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應用研究
脾切除對內毒素誘發肝臟脂多糖結合蛋白和TNFα基因表達的
Date: 2018-10-08 11:30:14
脾切除對內毒素誘發肝臟脂多糖結合蛋白和TNFα基因表達的影響
楊英祥，陸家齊，楊興東，冀振華，邱寶安，張志成
陸家齊，楊興東，冀振華，邱寶安，張志成，中國人民解放軍海軍總醫院肝膽外科 北京市 100037
楊英祥，男，1968-09-27生，安徽省青陽縣人，漢族，1993年第二軍醫大學畢業，1999年軍醫進修學院外科碩士，肝膽外科主治醫師.
項目負責人 楊英祥，100037，北京阜成路6號，中國人民解放軍海軍總醫院肝膽外科. zhhji@95777.com
電話: 010-66958512 傳真:010-68574422
收稿日期 2002-03-05 接受日期 2002-03-23
Effect of splenectomy on endotoxin-mediated lipopolysaccharide-binding protein and TNF-α gene expression
Ying-Xiang Yang, Jia-Qi Lu, Xing-Dong Yang, Zhen-Hua Ji, Bao-An Qiu, Zhi-Cheng Zhang
Ying-Xiang Yang, Jia-Qi Lu, Xing-Dong Yang, Zhen-Hua Ji, Bao-An Qiu, Zhi-Cheng Zhang, Department of Hepatobiliary Surgery, Chinese PLA Navy General Hospital, Beijing 100037, China.
Correspondence to: Ying-Xiang Yang, Department of HepatobiliarySurgery, Chinese PLA Navy General Hospital, Beijing 100037, China. zhhji@95777.com
Received 2002-03-05 Accepted 2002-03-23
Abstract
AIM:To investigate the role of lipopolysaccharide-binding protein (LBP) gene expression in the pathogenesis of endotoxemia-mediated systemic inflammation and organ damages after splenectomy.
METHODS:Total 112 male Wistar rats were randomly divided into two groups: control group (n=56), including omentectomy and mobilization of the spleen; s plenectomy group (n=56): including 10 minutes, 0.5, 1.5, 4, 12 and 24 hours subgroups after endotoxin injection. Tissue specimens from liver were obtained. Endotoxin concentrations in the tissue were measured with chromogenic limulus amebocyte lysate (LAL), which was modified by perchloric acid (PCA) pretreatment for samples. LBP and TNF-α mRNA expression were semi-quantitated by the reverse transcription polymerase chain reaction (RT-PCR) using (-actin as an internal standard. Enzyme-linked immunosorbent assay (ELISA) was performed for tumor necrosis factor-α(TNF-α) measurement. The biopsy were carried out for the histological examination.
RESULTS:After endotoxin administration,liver endotoxin concentrations were higher in splenectomized rats than those in controls at 10 minutes（21.5±4.3EU/g vs 10.9±3.3EU/g，P<0.01）. Maximal liver LBP mRNA and TNF-α mRNA expression was higher in animals with asplenia than those with eusplenia (LBP mRNA:1.45±0.31 vs 1.09±0.17 TNF mRNA 1.39±0.19 vs 0.97±0.16, P<0.05). Liver TNF-α levels increased markedly in splenectomy groups following endotoxin administration. There was a positive correlation between tissue TNF-αlevels and TNF-α mRNA expression. Similar result also obtained between TNF-α mRNA and LBP mRNA expression. H-E staining showed hepatic cell vacuolation in control groups and steatosis in model groups.
CONCLUSION:Splenectomy can lead to impairment of tissue clearance of endotoxin in liver. Endotoxin accumulated in the local site may markedly up-regulate LBP mRNA expression, and excessive LBP mRNA expression enhances synthesis and release of inflammatory factor. Thus, LBP increases LPS-induced production of inflammatory cytokines.
Yang YX, Lu JQ, Yang XD, Ji ZH, Qiu BA, Zhang ZC. Effect of splenectomy on endotoxin-mediated lipopolysaccharide-binding protein and TNF-α gene expression. Shijie Huaren Xiaohua Zazhi 2002;10(6):654-658
摘 要
目的:觀察脾切除動物受內毒素攻擊后肝臟脂多糖結合蛋白及腫瘤壞死因子基因表達的變化，探討脂多糖結合蛋白在脾切除后感染中的可能作用.
方法:♂Wister大鼠112只，隨機分為2組：假手術對照組(n=56只)：行大網膜切除；脾切除組(n=56只)：行脾切除術，每組根據時間點分為內毒素注射前、注射后1/6，0.5，1.5，4，12，24h組；各組均留取肝組織標本，采用改良過氯酸法預處理組織勻漿液、鱟試劑基質顯色法檢測組織內毒素水平；采用RT-PCR法檢測組織LBP，TNF-αmRNA表達水平，ELISA法測定組織TNF-α含量.對肝臟行常規病理檢查.
結果:靜脈注射內毒素后，脾切除動物肝組織內毒素較有脾動物明顯升高（21.5±4.3EU/g vs 10.9±3.3EU/g，P<0.01）.脾切除大鼠內毒素攻擊后肝組織LBP mRNA 及TNF-α mRNA表達峰值較對照組明顯增高（LBP mRNA：1.45±0.31 vs 1.09±0.17，TNF mRNA: 1.39±0.19 vs 0.97±0.16, P<0.05），且峰值持續時間延長，脾切除動物肝組織TNF水平較有脾動物明顯增高，相關分析表明，組織TNF-α水平與TNF-α mRNA表達呈顯著正相關，TNF-α mRNA表達與LBP mRNA表達顯著相關.HE染色光鏡下示有脾動物肝細胞空泡及脂肪變性，無脾大鼠可見肝小葉內散在小灶性壞死.
結論:脾切除后機體對內毒素清除延遲，內毒素在組織積聚，組織細胞LBP mRNA過量表達，增敏內毒素激活細胞，進而誘發炎癥因子大量產生和全身炎癥反應失控，造成器官明顯的損害.
楊英祥，陸家齊，楊興東，冀振華，邱寶安，張志成.脾切除對內毒素誘發肝臟脂多糖結合蛋白和TNFα基因表達的影響.世界華人消化雜志 2002;10(6): 654-658
0 引言
近年來，脂多糖結合蛋白(lipopolysaccharide-binding protein LBP)發現使人們在內毒素(lipopolysaccharide LPS)識別及激活細胞方面獲得了突破性進展，即認識到LBP可能是體內極為重要的內毒素雒糲低?，其增昧u慷瓤商岣000倍以上［1-15]，大量研究表明，創傷和膿毒癥時血清中LBP水平升高，組織內LBP mRNA表達也增加，局部組織內LBP介導的增敏效應可能在器官功能損害發病過程中有著重要的意義［16-27].脾切除術后兇險性感染(overwhelming postsplenectomy infection, OPSI)是指發生于脾切除后的暴發性細菌感染，起病突然，病程特點為迅速演變為休克、低灌注及多器官功能障礙［28-32].我們擬觀察脾切除動物受內毒素攻擊后肝臟脂多糖結合蛋白和腫瘤壞死因子基因表達及肝臟的組織學變化，以探討脂多糖結合蛋白在脾切除后感染中的可能作用.
1 材料和方法
1.1 材料 ♂Wistar大鼠112只，體質量200～250g，隨機分為2組：無脾組 (脾切除，56只)和有脾組 (假手術對照，56只).實驗前禁食過夜，自由飲水.10g/L 戊巴比妥鈉麻醉后.取左側肋緣下斜切口，行全脾切除術, 假手術對照組行部分大網膜切除術和脾移動.術后繼續飼養1wk.每組動物均由尾靜脈注射內毒素(E.coli O111 B4) 0.1mg/kg，分別于內毒素攻擊后1/6，0.5，1.5，4，12，24h處死動物，留取組織標本.置于10%甲醛液，HE染色，觀察普通病理變化.
1.2 方法
1.2.1 肝組織內毒素測定 采用改良過氯酸預處理組織勻漿液，用鱟試劑基質顯色法(LAL)測定組織內毒素含量.主要步驟：無菌取肝組織0.1g，加入3倍體積無熱原生理鹽水，冰浴勻漿.吸取組織勻漿液75μL，加入0.32mol/L PCA 150μL，搖勻加蓋后37℃水浴20min.離心(3000r/min，15min，4℃)后，取上清50μL加等量0.18mol/L NaOH，混勻后緩慢滴入鱟試劑50μL，加蓋37℃水浴22min.然后加入鱟三肽50μL，37℃水浴3min.加入亞硝酸鈉0.5mL中止反應，再依次加入氨基磺酸鈉0.5mL和萘乙二胺0.5mL，混勻后在DU-7型紫外可見光分光光度計上于波長545nm處測定吸光度值.
1.2.2 肝肺組織TNF-α蛋白含量 采用ELISA法測定，藥盒購自美國Biosource公司，操作方法按試劑盒說明書規定進行.試劑盒靈敏度為4ng/L，檢測范圍為4-1000ng/L，回收率為94-110%.
1.2.3 肝組織LBP及TNF-α mRNA測定 采用RT-PCR半定量法測定肝組織LBP及TNF撥mRNA表達.用異硫氰酸胍一步提取法提取組織總RNA，采用逆轉錄酶和隨機引物進行逆轉錄合成模板cDNA.通過PCR循環進行基因擴增.PCR步驟：分別向0.5mL離心管中加入10×buffer 2.5μL MgCl2 (20m mol/L) 2.0μL，dNTPs (5m mol/L) 1.0μL，3'5'(5μmol/L)引物1.0μL，逆轉錄產物3.0μL，去離子水補至25μL.加入石蠟油50μl，97℃變性5min，冷卻至88℃時加Taq酶(1U/管)進行PCR循環.大鼠LBP引物序列：5'-CAA ACT CTG CCA GTC ACA-3'，5'-GGA CAT TGG CAC CCA AGT-3'［33］.大鼠TNF-α引物序列：5'-AGA ACT CCA GGA GGT GTC TGT G-3'，5'-GT GGC AAA TCG GCT GAC GGT GT-3'［34］.PCR循環產物凝膠電泳，攝像，沖洗后采用LEICA Q-500IW圖像分析處理系統進行分析，以積分光密度比值表示mRNA相對表達量.
統計學處理 各項指標均以平均值±標準差(±s)表示，采用SAS(6.04版)統計軟件包、Excel(97版)統計函數，不同資料分別進行t檢驗、Q檢驗及相關與回歸分析等.
2 結果
2.1 注射內毒素前動物肝臟末見明顯異常 內毒素靜脈注射后12h，有脾動物肝中央靜脈周圍見炎細胞附壁及集聚，肝細胞水腫空泡變性，無脾大鼠可見肝竇內枯否氏細胞增生，肝小葉內散在小灶性壞死，其中有中性白細胞及淋巴細胞浸潤 (圖1).
A：對照組肝臟空泡變性
B：脾切除組見小灶性壞死
大鼠肝組織切片 HE×400
2.2 肝組織內毒素水平 內毒素注射前，脾切除和有脾大鼠肝組織含有少量內毒素，內毒素注射后10min，兩組動物肝組織內毒素水平均迅速增高，脾切除動物肝組織內毒素升高更明顯(21.5±4.3EU/g)，與有脾動物(10.9±3.3EU/g)相比差異顯著(P<0.01).肝組織內毒素4h后恢復至正常水平(表1).
表1 內毒素攻擊大鼠后肝組織內毒素和TNFα水平(x±s)
aP<0.01, bP<0.05, vs 注射前，cP<0.01,dP<0.05, vs 有脾組.
2.3 肝組織LBP mRNA表達 正常肝組織有少量LBP mRNA 表達，內毒素攻擊后肝組織LBP mRNA表達迅速增多，1.5h達峰值，脾切除大鼠峰值顯著高于有脾大鼠(P<0.05)，且峰值持續時間長，4h兩組動物仍有明顯差異(P<0.05),12h后恢復至傷前基礎值（表2）.
表2 內毒素攻擊后肝TNF-α及LBP mRNA表達(積分A比)
aP<0.01, vs 注射前，bP<0.05, vs有脾組.
2.4 肝組織TNF-α mRNA表達 正常肝組織有少量TNF-α mRNA表達；內毒素攻擊后0.5h，脾切除大鼠肝組織TNF-α mRNA表達迅速升高，1.5h達注射前的5倍，峰值更高(增加42.9%倍)，與有脾組動物有顯著差異(玃<0.05)，12h恢復至正常水平（表2）.
2.5 肝組織TNF-α水平 內毒素攻擊后0.5h肝組織TNF-α水平顯著升高，脾切除動物和有脾動物分別升高至注射前的2.62倍和2.25倍，1.5h肝組織TNF-α水平達峰值，脾切除大鼠峰值是有脾大鼠的1.34倍，兩組差異顯著(P<0.01，表1).肝組織TNF-α濃度與TNF-α，LBP mRNA表達均顯著相關(r=0.8547和0.7222，P<0.01)，肝組織TNF-α mRNA表達與LBP mRNA表達有明顯相關性(r=0.6715，P<0.01).
3 討論
本研究顯示內毒素攻擊后10min脾切除動物肝組織內毒素水平明顯增高，表明脾切除后組織內毒素的排泄減緩，組織內滯留量增加，提示肝臟在脾切除后器官損害的發生、發展中可能起著重要的調控作用［35-42］.LBP在介導細胞應答反應中起顯著的增敏作用，臨床OPSI病和特點為起病突然，兇猛病情迅速惡化，短期內陷入休克，一般認為革蘭氏陰性菌是OPSI的重要病原菌之一［29-31］，但LBP增敏系統是否參與脾切除后感染的過程，本實驗結果顯示，正常肝組織中存在少量LBP mRNA表達，小劑量內毒素(100ng/kg)靜脈注射后，無脾大鼠肝組織LBP mRNA表達即迅速增高，1.5h達峰值水平，4h后逐漸下降，12h恢復至正常范圍，無脾動物肝組織LBP mRNA表達峰值比有脾大鼠明顯增高，1.5h和4h時均存在顯著差異，表明脾切除動物感染時器官存在LBP過量表達.LBP具有“酶”的催化作用，可促進內毒素聚體形成單體和加速內毒素與CD14結合, 從而增強細胞對內毒素的敏感性.LBP基因缺陷動物在給予內毒素刺激時不出現急性炎癥反應，體內TNF-α水平不增高.大量研究表明, 創傷和膿毒癥時血清中LBP水平升高, 組織內LBP mRNA表達也持續增加, 局部組織內LBP介導的增敏效應可能在器官功能損害發病過程中有著重要的意義.
RT-PCR結果表明，無脾大鼠內毒素攻擊后肝組織TNF-α mRNA大量表達，說明脾切除可能造成動物免疫功能紊亂，使機體處于一種“預激狀態”，同時組織內毒素積聚及LBP mRNA表達增加，促使組織TNF-α mRNA大量表達，TNF-α合成和釋放增多.我們即往研究表明，脾切除后較大劑量內毒素(10mg/kg)攻擊可造成動物死亡率明顯增高，血清中TNF-α水平上升；而給予TNF-α單克隆抗體進行干預，血中TNF-α水平明顯降低且死亡率下降，可見TNF-α在無脾動物膿毒癥中起著重要的作用［43-45].本實驗結果顯示，脾切除動物遭受低劑量內毒素(100ng/kg)攻擊后30min，肝組織TNF-α水平升高，1.5h達峰值；而有脾大鼠注射內毒素后峰值比無脾大鼠明顯降低.同時相關分析表明，肝組織內TNF-α mRNA表達與組織TNF-α濃度顯著正相關(r=0.8547, P<0.01)，提示TNF-α的調節可能發生在轉錄水平，調節TNF-α基因轉錄或翻譯可能更有效地阻止脾切除后細菌內毒素的再次打擊.本結果還顯示，組織TNF-α濃度增高及TNF-α mRNA表達上調與LBP mRNA表達規律相似，相關分析進一步表明，肝組織LBP mRNA與TNF-α mRNA表達具有顯著相關性，提示無脾動物內毒素血癥時，體內內毒素與LBP形成復合體，廣泛激活不同組織合成并釋放大量炎癥因子，最終導致機體多器官損害.病理證實有脾大鼠受內毒素攻擊后，肝臟出現脂肪變性和枯否氏細胞增生，有研究認為這種改變為可逆性的損傷，機體對這種可逆性損傷是能夠代償的，但脾切除大鼠在受到相同劑量的內毒素攻擊時，則形成肝臟小灶性壞死，成為不可逆性變化.
我們認為脾切除后機體對內毒素清除延遲，致使內毒素在組織積聚，內毒素刺激組織細胞LBP mRNA表達，大量合成與分泌LBP，LBP-內毒素復合體可增敏內毒素激活細胞，進而誘發炎癥因子大量產生和全身炎癥反應失控，造成器官明顯的損害，最終可能發展成MODS.提示LBP增敏效應可能是臨床OPSI病程兇險發展的重要機制之一，通過早期阻抑LBP增敏效應，可能會減緩病情的發展，這為脾切除后兇險感染的預防提供新的思路.
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11 Vreugdenhil AC, Snoek AM, Greve JW, Buurman WA. Lipopolysaccharide-binding protein is vectorially secreted and transported by
cultured intestinal epithelial cells and is present in the intestinal mucus of mice. J Immunol 2000; 165: 4561-4566
12 Jeon YJ, Han SB, Ahn KS, Kim HM. Differential activation of murine macrophages by angelan and LPS. Immunopharmacology
2000; 49: 275-284
13 Dentener MA, Vreugdenhil AC, Hoet PH, Vernooy JH, Nieman FH, Heumann D, Janssen YM, Buurman WA, Wouters EF. Production
of the acute-phase protein lipopolysaccharide-binding protein by respiratory type II epithelial cells: implications for local defense to
bacterial endotoxins. Am J Respir Cell Mol Biol 2000; 23: 146-153
14 Vesy CJ, Kitchens RL， Wolfbauer G, Albers JJ, Munford RS. Lipopolysaccharide-binding protein and phospholipid transfer protein
release lipopolysaccharides from gram-negative bacterial membranes. Infect Immun 2000; 68: 2410-2417
15 Mayeux PR.Pathobiology of lipopolysaccharide. J Toxicol Environ Health 1997; 51: 415-435
16 Yao YM, Yu Y, Fang WH, Zhe HX, Shi ZG, Sheng ZY, Schlag G. The significance of lipopolysaccharide-binding protein and
lipopolysac charide receptor CD14 in increasing sensitivity toendotoxin response and multiple organ damage following trauma.
Chin Critical Care Med 1998; 10: 712-718
17 Hou YF, Mao BL, Qian GS, Xv J. Expression of lipopolysaccharide binding protein mRNA in pulmonary tissues of rat with "two-hit"
acute lung injury and the protective effects of anisodamine in this event. Disan Junyi Daxue Xuebao 2001; 23: 824-826
18 Fang WH, Yao YM, SHI ZG, Yu Y, Wu Y, Lu LR. The significance of the expressions of lipopolysaccharide binding prot ein mRNA and
lipopolysaccharide receptor CD14 mRNA in the liver of burned rat. Chin J Burns 2000; 16: 157-160
19 Wiezer MJ, Meijer C, Sietses C, Prins HA, Cuesta MA Beelen RH, Meijer S, vanLeeuwen PA. Bactericidal/permeability-increasing
protein preserves leukocyte functions after major liver resection. Ann Surg 2000; 232: 208-215
20 Fujimoto M, Uemura M, Nakatani Y, Tsujita S, Hoppo K, Tamagawa T, Kitano H, Kikukawa M, Ann T, Ishii Y, Kojima H, Sakurai S ,
Tanaka R, Namisaki T, Noguchi R, Higashino T, Kikuchi E, Nishimura K, Takaya A, Fukui H. Plasma endotoxin and serum cytokine
levels in patients with alcoholic hepatitis: relation to severity of liver disturbance. Alcohol Clin Exp Res 2000; 24: 48S-54S
21 Kimmings AN, vanDeventer SJ, Obertop H, Rauws EA, Huibregtse K, Gouma DJ.Endotoxin, cytokines, and endotoxin binding
proteins in obstructive jaundice and after preoperative biliary drainage. Gut 2000; 46: 725-731
22 Erwin PJ, Lewis H, Dolan S, Tobias PS, Schumann RR, Lamping N, Wisdom GB, Rowlands BJ, Halliday MI. Lipopolysaccharide
binding protein in acute pancreatitis. Crit Care Med 2000; 28: 104-109
23 Zweigner J, Gramm HJ, Singer OC, Wegscheider K, Schumann RR. High concentrations of lipopolysaccharide-binding protein
in serum of patients with severe sepsis or septic shock inhibit the lipopolysaccharide response in human monocytes.
Blood 2001; 98: 3800-3808
24 Sablotzki A, Borgermann J, Baulig W, Friedrich I, Spillner J, Silber RE, Czeslick E. Lipopolysaccharide-binding protein (LBP) and
markers of acute-phase response in patients with multiple organ dysfunction syndrome (MODS) following open heart surgery.
Thorac Cardiovasc Surg 2001; 49: 273-278
25 Haudek SB, Natmessnig BE, Redl H, Schlag G, Hatlen LE, Tobias PS. Isolation, partial characterization, and concentration in
experimental sepsis of baboon lipopolysaccharide-binding protein. J Lab Clin Med 2000; 136: 363-370
26 Kimmings AN, Vandeventer SJ, Rauws EAJ, Huibregtse K, Gouma DJ. Systemic inflammatory response in acute cholangitis and
after subsequent treatment. Eur J Surg 2000; 166: 700-705
27 Schroder NW, Opitz B, Lamping N, Michelsen KS, Zahringer U, Gobel UB, Schumann RR. Involvement of lipopolysaccharide
binding protein, CD14, and Toll-like receptors in the initiation of innate immune responses by Treponema glycolipids.
J Immunol 2000; 165: 2683-2693
28 Yang YX. The progress in the pathogenesis of overwhelming postsplenectomy infection. Mod Surg 1999;5:59-62
29 Werner AM, Solis MM, Vogel R, Southerland SS, Ashley AV, Floyd JC, Brown C, Ashley DW. Improved antibody responses to
delayed pneumococcal vaccination in splenectomized rats. Am Surg 1999; 65: 844-847
30 Brigden ML, Pattullo AL. Prevention and management of overwhelming postsplenectomy infection--an update.
Crit Care Med 1999; 27: 836-842
31 Waghorn DJ. Overwhelming infection in asplenic patients: current best practice preventive measures are not being followed.
J Clin Pathol 2001; 54: 214-218
32 Yang YX, Lu JQ, Yao YM, Jiao HB, Yu Y, Fu J. The effect of splenectomy on circulating endotoxin clearance and tissue distribution
of endotoxin in rats. Chin J Surg 2000; 38: 787-789
33 Su GL, Freeswick PD, Geller DA. Molecular cloning, characterization, and tissue distribution of rat lipopolysaccharide binding protein,
J Immunol 1994; 153:743-752
34 Estler HC, Grewe M, Gaussling R. Rat tumor necrosis factor-α: transcription in rat kupffer cells and in vitro posttranslational
processing based on a PCR derived cDNA. Biol Chem Hoppe Seyler 1992; 373: 271-281
35 Wang SB, Lu JQ, YU JQ, Lu LR, Yu Y, Yiao YM. The relationship between plasma endotoxin levels and systemic inflammatory
response syndrome in portal hypertensive cirrhosis patients after splenectomy. Chin Crit Care Med 1999;11: 538-540
36 Zhang GF, Zhang MA, Cheng YR, Wang L. The potential role of endothelin and nitric oxide in endotoxin-induced gastric mucosal
damage.Shijie Huaren Xiaohua Zhazhi 2000; 8: 24
37 Liu YQ, Li KY. The characteristic of gastric mucosa in patients with cirrhosis. Shijie Huaren Xiaohua Zhazhi 2000; (Suppl 8): 36
38 Li X, Wang YW. Progress in the study on endotoxemia. Shijie Huaren Xiaohua Zhazhi 2000; (Suppl 8): 114
39 Chi XL, Wang BE, Zhang SW, Zhang NN. Alterations of gastrointestina motility and mucosal barrier in shock rat model induced
by endotoxin plus TNF-α.Shijie Huaren Xiaohua Zhazhi 1999;7:510-512
40 Zhao LF, Han DW. Clinical significance of endotoxemia in liver diseases. Shijie Huaren Xiaohua Zhazhi 1999; 7: 391-393
41 Zhou RR, Liang B. The effect of lipopolysaccharide on hepatocytes in rats in vitro. Shijie Huaren Xiaohua Zhazhi 1999;7:424-425
42 Zhou RR, Liang B. The effect of lipopolysaccharide on hepatocytes cultured at various intervals.
Shijie Huaren Xiaohua Zhazhi 1999;7:431-432
43 Lu JQ, Yang YX, Yiao YM, Yuan SC, Jiao HB, Yu Y. The effect of splenectomy on endotoxin induced tumor necrosis factor
mRNA expression in liver and pulmonary tissue.Chin Crit Care Med 2000; 12: 338-340
44 Yang YX, Lu JQ, Yiao YM, Jiao HB, Yu Y, Fu J. Effect of splenectomy on endotoxin mediated tumor necrosis factor and
interleukin-6 in rats. Chin Crit Care Med 1999; 11: 712-714
45 Jiao HB，Lu JQ，Lu LY, Yu JQ, Wang SB, LI JY. The effect of slenectomy and Escherichia coli endotoxin insult on the plasma
endotoxin TNF and NO levels in rats. Chin J Gen Surg, 1997; 6:78-82
2.4 肝組織TNF-α mRNA表達 正常肝組織有少量TNF-α mRNA表達；內毒素攻擊后0.5h，脾切除大鼠肝組織TNF-α mRNA表達迅速升高，1.5h達注射前的5倍，峰值更高(增加42.9%倍)，與有脾組動物有顯著差異(玃<0.05)，12h恢復至正常水平（表2）.
2.5 肝組織TNF-α水平 內毒素攻擊后0.5h肝組織TNF-α水平顯著升高，脾切除動物和有脾動物分別升高至注射前的2.62倍和2.25倍，1.5h肝組織TNF-α水平達峰值，脾切除大鼠峰值是有脾大鼠的1.34倍，兩組差異顯著(P<0.01，表1).肝組織TNF-α濃度與TNF-α，LBP mRNA表達均顯著相關(r=0.8547和0.7222，P<0.01)，肝組織TNF-α mRNA表達與LBP mRNA表達有明顯相關性(r=0.6715，P<0.01).
3 討論
本研究顯示內毒素攻擊后10min脾切除動物肝組織內毒素水平明顯增高，表明脾切除后組織內毒素的排泄減緩，組織內滯留量增加，提示肝臟在脾切除后器官損害的發生、發展中可能起著重要的調控作用［35-42］.LBP在介導細胞應答反應中起顯著的增敏作用，臨床OPSI病和特點為起病突然，兇猛病情迅速惡化，短期內陷入休克，一般認為革蘭氏陰性菌是OPSI的重要病原菌之一［29-31］，但LBP增敏系統是否參與脾切除后感染的過程，本實驗結果顯示，正常肝組織中存在少量LBP mRNA表達，小劑量內毒素(100ng/kg)靜脈注射后，無脾大鼠肝組織LBP mRNA表達即迅速增高，1.5h達峰值水平，4h后逐漸下降，12h恢復至正常范圍，無脾動物肝組織LBP mRNA表達峰值比有脾大鼠明顯增高，1.5h和4h時均存在顯著差異，表明脾切除動物感染時器官存在LBP過量表達.LBP具有“酶”的催化作用，可促進內毒素聚體形成單體和加速內毒素與CD14結合, 從而增強細胞對內毒素的敏感性.LBP基因缺陷動物在給予內毒素刺激時不出現急性炎癥反應，體內TNF-α水平不增高.大量研究表明, 創傷和膿毒癥時血清中LBP水平升高, 組織內LBP mRNA表達也持續增加, 局部組織內LBP介導的增敏效應可能在器官功能損害發病過程中有著重要的意義.
RT-PCR結果表明，無脾大鼠內毒素攻擊后肝組織TNF-α mRNA大量表達，說明脾切除可能造成動物免疫功能紊亂，使機體處于一種“預激狀態”，同時組織內毒素積聚及LBP mRNA表達增加，促使組織TNF-α mRNA大量表達，TNF-α合成和釋放增多.我們即往研究表明，脾切除后較大劑量內毒素(10mg/kg)攻擊可造成動物死亡率明顯增高，血清中TNF-α水平上升；而給予TNF-α單克隆抗體進行干預，血中TNF-α水平明顯降低且死亡率下降，可見TNF-α在無脾動物膿毒癥中起著重要的作用［43-45].本實驗結果顯示，脾切除動物遭受低劑量內毒素(100ng/kg)攻擊后30min，肝組織TNF-α水平升高，1.5h達峰值；而有脾大鼠注射內毒素后峰值比無脾大鼠明顯降低.同時相關分析表明，肝組織內TNF-α mRNA表達與組織TNF-α濃度顯著正相關(r=0.8547, P<0.01)，提示TNF-α的調節可能發生在轉錄水平，調節TNF-α基因轉錄或翻譯可能更有效地阻止脾切除后細菌內毒素的再次打擊.本結果還顯示，組織TNF-α濃度增高及TNF-α mRNA表達上調與LBP mRNA表達規律相似，相關分析進一步表明，肝組織LBP mRNA與TNF-α mRNA表達具有顯著相關性，提示無脾動物內毒素血癥時，體內內毒素與LBP形成復合體，廣泛激活不同組織合成并釋放大量炎癥因子，最終導致機體多器官損害.病理證實有脾大鼠受內毒素攻擊后，肝臟出現脂肪變性和枯否氏細胞增生，有研究認為這種改變為可逆性的損傷，機體對這種可逆性損傷是能夠代償的，但脾切除大鼠在受到相同劑量的內毒素攻擊時，則形成肝臟小灶性壞死，成為不可逆性變化.
我們認為脾切除后機體對內毒素清除延遲，致使內毒素在組織積聚，內毒素刺激組織細胞LBP mRNA表達，大量合成與分泌LBP，LBP-內毒素復合體可增敏內毒素激活細胞，進而誘發炎癥因子大量產生和全身炎癥反應失控，造成器官明顯的損害，最終可能發展成MODS.提示LBP增敏效應可能是臨床OPSI病程兇險發展的重要機制之一，通過早期阻抑LBP增敏效應，可能會減緩病情的發展，這為脾切除后兇險感染的預防提供新的思路.
4 REFERENCES
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cultured intestinal epithelial cells and is present in the intestinal mucus of mice. J Immunol 2000; 165: 4561-4566
12 Jeon YJ, Han SB, Ahn KS, Kim HM. Differential activation of murine macrophages by angelan and LPS. Immunopharmacology
2000; 49: 275-284
13 Dentener MA, Vreugdenhil AC, Hoet PH, Vernooy JH, Nieman FH, Heumann D, Janssen YM, Buurman WA, Wouters EF. Production
of the acute-phase protein lipopolysaccharide-binding protein by respiratory type II epithelial cells: implications for local defense to
bacterial endotoxins. Am J Respir Cell Mol Biol 2000; 23: 146-153
14 Vesy CJ, Kitchens RL， Wolfbauer G, Albers JJ, Munford RS. Lipopolysaccharide-binding protein and phospholipid transfer protein
release lipopolysaccharides from gram-negative bacterial membranes. Infect Immun 2000; 68: 2410-2417
15 Mayeux PR.Pathobiology of lipopolysaccharide. J Toxicol Environ Health 1997; 51: 415-435
16 Yao YM, Yu Y, Fang WH, Zhe HX, Shi ZG, Sheng ZY, Schlag G. The significance of lipopolysaccharide-binding protein and
lipopolysac charide receptor CD14 in increasing sensitivity toendotoxin response and multiple organ damage following trauma.
Chin Critical Care Med 1998; 10: 712-718
17 Hou YF, Mao BL, Qian GS, Xv J. Expression of lipopolysaccharide binding protein mRNA in pulmonary tissues of rat with "two-hit"
acute lung injury and the protective effects of anisodamine in this event. Disan Junyi Daxue Xuebao 2001; 23: 824-826
18 Fang WH, Yao YM, SHI ZG, Yu Y, Wu Y, Lu LR. The significance of the expressions of lipopolysaccharide binding prot ein mRNA and
lipopolysaccharide receptor CD14 mRNA in the liver of burned rat. Chin J Burns 2000; 16: 157-160
19 Wiezer MJ, Meijer C, Sietses C, Prins HA, Cuesta MA Beelen RH, Meijer S, vanLeeuwen PA. Bactericidal/permeability-increasing
protein preserves leukocyte functions after major liver resection. Ann Surg 2000; 232: 208-215
20 Fujimoto M, Uemura M, Nakatani Y, Tsujita S, Hoppo K, Tamagawa T, Kitano H, Kikukawa M, Ann T, Ishii Y, Kojima H, Sakurai S ,
Tanaka R, Namisaki T, Noguchi R, Higashino T, Kikuchi E, Nishimura K, Takaya A, Fukui H. Plasma endotoxin and serum cytokine
levels in patients with alcoholic hepatitis: relation to severity of liver disturbance. Alcohol Clin Exp Res 2000; 24: 48S-54S
21 Kimmings AN, vanDeventer SJ, Obertop H, Rauws EA, Huibregtse K, Gouma DJ.Endotoxin, cytokines, and endotoxin binding
proteins in obstructive jaundice and after preoperative biliary drainage. Gut 2000; 46: 725-731
22 Erwin PJ, Lewis H, Dolan S, Tobias PS, Schumann RR, Lamping N, Wisdom GB, Rowlands BJ, Halliday MI. Lipopolysaccharide
binding protein in acute pancreatitis. Crit Care Med 2000; 28: 104-109
23 Zweigner J, Gramm HJ, Singer OC, Wegscheider K, Schumann RR. High concentrations of lipopolysaccharide-binding protein
in serum of patients with severe sepsis or septic shock inhibit the lipopolysaccharide response in human monocytes.
Blood 2001; 98: 3800-3808
24 Sablotzki A, Borgermann J, Baulig W, Friedrich I, Spillner J, Silber RE, Czeslick E. Lipopolysaccharide-binding protein (LBP) and
markers of acute-phase response in patients with multiple organ dysfunction syndrome (MODS) following open heart surgery.
Thorac Cardiovasc Surg 2001; 49: 273-278
25 Haudek SB, Natmessnig BE, Redl H, Schlag G, Hatlen LE, Tobias PS. Isolation, partial characterization, and concentration in
experimental sepsis of baboon lipopolysaccharide-binding protein. J Lab Clin Med 2000; 136: 363-370
26 Kimmings AN, Vandeventer SJ, Rauws EAJ, Huibregtse K, Gouma DJ. Systemic inflammatory response in acute cholangitis and
after subsequent treatment. Eur J Surg 2000; 166: 700-705
27 Schroder NW, Opitz B, Lamping N, Michelsen KS, Zahringer U, Gobel UB, Schumann RR. Involvement of lipopolysaccharide
binding protein, CD14, and Toll-like receptors in the initiation of innate immune responses by Treponema glycolipids.
J Immunol 2000; 165: 2683-2693
28 Yang YX. The progress in the pathogenesis of overwhelming postsplenectomy infection. Mod Surg 1999;5:59-62
29 Werner AM, Solis MM, Vogel R, Southerland SS, Ashley AV, Floyd JC, Brown C, Ashley DW. Improved antibody responses to
delayed pneumococcal vaccination in splenectomized rats. Am Surg 1999; 65: 844-847
30 Brigden ML, Pattullo AL. Prevention and management of overwhelming postsplenectomy infection--an update.
Crit Care Med 1999; 27: 836-842
31 Waghorn DJ. Overwhelming infection in asplenic patients: current best practice preventive measures are not being followed.
J Clin Pathol 2001; 54: 214-218
32 Yang YX, Lu JQ, Yao YM, Jiao HB, Yu Y, Fu J. The effect of splenectomy on circulating endotoxin clearance and tissue distribution
of endotoxin in rats. Chin J Surg 2000; 38: 787-789
33 Su GL, Freeswick PD, Geller DA. Molecular cloning, characterization, and tissue distribution of rat lipopolysaccharide binding protein,
J Immunol 1994; 153:743-752
34 Estler HC, Grewe M, Gaussling R. Rat tumor necrosis factor-α: transcription in rat kupffer cells and in vitro posttranslational
processing based on a PCR derived cDNA. Biol Chem Hoppe Seyler 1992; 373: 271-281
35 Wang SB, Lu JQ, YU JQ, Lu LR, Yu Y, Yiao YM. The relationship between plasma endotoxin levels and systemic inflammatory
response syndrome in portal hypertensive cirrhosis patients after splenectomy. Chin Crit Care Med 1999;11: 538-540
36 Zhang GF, Zhang MA, Cheng YR, Wang L. The potential role of endothelin and nitric oxide in endotoxin-induced gastric mucosal
damage.Shijie Huaren Xiaohua Zhazhi 2000; 8: 24
37 Liu YQ, Li KY. The characteristic of gastric mucosa in patients with cirrhosis. Shijie Huaren Xiaohua Zhazhi 2000; (Suppl 8): 36
38 Li X, Wang YW. Progress in the study on endotoxemia. Shijie Huaren Xiaohua Zhazhi 2000; (Suppl 8): 114
39 Chi XL, Wang BE, Zhang SW, Zhang NN. Alterations of gastrointestina motility and mucosal barrier in shock rat model induced
by endotoxin plus TNF-α.Shijie Huaren Xiaohua Zhazhi 1999;7:510-512
40 Zhao LF, Han DW. Clinical significance of endotoxemia in liver diseases. Shijie Huaren Xiaohua Zhazhi 1999; 7: 391-393
41 Zhou RR, Liang B. The effect of lipopolysaccharide on hepatocytes in rats in vitro. Shijie Huaren Xiaohua Zhazhi 1999;7:424-425
42 Zhou RR, Liang B. The effect of lipopolysaccharide on hepatocytes cultured at various intervals.
Shijie Huaren Xiaohua Zhazhi 1999;7:431-432
43 Lu JQ, Yang YX, Yiao YM, Yuan SC, Jiao HB, Yu Y. The effect of splenectomy on endotoxin induced tumor necrosis factor
mRNA expression in liver and pulmonary tissue.Chin Crit Care Med 2000; 12: 338-340
44 Yang YX, Lu JQ, Yiao YM, Jiao HB, Yu Y, Fu J. Effect of splenectomy on endotoxin mediated tumor necrosis factor and
interleukin-6 in rats. Chin Crit Care Med 1999; 11: 712-714
45 Jiao HB，Lu JQ，Lu LY, Yu JQ, Wang SB, LI JY. The effect of slenectomy and Escherichia coli endotoxin insult on the plasma
endotoxin TNF and NO levels in rats. Chin J Gen Surg, 1997; 6:78-82
