吸烟史的健康志愿者20例(除外心肺疾病)，男11例，女9例，平均年龄(53.4±11.3)岁。
3．仪器、试剂
(1)．全自动化学发光酶免疫分析仪IMMULITE(美国DPC公司产品)
(2)．IL-6、IL-8、TNF-α包被株(均为美国DPC公司产
品）
oXBET－32型细菌内毒素测定仪（天津大学无线电厂制
造）
KX内毒素检测用超纯水，批号：010208（湛江海洋生物制品
厂）
KX内毒素检测用鲨试剂，批号刃10120（湛江海洋生物制品
厂）
＊X电热鼓风干燥机（中国天津泰斯特仪器有限公司）
（）．GZJ多用振荡器H京冰箱电机厂）
uX恒温水浴箱（余姚电讯仪表实业公司）
二、方法
1．标本采集：晨起空腹取肘正中静脉血5毫升。3毫升放人
常规试管中，立即离心取血清，放置于－30℃以下保存，待测几－
6＊L－8、TNF－a。另 2毫升放人 0．互毫升肝素抗凝的无热源试
管内，封口，混匀，立即离心取上清，放人无热源试管内，于－20℃
以下保存，待测内毒素。
2．全自动化学发光酶免疫分析仪测定细胞因子
O卜双抗体夹心法间接法测定血浆IL－6
闷）．双抗体夹心法直接法测定血浆IL－8＊NF－a
3．BET－32B型细菌内毒素测定仪采用鲨试验动态法比浊法
测定血浆内毒素
4．统计学处理
结果以 hi 8表示。采用 SHARP计算器计算均数和标准差，
并用t检验做统计学处理、用EXcel软件做直线相关分析。
结 果
一人OPD组与对照组内毒素＊L—6＊L—8、TNF一。水平比
较（见表1）
·2·
表ICOPD组与对照组血浆内毒素、IL－6、IL－8、TNF－a水平比较（i。。）
——
一
组 拈5｝9J且．二！．017刊．皿“”7 叨j．删”互二．416d．1历“”9，…工！33””
NNt o 53．7c12．4 0．715c0．2I6 5．270【0．e 5．815【1．3po 4．MOO．883
— —
伎＊OPD组与对刃组比玫．P＜叭山，．．于＜0．01
l．COPD组内毒素水平明显高于对照组u＜0．0门，两者之间
差异显著。
2．COPD组几—6水平明显高于对照组u＜0．05人两者之间
差异显著。
3．＊0＊D组IL－8水平明显高于对照组u＜0．01入两者之间
差异显著。
4．m＊ 组＊＊F一。水平明显高于对照组（P＜0．01），两者之
间差异显著。
H人OPD患者急性发作期血浆内毒素与兀－6、IL－8、TNF－
a相关分析
1．COPD患者血浆内毒素水平与IL－6水平不相关u＞0．
05）。
2．＊OPD患者血浆内毒素水平与IL－8水平正相关（P＜o．
01）。
3．m皿患者血浆内毒素水平与TN F－a水平正相关K＜o．
01）。
讨 论
内毒素是细菌胞壁外膜的脂多糖，是一种毒性很强的致病物
质，它可引起动物和人一系列的病理生理反应。细胞因子是机体
的各种细胞合成和分泌的多肽类因子，它通过结合到免疫细胞表
面的受体来传递复杂的分子信号，从而控制免疫细胞的活动、增
殖、分化及效应。在细胞因子中IL－6＊L－8、TNF－。是重要的
·3·
炎性介质，参与介导内毒素性肺损伤。研究COPD患者急性发作
期血浆内毒素＊L－6＊L－8＼TNF－a水平，可以推断它们在
COPD急性发作期的致病作用。
本研究表明，COPD急性发作期血浆内毒素水平明显高于对
照组，这说明COPD患者急性发作期存在着内毒素血症。内毒素
性肺损伤模型镜下组织学改变，主要是中性粒细胞和单核细胞出
现在肺毛细血管内，出现大范围的肺实质炎性改变，这与COPD病
理学改变各种炎性细胞的浸润相符。炎性细胞浸润可致机体动脉
血氧分压下降，肺内分流和气道内峰压力增加，肺顺应性下降，呼
吸道粘膜充血水肿，引起咳喘等症状，这又与临床观察到的COPD
患者急性发作期咳、痰、喘加重的症状相符合。以上这些表明内毒
素血症与COPD患者的肺损伤之间存在着某种联系
Preface
Chronic obstructive pulmonary disease ( COPD ) is a very common respiratory disease. It is a chronic inflammatory disease characterized by progressive, irreversible obstruction in the air way and usually develops into pulmonary heart disease in the late stage. The etiology and the pathogenesis of COPD are not completely clear by now, but a lot of studies have shown that endotoxin and some cytokines play a very important role in the development and progression of COPD. During the acute episode of COPD, the level of endotoxin, interleukin
- 6 (IL - 6) , interleukin - 8 (IL - 8 ) and tumor necrosis factor alpha (TNF - ) in patients' plasma is markedly increased, but there is few report on the correlation of endotoxin with IL - 6, IL - 8 and TNF
- a . Our study , by detecting the plasmatic level of endotoxin, IL -6, IL - 8 and TNF - , was to investigate the correlation of endotoxin with IL - 6, IL - 8 and TNF - in the plasma of COPD patients during acute episode and their role in the pathogenesis of COPD, and furthermore to provide suitable methods for treatment.
Materials and Methods
1. Materials
All the study objects were divided into two groups: COPD group and control group. COPD group consisted of 32 patients in acute episode who had been diagnosed definitely according to the criteria formulated by Chinese Medicine Association, and their average age is 58.9 11. 1. The control group was composed of 20 healthy volunteers who had no history of smoking and their average age is 53. 4 ?11.3.
2. Methods
(1) Collection of specimen; 5ml venous blood was aspirated in the morning when the patients were in fast. 3ml of the blood was placed in ordinary tubes. After centrifuged, the specimen was conserved under - 30 for later detection of IL - 6, IL - 8 and TNF -a. Another 2ml was placed in a heparin - anticoagulated sterilized tubes. After homogenized and centrifuged, the plasma was put into another sterilized tubes, being conserved under - 201 for later detection of endotoxin.
(2) Detection of the cytokines automatic chemical fluorescent enzyme immune analysis instrument. (1)detecting the level of IL - 6 with indirect method. (2)detecting the level of IL - 8 and TNF - a with direct method.
(3) Detection of the level of endotoxin with kinetic turbimetric Assay method.
3. Statistical analysis: t test was performed and correlation analysis was performed through Excel software.
Results
1. Comparison of the level of endotoxin, IL -6, IL -8 and TNF - between COPD group and control group
(1)The level of endotoxin in COPD group was significantly higher than that of control group.
(2)The level of IL - 6 in COPD group was significantly higher than that of control group.
(3)The level of IL - 8 in COPD group was significantly higher than that of control group.
(4)The level of TNF - a in COPD group was significantly higher than that of control group.
2. correlation analysis of the level of endotoxin with IL - 6, IL -8 and TNF - a in patients of acute episode
(1)The level of endotoxin in the plasma of COPD patients was of no correlation with that of IL - 6. ( P > 0.05 )
(2)The level of endotoxin in the plasma of COPD patients was of positive correlation with that ofIL-8. (P<0.01)
(3)The level of endotoxin in the plasma of COPD patients was of positive correlation with that of TNF -a (P<0.01)
Discussion
Endotoxin is a kind of polysaccharide lying in the cell membrane of bacteria. It is a pathogenic material of strong toxicity and may cause a series of pathophysiological changes in both animals and human beings. Cytokines are polypeptides produced and secreted by body cells. They could transmit complex molecular massage by combining with the specific receptors on the surface of immune cells so as to control the
movement, multiplication, differentiation and the reactivity of immune cells. Among all the cytokines, IL - 6, IL - 8 and TNF - a are the main important inflammatory mediators, and they all take part in the endotoxic injury to the lung.
慢性阻塞性肺疾病(COPD)是呼吸系统常见病、多发病，是一种以进行性、不可逆性气流阻塞为特征的慢性炎症性疾病。由于气道阻塞是不可逆的，最终多发展为肺心病。COPD的病因和发病机制尚未完全明了，国内外大量研究已证实：内毒素和细胞因子等多种炎性介质在COPD发生发展进程中起重要作用，COPD病人急性发作期血浆内毒素、IL-6、IL-8、TNF-α水平均明显升高，但其血浆内毒素水平与血浆IL-6、IL-8、TNF-α水平之间是否存在相关性，目前鲜有报道。本实验主要是通过检测COPD急性发作期病人血浆内毒素、IL-6、IL-8、TNF-α水平，探讨COPD急性发作期内毒素与IL-6、IL-8、TNF-α的相关性，进而探讨它们在COPD发病机制中的作用，为COPD急性发作期的合理治疗提供理论依据。
资料与方法
一、资料
1．COPD组：急性发作期患者32例，男15例，女17例，平均年龄(58.9±11.1)岁，诊断符合中华医学会呼吸病学会制定的《慢性阻塞性肺疾病(COPD)诊治规范(草案)》。
2．正常对照组：无吸烟史的健康志愿者20例(除外心肺疾病)，男11例，女9例，平均年龄(53.4±11.3)岁。
3．仪器、试剂
(1)．全自动化学发光酶免疫分析仪IMMULITE(美国DPC公司产品)
(2)．IL-6、IL-8、TNF-α包被株(均为美国DPC公司产
品）
oXBET－32型细菌内毒素测定仪（天津大学无线电厂制
造）
KX内毒素检测用超纯水，批号：010208（湛江海洋生物制品
厂）
KX内毒素检测用鲨试剂，批号刃10120（湛江海洋生物制品
厂）
＊X电热鼓风干燥机（中国天津泰斯特仪器有限公司）
（）．GZJ多用振荡器H京冰箱电机厂）
uX恒温水浴箱（余姚电讯仪表实业公司）
二、方法
1．标本采集：晨起空腹取肘正中静脉血5毫升。3毫升放人
常规试管中，立即离心取血清，放置于－30℃以下保存，待测几－
6＊L－8、TNF－a。另 2毫升放人 0．互毫升肝素抗凝的无热源试
管内，封口，混匀，立即离心取上清，放人无热源试管内，于－20℃
以下保存，待测内毒素。
2．全自动化学发光酶免疫分析仪测定细胞因子
O卜双抗体夹心法间接法测定血浆IL－6
闷）．双抗体夹心法直接法测定血浆IL－8＊NF－a
3．BET－32B型细菌内毒素测定仪采用鲨试验动态法比浊法
测定血浆内毒素
4．统计学处理
结果以 hi 8表示。采用 SHARP计算器计算均数和标准差，
并用t检验做统计学处理、用EXcel软件做直线相关分析。
结 果
一人OPD组与对照组内毒素＊L—6＊L—8、TNF一。水平比
较（见表1）
·2·
表ICOPD组与对照组血浆内毒素、IL－6、IL－8、TNF－a水平比较（i。。）
——
一
组 拈5｝9J且．二！．017刊．皿“”7 叨j．删”互二．416d．1历“”9，…工！33””
NNt o 53．7c12．4 0．715c0．2I6 5．270【0．e 5．815【1．3po 4．MOO．883
— —
伎＊OPD组与对刃组比玫．P＜叭山，．．于＜0．01
l．COPD组内毒素水平明显高于对照组u＜0．0门，两者之间
差异显著。
2．COPD组几—6水平明显高于对照组u＜0．05人两者之间
差异显著。
3．＊0＊D组IL－8水平明显高于对照组u＜0．01入两者之间
差异显著。
4．m＊ 组＊＊F一。水平明显高于对照组（P＜0．01），两者之
间差异显著。
H人OPD患者急性发作期血浆内毒素与兀－6、IL－8、TNF－
a相关分析
1．COPD患者血浆内毒素水平与IL－6水平不相关u＞0．
05）。
2．＊OPD患者血浆内毒素水平与IL－8水平正相关（P＜o．
01）。
3．m皿患者血浆内毒素水平与TN F－a水平正相关K＜o．
01）。
讨 论
内毒素是细菌胞壁外膜的脂多糖，是一种毒性很强的致病物
质，它可引起动物和人一系列的病理生理反应。细胞因子是机体
的各种细胞合成和分泌的多肽类因子，它通过结合到免疫细胞表
面的受体来传递复杂的分子信号，从而控制免疫细胞的活动、增
殖、分化及效应。在细胞因子中IL－6＊L－8、TNF－。是重要的
·3·
炎性介质，参与介导内毒素性肺损伤。研究COPD患者急性发作
期血浆内毒素＊L－6＊L－8＼TNF－a水平，可以推断它们在
COPD急性发作期的致病作用。
本研究表明，COPD急性发作期血浆内毒素水平明显高于对
照组，这说明COPD患者急性发作期存在着内毒素血症。内毒素
性肺损伤模型镜下组织学改变，主要是中性粒细胞和单核细胞出
现在肺毛细血管内，出现大范围的肺实质炎性改变，这与COPD病
理学改变各种炎性细胞的浸润相符。炎性细胞浸润可致机体动脉
血氧分压下降，肺内分流和气道内峰压力增加，肺顺应性下降，呼
吸道粘膜充血水肿，引起咳喘等症状，这又与临床观察到的COPD
患者急性发作期咳、痰、喘加重的症状相符合。以上这些表明内毒
素血症与COPD患者的肺损伤之间存在着某种联系
Preface
Chronic obstructive pulmonary disease ( COPD ) is a very common respiratory disease. It is a chronic inflammatory disease characterized by progressive, irreversible obstruction in the air way and usually develops into pulmonary heart disease in the late stage. The etiology and the pathogenesis of COPD are not completely clear by now, but a lot of studies have shown that endotoxin and some cytokines play a very important role in the development and progression of COPD. During the acute episode of COPD, the level of endotoxin, interleukin
- 6 (IL - 6) , interleukin - 8 (IL - 8 ) and tumor necrosis factor alpha (TNF - ) in patients' plasma is markedly increased, but there is few report on the correlation of endotoxin with IL - 6, IL - 8 and TNF
- a . Our study , by detecting the plasmatic level of endotoxin, IL -6, IL - 8 and TNF - , was to investigate the correlation of endotoxin with IL - 6, IL - 8 and TNF - in the plasma of COPD patients during acute episode and their role in the pathogenesis of COPD, and furthermore to provide suitable methods for treatment.
Materials and Methods
1. Materials
All the study objects were divided into two groups: COPD group and control group. COPD group consisted of 32 patients in acute episode who had been diagnosed definitely according to the criteria formulated by Chinese Medicine Association, and their average age is 58.9 11. 1. The control group was composed of 20 healthy volunteers who had no history of smoking and their average age is 53. 4 ?11.3.
2. Methods
(1) Collection of specimen; 5ml venous blood was aspirated in the morning when the patients were in fast. 3ml of the blood was placed in ordinary tubes. After centrifuged, the specimen was conserved under - 30 for later detection of IL - 6, IL - 8 and TNF -a. Another 2ml was placed in a heparin - anticoagulated sterilized tubes. After homogenized and centrifuged, the plasma was put into another sterilized tubes, being conserved under - 201 for later detection of endotoxin.
(2) Detection of the cytokines automatic chemical fluorescent enzyme immune analysis instrument. (1)detecting the level of IL - 6 with indirect method. (2)detecting the level of IL - 8 and TNF - a with direct method.
(3) Detection of the level of endotoxin with kinetic turbimetric Assay method.
3. Statistical analysis: t test was performed and correlation analysis was performed through Excel software.
Results
1. Comparison of the level of endotoxin, IL -6, IL -8 and TNF - between COPD group and control group
(1)The level of endotoxin in COPD group was significantly higher than that of control group.
(2)The level of IL - 6 in COPD group was significantly higher than that of control group.
(3)The level of IL - 8 in COPD group was significantly higher than that of control group.
(4)The level of TNF - a in COPD group was significantly higher than that of control group.
2. correlation analysis of the level of endotoxin with IL - 6, IL -8 and TNF - a in patients of acute episode
(1)The level of endotoxin in the plasma of COPD patients was of no correlation with that of IL - 6. ( P > 0.05 )
(2)The level of endotoxin in the plasma of COPD patients was of positive correlation with that ofIL-8. (P<0.01)
(3)The level of endotoxin in the plasma of COPD patients was of positive correlation with that of TNF -a (P<0.01)
Discussion
Endotoxin is a kind of polysaccharide lying in the cell membrane of bacteria. It is a pathogenic material of strong toxicity and may cause a series of pathophysiological changes in both animals and human beings. Cytokines are polypeptides produced and secreted by body cells. They could transmit complex molecular massage by combining with the specific receptors on the surface of immune cells so as to control the
movement, multiplication, differentiation and the reactivity of immune cells. Among all the cytokines, IL - 6, IL - 8 and TNF - a are the main important inflammatory mediators, and they all take part in the endotoxic injury to the lung.
引文
1．中华医学会呼吸病学会分会．慢性阻塞性肺疾病(COPD)诊治规范．中华呼吸结核杂志，1997，20：99-106
2. Kubo K, Amari T, Kaneki T, et al. A 21-aminosteroid, U-74006F, attenuates endotoxin-induced lung injury in awake sheep. Respirology, 1999,4:167-172
3. Adembri C, Domenici-Lombardo L, Meucci M, et al. Morphologic and biochemical kinetics of the pathogenesis of acute lung injury by endotoxin in rats. Minerva-Anestesiol, 1998,64:281-287
4. Murakami K, Okajima K, Uchiba M, et al. A novel platelet activating factor antagonist, SM-12502, attenuates endotoxin-induced disseminated intravascular coagulation and acute pulmonary vascular injury by inhibiting TNF production in rats. Thromb Haemost, 1996,75: 965-970
5. Pulido EJ, et al. Cardiotrophin-lattenuates endotoxin-induced acute lung injury. J Surg Res,1999,84(2) :240
6. Gamble JR, et al. Stimulation of the adherencs of neutrophils to umbilical vein endothelium by human recombinat tumor necrosis factor. Proc Natl Acad Sci USA, 1985,82:8667
7. Coodman RB, Forstrom JY, Osborn SG, et al. Identificafien of two neutrophil chemotactic peptides produced by porcine alveolar macrophages. J Biol Chem, 1991,266:8455
8. Fry DE. Multiple system organ failure. Surg Clin North Am. 1998, 68:107
9. Shapiro L, Gelfand JA. Cytokines and sepsis: pathophysiology and therapy. New-Horiz, 1993,1 (1):13
10. Miller LS, Morita Y, Rangan U et al. Suppression of cytokine-in-
duced neutrophil accumulation in rat mesenteric venules in vivo by general anesthesia. Int-J-Microcirc-Clin-Exp, 1996,16:54
11. Carter AB, Monick MM, Hunninghake GW. Lipopolysaccharide-induced NF-kappa B activation and eytokine release in human alveolar macrophages is PKC-independent and TK-and PC-PLC-dependent. Am-J-Respir-Cell-Mol-Biol, 1998,18:384-391
12. Caty MG, Guice KS, Oldham KT, et al. Evidence for tumor necrosis factor-induced pulmonary microvascular injury after intestinal ischemia-reperfusion injury Ann Surg, 1990,212:694
2. Kubo K, Amari T, Kaneki T, et al. A 21-aminosteroid, U-74006F, attenuates endotoxin-induced lung injury in awake sheep. Respirology, 1999,4:167-172
3. Adembri C, Domenici-Lombardo L, Meucci M, et al. Morphologic and biochemical kinetics of the pathogenesis of acute lung injury by endotoxin in rats. Minerva-Anestesiol, 1998,64:281-287
4. Murakami K, Okajima K, Uchiba M, et al. A novel platelet activating factor antagonist, SM-12502, attenuates endotoxin-induced disseminated intravascular coagulation and acute pulmonary vascular injury by inhibiting TNF production in rats. Thromb Haemost, 1996,75: 965-970
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