肝移植缺血再灌注后Kupffer细胞CD14的表达机制
彭勇,龚建平, 刘长安,李生伟, 刘海忠,李寿柏,重庆医科大学附属第二医院肝胆外科 重庆市 400010
彭勇,男, 1969-07-25生,四川省眉山县人,汉族, 博士,副教授, 主要从事肝移植相关研究.
国家自然科学基金资助项目,No. 30300337; 30200278; 30170919
项目负责人:彭勇, 400010, 重庆市渝中区临江路76号,重庆医科大学附属第二医院肝胆外科. pengyong725@sina.com
电话:023-63784264 传真:023-63829191
收稿日期:2004-01-15 接受日期:2004-02-24
CD14 expression in Kupffer cells of ischemia-reperfusion injury afterrat liver transplantation
Yong Peng, Jian-Ping Gong, Chang-An Liu, Sheng-Wei Li, Hai-Zhong Liu,Shou-Bai Li
Yong Peng, Jian-Ping Gong, Chang-An Liu, Sheng-Wei Li, Hai-Zhong Liu,Shou-Bai Li, Department of Hepatobiliary Surgery, Chongqing University ofMedical Sciences, Chongqing 400010, China
Supported by the National Natural Science Foundation of China, No.30300337; 30200278; 30170919
Correspondence to: Dr. Yong Peng, Department of HepatobiliarySurgery, the Second Affiliated Hospital, Chongqing University of MedicalSciences, 74 Linjiang Road, Chongqing 400010, China. pengyong725@sina.com
Received: 2004-01-15 Accepted: 2004-02-24
AbstractAIM: To study the expression of lipopolysaccharide receptor CD14 mRNAand protein in Kupffer cells and its role in ischemia-reperfusion injury (IRI)in rat liver graft.
METHODS: The Kupffer cells were isolated and divided into control,ischemia-reperfusion (IR), and anti CD14 antibody groups. The CD14 mRNA,CD14 protein, nuclear factor kappa B (NF-kB)activity, and TNF-aand IL-1 level in the culture supernatant were measured.
RESULTS: The CD14 mRNA, and protein in IR group were significantly higherthan those in control group (mRNA 1.28±0.12vs 0.42±0.02,protein 23.7±2.36vs 6.3±1.27,P <0.01). The NF-kBactivity, TNF-aand IL-1 level in IR group were significantly higher than those incontrol group (NF-kB2.79±0.48vs 0.27±0.01,TNF-a205.9±12.04ng/L vs 57.4±4.35ng/L, IL-1 176.8±8.94ng/L vs 37.6±3.47ng/L, P <0.01), and they greatly decreased after anti-CD14antibody treatment compared with IR group (NF-kB1.34±0.24vs 2.79±0.48,TNF-a129.6±6.48ng/L vs 205.9±12.04ng/L, IL-1 103.4±5.74ng/L vs 176.8±8.94ng/L, P <0.05), but still significantly higher than those incontrol group (NF-kB1.34±0.24vs 0.27±0.01,TNF-a129.6±6.48ng/L vs 57.4±4.35ng/L, IL-1 103.4±5.74ng/L vs 37.6±3.47ng/L, P <0.01).
CONCLUSION:LPS following IR can up-regulate the expression of CD14 mRNA and proteinin Kupffer cells, and subsequently activate NF-kBto produce cytokines. But other signal transduction pathways might alsoparticipate in the NF-kBactivation and IRI.
Peng Y, Gong JP, Liu CA, Li SW, Liu HZ, Li SB. CD14 expression in Kupffercells of ischemia-reperfusion injury after rat liver transplantation.Shijie Huaren XiaohuaZazhi 2004;12(6):1333-1336
摘要
目的: 研究大鼠肝移植缺血再灌注后Kupffer细胞脂多糖受体CD14表达即其参与缺血再灌注损伤的机制.
方法:分离培养大鼠Kupffer细胞，分为正常对照组，肝移植缺血再灌注组，抗CD14抗体组，检测Kupffer细胞CD14mRNA、膜蛋白表达，核转录因子kB活性、以及培养上清TNF-a、IL-1的分泌量.
结果:再灌注后Kupffer细胞CD14mRNA和膜蛋白表达明显高于对照组(mRNA1.28±0.12vs 0.42±0.02;膜蛋白23.7±2.36vs 6.3±1.27，P<0.01); 再灌注后核转录因子kB活性、培养上清TNF-a、IL-1表达量明显高于对照组(NF-kB2.79±0.48vs 0.27±0.01;TNF-a205.9±12.04ng/L vs 57.4±4.35ng/L; IL-1 176.8±8.94ng/L vs 37.6±3.47ng/L，P<0.01); 用抗CD14抗体后NF-kB活性、TNF-a、IL-1表达量与再灌注相比明显下降(NF-kB1.34±0.24vs 2.79±0.48;TNF-a129.6±6.48ng/L vs 205.9±12.04ng/L; IL-1 103.4±5.74ng/L vs 176.8±8.94ng/L; P <0.05)，但仍然高于对照组(NF-kB1.34±0.24vs 0.27±0.01;TNF-a129.6±6.48ng/L vs 57.4±4.35ng/L; IL-1 103.4±5.74ng/L vs 37.6±3.47ng/L，P<0.01).
结论:缺血再灌注时LPS能够上调Kupffer细胞CD14表达，激活NF-kB，启动细胞因子的转录和分泌，但尚存在除CD14以外的其他信号途径参与了NF-kB的激活和缺血再灌注损伤.
彭勇,龚建平, 刘长安,李生伟, 刘海忠,李寿柏. 肝移植缺血再灌注后Kupffer细胞CD14的表达机制.世界华人消化杂志 2004;12(6):1333-1336
0 引言脂多糖(LPS)受体CD14是存在于单核-巨噬细胞表面的一种膜蛋白，主要功能是识别LPS-LBP(脂多糖结合蛋白)复合物[1-3].LPS-LBP与CD14结合后，促使单核-巨噬细胞活化并释放多种前炎症因子，在内毒素血症、酒精性肝病、肝硬化等疾病中扮演重要角色[4-8].Kupffer细胞作为肝脏内一种独特的单核-巨噬细胞是否也表达CD14，CD14是否参与肝移植缺血再灌注损伤(ischemia-reperfusioninjury，IRI)，引起IRI的细胞因子是否由CD14信号传导通路产生，这些问题到目前为止均未得到解决.我们通过分离培养肝移植缺血再灌注(IR)后的Kupffer细胞，观察CD14mRNA和蛋白的表达，CD14胞内信号传导途径中NF-kB活性以及细胞因子的产生情况，探讨其引起IRI的确切机制.
1 材料和方法
1.1 材料 实验分三组(每组30例)，对照组，取正常大鼠Kupffer细胞为实验对象;再灌注(IR)组，取再灌注后1h Kupffer细胞为研究对象;抗CD14抗体组，取再灌注后1h Kupffer细胞，并在培养液中加入CD14抗体0.2mL共同培养12h. 健康♂Wistar大鼠，质量210-250g (重庆医科大学实验动物中心提供).参照Peng etal [9]的改进Kamada袖套法，建立大鼠原位肝移植动物模型.供体手术: 游离肝周韧带和血管，切取供肝，在水浴中完成门静脉、肝下下腔静脉袖套准备，胆管内支架插管.受体手术: 切除原肝，原位植入供肝，先完成肝上下腔静脉连续缝合，门静脉袖套吻合，结束无肝期，以此时作为再灌注开始.完成肝下下腔静脉袖套吻合和胆道重建.再灌注后1 h，参照Gonget al介绍的胶原酶肝脏原位灌注法分离Kupffer细胞[3].经门静脉插管，50g/L IV型胶原酶(Sigma)体外循环灌注消化肝脏，不连续Percoll(Pharmacia)密度梯度离心分离Kupffer细胞，用含100mL/L小牛血清的RPMI1640培养液，37°C，50mL/L CO2培养6h后，洗去未贴壁细胞，重悬贴壁细胞，调整细胞浓度为1×106待用.测其纯度大于90%，活力大于95%.
1.2 方法
1.2.1 Kupffer细胞CD14mRNA检测 采用Trizol试剂盒(lifeTechnologies)提取Kupffer细胞总RNA，取0.5mL RNA产物，用RT-PCR试剂盒(Roche)将其逆转录为互补DNA(cDNA)，-70°C保存待用.以b-actin作为内参对照进行PCR反应(表1)，CD14以及b-actin引物由上海生工合成.PCR循环条件为:94 °C 1 min，58°C 1 min，72°C 1 min，30个循环，72°C延长7min. 用15 g/L琼脂糖凝胶电泳PCR产物，EB染色，凝胶呈像系统和图像分析系统观察并半定量计算PCR产物的相对表达量，结果以CD14/b-actin的灰度比值表示.
1.2.2 Kupffer细胞CD14蛋白的检测 采用Westernblot 检测Kupffer细胞CD14蛋白的表达.Kupffer细胞蛋白提取物50mg用100g/L SDS-PAGE进行电泳分离，电泳后的蛋白质转移至硝酸纤维膜4°C过夜，20g/L脱脂奶粉封闭1h，与抗CD14多克隆抗体(一抗，SantaCruz)反应2 h，PBST洗涤3次去除封闭液和一抗，与辣根过氧化酶标记的二抗(生物晶美公司)反应2h，PBST洗涤3次去除二抗.最后加入增强化学发光剂自显影，凝胶成像系统进行密度扫描，图像分析软件分析蛋白区带，CD14蛋白表达量用蛋白区带积分吸光度表示.
表1 RT-PCR检测CD14引物设计
1.2.3Kupffer细胞 NF-kB活性检测 应用凝胶迁移变动分析(EMSA)检测Kupffer细胞NF-kB活性.提取KC细胞核蛋白，NF-kB寡核苷酸探针序列为5’GCCTCC AAT GTT CGC GAA CTT TCG 3’(Santa Cruz产品)，32P标记，进行凝胶阻滞分析，放射自显影，测定每条电泳滞后带的吸光度值，所得结果表示NF-kB的相对活性.
1.2.4 细胞培养上清液TNF-a和IL-1检测 采用ELISA检测试剂盒(Sigma)测定上清液TNF-a和IL-1(操作步骤参见说明书).
统计学处理 实验数据以均数±标准差(mean±SD)表示，用SPSS9.0进行统计分析，以P<0.05为差别有显著性.
2 结果
2.1 Kupffer细胞CD14mRNA表达 对照组有微量CD14mRNA 表达，再灌注后CD14mRNA表达明显升高(CD14/b-actin1.28±0.12 vs 0.42±0.02，P<0.01).
2.2 Kupffer细胞 CD14蛋白表达 再灌注后Kupffer细胞CD14蛋白明显表达，而对照组仅有微量的CD14蛋白表达(23.7±2.36vs 6.3±1.27，P<0.01，图1).
2.3 Kupffer细胞 NF-kB活性改变 再灌注后NF-kB活性明显高于对照组(2.79±0.48vs 0.27±0.01，P<0.01)，应用抗CD14抗体后，NF-kB活性与IR相比显著降低(1.34±0.24vs 2.79±0.48，P<0.05)，但仍然高于对照组(1.34±0.24vs 0.27±0.01，P<0.01，图2).
2.4 TNF-a和IL-1表达量 IR组和抗CD14治疗组TNF-a和IL-1均明显高于对照组(P<0.01)，但抗CD14治疗组又明显低于IR组(P<0.05，图3).
图1(PDF)CD14蛋白Westernblot检测结果.
图2(PDF)NF-kBEMSA放射自显影图像.1: 对照组; 2:IR组; 3: 抗CD14组.
图3(PDF)Kupffer细胞培养上清中TNF-a和IL-1分泌量.
3 讨论CD14在介导内毒素引起的单核巨噬细胞活化过程中起启动作用[2,10-12]，Kupffer细胞是肝移植缺血再灌注损伤的主要参与者[13-16]，因此研究Kupffer细胞上CD14的表达以及其后的胞内信号传导途径，对于阐明肠道内毒素激活Kupffer细胞引起缺血再灌注损伤的机制意义重大[17-20].我们发现，正常处于静息状态下的Kupffer细胞有少量的CD14表达，这可能对于维持正常Kupffer细胞功能，使之处于应激状态是必需的[15,21-23]. 但再灌注后，大量内毒素由门静脉入肝，立即引起CD14mRNA以及蛋白的表达大量增多，启动Kupffer细胞的激活[24-27].
缺血再灌注损伤的最终形式表现为大量有害细胞因子(如TNF-a、IL-1等)对移植物的攻击和损伤[28-31].Kupffer细胞CD14表达增加是否与细胞因子分泌增多存在因果关系？我们通过研究CD14以后的胞内信号传导途径，特别是处于CD14与细胞因子之间的NF-kB的活性变化，阐明了他们之间的联系.再灌注后NF-kB活性明显高于对照，TNF-a等细胞因子的产生也较对照明显升高.上述一系列连续的变化过程表明，LPS-CD14-NF-kB-细胞因子是存在于再灌注损伤中的一条重要信号传导途径，CD14对其下游的NF-kB活化及细胞因子的产生存在必然关系.但我们同时也发现，CD14并非激活NF-kB惟一上游信号，通过用抗CD14抗体阻断CD14功能后，发现NF-kB活性以及细胞因子的产生有所降低，但仍然高于正常，证明除CD14途径外，尚有其他信号途径可以激活NF-kB.
总之，我们的研究表明Kupffer有CD14基因以及膜蛋白的表达，缺血再灌注后，CD14表达增强，内毒素通过与CD14的结合，启动Kupffer细胞激活的信号传导，信号通过多级酶联反应由胞外传到胞内，激活NF-kB，启动TNF-a等细胞因子的转录和分泌，最终造成对移植物的攻击和损害.但尚需进一步研究CD14至NF-kB之间的准确信号传导过程，以及除CD14以外的其他激活NF-kB的信号途径，才能完整阐明参与缺血再灌注损伤的整个信号传导通路.
致谢:本研究是在重庆市肝胆外科重点实验室完成，在此感谢!
4 参考文献1 Heumann D, Lauener R, Ryffel B.The dual role of LBP and CD14 inresponse to Gram-negative bacteria or Gram-negative
compounds. J Endotoxin Res 2003;9:381-384
2 Latz E, Visintin A, Lien E, Fitzgerald KA, Espevik T, Golenbock DT.The LPS receptor generates inflammatory signals from
the cell surface. J Endotoxin Res 2003;9:375-380
3 Lin M, Rikihisa Y.Ehrlichia chaffeensis downregulates surfaceToll-like receptors 2/4, CD14 and transcription factors PU.1
and inhibits lipopolysaccharide activationof NF-kappa B, ERK 1/2 and p38 MAPK in host monocytes. Cell Microbiol
2004;6:175-186
4 Gong JP, Wu CX, Liu CA, Li SW, Shi YJ, Yang K, Li Y, LiXH.Intestinal damage mediated by Kupffer cells in rats with
endotoxemia. World J Gastroenterol 2002;8:923-927
5 Olszyna DP, Verbon A, Pribble JP, Turner T, Axtelle T, van DeventerSJ, van der Poll T. Effect of IC14, an anti-CD14
antibody, on plasma and cell-associatedchemokines during human endotoxemia. Eur Cytokine Netw 2003;14:158-162
6 Takeda Y, Arii S, Mori A, Imamura M. High expression of the CD14gene and interleukin-1beta gene in the liver and lungs
of cirrhotic rats after partial hepatectomy.J Surg Res 2003;115:9-17
7 Nagy LE. Recent insights into the role of the innate immune systemin the development of alcoholic liver disease. Exp Biol
Med (Maywood) 2003;228:882-890
8 Dai LL, Gong JP, Zuo GQ, Wu CX, Shi YJ, Li XH, Peng Y, Deng W, LiSW, Liu CA. Synthesis of endotoxin receptor CD14
protein in Kupffer cells and its role inalcohol-induced liver disease. World J Gastroenterol 2003;9:622-626
9 Peng Y, Gong JP, Yan LN, Li SB, Li XH. Improved two-cuff techniquefor orthotopic liver transplantation in rat.
Hepatob-iliary Pancreat Dis Int 2004;3:33-37
10 Vives-Pi M, Somoza N, Fernandez-Alvarez J, Vargas F, Caro P, AlbaA, Gomis R, Labeta MO, Pujol-Borrell R. Evidence of
expression of endotoxin receptors CD14,toll-like receptors TLR4 and TLR2 and associated molecule MD-2 andof
sensitivity to endotoxin (LPS) in isletbeta cells. Clin Exp Immunol 2003;133:208-218
11 Opal SM, Palardy JE, Parejo N, Jasman RL. Effect of anti-CD14monoclonal antibody on clearance of Escherichia coli
bacteremia and endotoxemia. Crit Care Med 2003;31:929-932
12 Jiang JX, Chen YH, Xie GQ, Ji SH, Liu DW, Qiu J, Zhu PF, Wang ZG.Intrapulmonary expression of scavenger receptor and
CD14 and their relation to localinflammatory responses to endotoxemia in mice. World J Surg 2003;27:1-9
13 Vajdova K, Heinrich S, Tian Y, Graf R, Clavien PA. Ischemicpreconditioning and intermittent clamping improve murine
hepatic microcirculation and Kupffer cellfunction after ischemic injury. Liver Transpl 2004;10:520-528
14 Schauer RJ, Gerbes AL, Vonier D, Meissner H, Michl P, Leiderer R,Schildberg FW, Messmer K, Bilzer M. Glutathione
protects the rat liver against reperfusioninjury after prolonged warm ischemia. Ann Surg 2004;239:220-231
15 Cavalieri B, Perrelli MG, Aragno M, Ramadori P, Poli G, Cutrin JC.Ischaemic preconditioning modulates the activity of
Kupffer cells during in vivo reperfusioninjury of rat liver. Cell Biochem Funct 2003;21:299-305
16 Giakoustidis DE, Iliadis S, Tsantilas D, Papageorgiou G, Kontos N,Kostopoulou E, Botsoglou NA, Gerasimidis T, Dimitriadou
A. Blockade of Kupffer cells by gadoliniumchloride reduces lipid peroxidation and protects liver from ischemia/reperfusion
injury. Hepatogastroenterology 2003;50:1587-1592
17 Tsoulfas G, Takahashi Y, Ganster RW, Yagnik G, Guo Z, Fung JJ,Murase N, Geller DA. Activation of the lipopolysaccharide
signaling pathway in hepatictransplantation preservation injury. Transplantation 2002;74:7-13
18 Manigold T, Bocker U, Hanck C, Gundt J, Traber P, Antoni C, RossolS. Differential expression of toll-like receptors 2 and 4
in patients with liver cirrhosis. Eur JGastroenterol Hepatol 2003;15:275-282
19 Paik YH, Schwabe RF, Bataller R, Russo MP, Jobin C, Brenner DA.Toll-likereceptor 4 mediates inflammatory signaling by
bacterial lipopolysaccharide in humanhepatic stellate cells. Hepatology 2003;37:1043-1055
20 Su GL, Goyert SM, Fan MH, Aminlari A, Gong KQ, Klein RD, Myc A,Alarcon WH, Steinstraesser L, Remick DG, Wang
SC.Activation of human and mouse Kupffercells by lipopolysaccharide is mediated by CD14. Am J Physiol Gastrointest
Liver Physiol 2002;283:G640-645
21 Wheeler MD, Thurman RG. Up-regulation of CD14 in liver caused byacute ethanol involves oxidant-dependent AP-1
pathway. J Biol Chem 2003;278:8435-8441
22 Su GL. Lipopolysaccharides in liver injury: molecular mechanisms ofKupffer cell activation. Am J Physiol Gastrointest Liver
Physiol 2002;283:G256-265
23 Enomoto N, Takei Y, Hirose M, Ikejima K, Miwa H, Kitamura T, SatoN. Thalidomide prevents alcoholic liver injury in rats
through suppression of Kupffer cellsensitization and TNF-alpha production. Gastroenterology 2002;123:291-300
24 van Oosten M, van Amersfoort ES, van Berkel TJ, Kuiper J.Scavengerreceptor-like receptors for the binding of
lipopolysaccharide and lipoteichoic acid toliver endothelial and Kupffer cells. J Endotoxin Res 2001;7:381-384
25 Van Amersfoort ES, Van Berkel TJ, Kuiper J. Receptors, mediators,and mechanisms involved in bacterial sepsis and septic
shock. Clin Microbiol Rev 2003;16:379-414
26 Zipfel A, Schenk M, Metzdorf B, Bode C, Viebahn R. Release of TNF-alphafrom lipopolysaccharide (LPS)-stimulated Kupffer
cells in serum- and nutrient-free medium.Inflammation 2001;25:287-92
27 Enomoto N, Schemmer P, Ikejima K, Takei Y, Sato N, Brenner DA,Thurman RG. Long-term alcohol exposure changes
sensitivity of rat Kupffer cells tolipopolysaccharide. Alcohol Clin Exp Res 2001;25:1360-1367
28 Kozar RA, Schultz SG, Bick RJ, Poindexter BJ, DeSoignie R, MooreFA. Enteral glutamine but not alanine maintains small
bowel barrier function after ischemia/reperfusioninjury in rats. Shock 2004;21:433-437
29 Arumugam TV, Shiels IA, Woodruff TM, Granger DN, Taylor SM. Therole of the complement system in
ischemia-reperfusion injury. Shock 2004;21:401-409
30 Que X, Debonera F, Xie J, Furth EE, Aldeguer X, Gelman AE, OlthoffKM. Pattern of ischemia reperfusion injury in a mouse
orthotopic liver transplant model. J SurgRes 2004;116:262-268
31 Takeuchi D, Yoshidome H, Kato A, Ito H, Kimura F, Shimizu H,Ohtsuka M, Morita Y, Miyazaki M. Interleukin 18 causes
hepatic ischemia/reperfusion injury bysuppressing anti-inflammatory cytokine expression in mice. Hepatology
2004;39:699-710, 百拇医药( 彭 勇, 龚建平, 刘长安, 李生伟, 刘海忠, 李寿柏)
彭勇,男, 1969-07-25生,四川省眉山县人,汉族, 博士,副教授, 主要从事肝移植相关研究.
国家自然科学基金资助项目,No. 30300337; 30200278; 30170919
项目负责人:彭勇, 400010, 重庆市渝中区临江路76号,重庆医科大学附属第二医院肝胆外科. pengyong725@sina.com
电话:023-63784264 传真:023-63829191
收稿日期:2004-01-15 接受日期:2004-02-24
CD14 expression in Kupffer cells of ischemia-reperfusion injury afterrat liver transplantation
Yong Peng, Jian-Ping Gong, Chang-An Liu, Sheng-Wei Li, Hai-Zhong Liu,Shou-Bai Li
Yong Peng, Jian-Ping Gong, Chang-An Liu, Sheng-Wei Li, Hai-Zhong Liu,Shou-Bai Li, Department of Hepatobiliary Surgery, Chongqing University ofMedical Sciences, Chongqing 400010, China
Supported by the National Natural Science Foundation of China, No.30300337; 30200278; 30170919
Correspondence to: Dr. Yong Peng, Department of HepatobiliarySurgery, the Second Affiliated Hospital, Chongqing University of MedicalSciences, 74 Linjiang Road, Chongqing 400010, China. pengyong725@sina.com
Received: 2004-01-15 Accepted: 2004-02-24
AbstractAIM: To study the expression of lipopolysaccharide receptor CD14 mRNAand protein in Kupffer cells and its role in ischemia-reperfusion injury (IRI)in rat liver graft.
METHODS: The Kupffer cells were isolated and divided into control,ischemia-reperfusion (IR), and anti CD14 antibody groups. The CD14 mRNA,CD14 protein, nuclear factor kappa B (NF-kB)activity, and TNF-aand IL-1 level in the culture supernatant were measured.
RESULTS: The CD14 mRNA, and protein in IR group were significantly higherthan those in control group (mRNA 1.28±0.12vs 0.42±0.02,protein 23.7±2.36vs 6.3±1.27,P <0.01). The NF-kBactivity, TNF-aand IL-1 level in IR group were significantly higher than those incontrol group (NF-kB2.79±0.48vs 0.27±0.01,TNF-a205.9±12.04ng/L vs 57.4±4.35ng/L, IL-1 176.8±8.94ng/L vs 37.6±3.47ng/L, P <0.01), and they greatly decreased after anti-CD14antibody treatment compared with IR group (NF-kB1.34±0.24vs 2.79±0.48,TNF-a129.6±6.48ng/L vs 205.9±12.04ng/L, IL-1 103.4±5.74ng/L vs 176.8±8.94ng/L, P <0.05), but still significantly higher than those incontrol group (NF-kB1.34±0.24vs 0.27±0.01,TNF-a129.6±6.48ng/L vs 57.4±4.35ng/L, IL-1 103.4±5.74ng/L vs 37.6±3.47ng/L, P <0.01).
CONCLUSION:LPS following IR can up-regulate the expression of CD14 mRNA and proteinin Kupffer cells, and subsequently activate NF-kBto produce cytokines. But other signal transduction pathways might alsoparticipate in the NF-kBactivation and IRI.
Peng Y, Gong JP, Liu CA, Li SW, Liu HZ, Li SB. CD14 expression in Kupffercells of ischemia-reperfusion injury after rat liver transplantation.Shijie Huaren XiaohuaZazhi 2004;12(6):1333-1336
摘要
目的: 研究大鼠肝移植缺血再灌注后Kupffer细胞脂多糖受体CD14表达即其参与缺血再灌注损伤的机制.
方法:分离培养大鼠Kupffer细胞，分为正常对照组，肝移植缺血再灌注组，抗CD14抗体组，检测Kupffer细胞CD14mRNA、膜蛋白表达，核转录因子kB活性、以及培养上清TNF-a、IL-1的分泌量.
结果:再灌注后Kupffer细胞CD14mRNA和膜蛋白表达明显高于对照组(mRNA1.28±0.12vs 0.42±0.02;膜蛋白23.7±2.36vs 6.3±1.27，P<0.01); 再灌注后核转录因子kB活性、培养上清TNF-a、IL-1表达量明显高于对照组(NF-kB2.79±0.48vs 0.27±0.01;TNF-a205.9±12.04ng/L vs 57.4±4.35ng/L; IL-1 176.8±8.94ng/L vs 37.6±3.47ng/L，P<0.01); 用抗CD14抗体后NF-kB活性、TNF-a、IL-1表达量与再灌注相比明显下降(NF-kB1.34±0.24vs 2.79±0.48;TNF-a129.6±6.48ng/L vs 205.9±12.04ng/L; IL-1 103.4±5.74ng/L vs 176.8±8.94ng/L; P <0.05)，但仍然高于对照组(NF-kB1.34±0.24vs 0.27±0.01;TNF-a129.6±6.48ng/L vs 57.4±4.35ng/L; IL-1 103.4±5.74ng/L vs 37.6±3.47ng/L，P<0.01).
结论:缺血再灌注时LPS能够上调Kupffer细胞CD14表达，激活NF-kB，启动细胞因子的转录和分泌，但尚存在除CD14以外的其他信号途径参与了NF-kB的激活和缺血再灌注损伤.
彭勇,龚建平, 刘长安,李生伟, 刘海忠,李寿柏. 肝移植缺血再灌注后Kupffer细胞CD14的表达机制.世界华人消化杂志 2004;12(6):1333-1336
0 引言脂多糖(LPS)受体CD14是存在于单核-巨噬细胞表面的一种膜蛋白，主要功能是识别LPS-LBP(脂多糖结合蛋白)复合物[1-3].LPS-LBP与CD14结合后，促使单核-巨噬细胞活化并释放多种前炎症因子，在内毒素血症、酒精性肝病、肝硬化等疾病中扮演重要角色[4-8].Kupffer细胞作为肝脏内一种独特的单核-巨噬细胞是否也表达CD14，CD14是否参与肝移植缺血再灌注损伤(ischemia-reperfusioninjury，IRI)，引起IRI的细胞因子是否由CD14信号传导通路产生，这些问题到目前为止均未得到解决.我们通过分离培养肝移植缺血再灌注(IR)后的Kupffer细胞，观察CD14mRNA和蛋白的表达，CD14胞内信号传导途径中NF-kB活性以及细胞因子的产生情况，探讨其引起IRI的确切机制.
1 材料和方法
1.1 材料 实验分三组(每组30例)，对照组，取正常大鼠Kupffer细胞为实验对象;再灌注(IR)组，取再灌注后1h Kupffer细胞为研究对象;抗CD14抗体组，取再灌注后1h Kupffer细胞，并在培养液中加入CD14抗体0.2mL共同培养12h. 健康♂Wistar大鼠，质量210-250g (重庆医科大学实验动物中心提供).参照Peng etal [9]的改进Kamada袖套法，建立大鼠原位肝移植动物模型.供体手术: 游离肝周韧带和血管，切取供肝，在水浴中完成门静脉、肝下下腔静脉袖套准备，胆管内支架插管.受体手术: 切除原肝，原位植入供肝，先完成肝上下腔静脉连续缝合，门静脉袖套吻合，结束无肝期，以此时作为再灌注开始.完成肝下下腔静脉袖套吻合和胆道重建.再灌注后1 h，参照Gonget al介绍的胶原酶肝脏原位灌注法分离Kupffer细胞[3].经门静脉插管，50g/L IV型胶原酶(Sigma)体外循环灌注消化肝脏，不连续Percoll(Pharmacia)密度梯度离心分离Kupffer细胞，用含100mL/L小牛血清的RPMI1640培养液，37°C，50mL/L CO2培养6h后，洗去未贴壁细胞，重悬贴壁细胞，调整细胞浓度为1×106待用.测其纯度大于90%，活力大于95%.
1.2 方法
1.2.1 Kupffer细胞CD14mRNA检测 采用Trizol试剂盒(lifeTechnologies)提取Kupffer细胞总RNA，取0.5mL RNA产物，用RT-PCR试剂盒(Roche)将其逆转录为互补DNA(cDNA)，-70°C保存待用.以b-actin作为内参对照进行PCR反应(表1)，CD14以及b-actin引物由上海生工合成.PCR循环条件为:94 °C 1 min，58°C 1 min，72°C 1 min，30个循环，72°C延长7min. 用15 g/L琼脂糖凝胶电泳PCR产物，EB染色，凝胶呈像系统和图像分析系统观察并半定量计算PCR产物的相对表达量，结果以CD14/b-actin的灰度比值表示.
1.2.2 Kupffer细胞CD14蛋白的检测 采用Westernblot 检测Kupffer细胞CD14蛋白的表达.Kupffer细胞蛋白提取物50mg用100g/L SDS-PAGE进行电泳分离，电泳后的蛋白质转移至硝酸纤维膜4°C过夜，20g/L脱脂奶粉封闭1h，与抗CD14多克隆抗体(一抗，SantaCruz)反应2 h，PBST洗涤3次去除封闭液和一抗，与辣根过氧化酶标记的二抗(生物晶美公司)反应2h，PBST洗涤3次去除二抗.最后加入增强化学发光剂自显影，凝胶成像系统进行密度扫描，图像分析软件分析蛋白区带，CD14蛋白表达量用蛋白区带积分吸光度表示.
表1 RT-PCR检测CD14引物设计
| 引物序列 | 长度(bp) | |
| CD14 | 5’CTCAACCTAGAGCCGTTTCT 3’ | 267 |
| 5’CAGGATTGTCAGACAGGTCT 3’ | ||
| b-actin | 5’ATCATGTTTGAGACCTTCAACA 3’ | 300 |
| 5’CATCTCTTGCTCGAAGTCCA 3’ |
1.2.3Kupffer细胞 NF-kB活性检测 应用凝胶迁移变动分析(EMSA)检测Kupffer细胞NF-kB活性.提取KC细胞核蛋白，NF-kB寡核苷酸探针序列为5’GCCTCC AAT GTT CGC GAA CTT TCG 3’(Santa Cruz产品)，32P标记，进行凝胶阻滞分析，放射自显影，测定每条电泳滞后带的吸光度值，所得结果表示NF-kB的相对活性.
1.2.4 细胞培养上清液TNF-a和IL-1检测 采用ELISA检测试剂盒(Sigma)测定上清液TNF-a和IL-1(操作步骤参见说明书).
统计学处理 实验数据以均数±标准差(mean±SD)表示，用SPSS9.0进行统计分析，以P<0.05为差别有显著性.
2 结果
2.1 Kupffer细胞CD14mRNA表达 对照组有微量CD14mRNA 表达，再灌注后CD14mRNA表达明显升高(CD14/b-actin1.28±0.12 vs 0.42±0.02，P<0.01).
2.2 Kupffer细胞 CD14蛋白表达 再灌注后Kupffer细胞CD14蛋白明显表达，而对照组仅有微量的CD14蛋白表达(23.7±2.36vs 6.3±1.27，P<0.01，图1).
2.3 Kupffer细胞 NF-kB活性改变 再灌注后NF-kB活性明显高于对照组(2.79±0.48vs 0.27±0.01，P<0.01)，应用抗CD14抗体后，NF-kB活性与IR相比显著降低(1.34±0.24vs 2.79±0.48，P<0.05)，但仍然高于对照组(1.34±0.24vs 0.27±0.01，P<0.01，图2).
2.4 TNF-a和IL-1表达量 IR组和抗CD14治疗组TNF-a和IL-1均明显高于对照组(P<0.01)，但抗CD14治疗组又明显低于IR组(P<0.05，图3).
图1(PDF)CD14蛋白Westernblot检测结果.
图2(PDF)NF-kBEMSA放射自显影图像.1: 对照组; 2:IR组; 3: 抗CD14组.
图3(PDF)Kupffer细胞培养上清中TNF-a和IL-1分泌量.
3 讨论CD14在介导内毒素引起的单核巨噬细胞活化过程中起启动作用[2,10-12]，Kupffer细胞是肝移植缺血再灌注损伤的主要参与者[13-16]，因此研究Kupffer细胞上CD14的表达以及其后的胞内信号传导途径，对于阐明肠道内毒素激活Kupffer细胞引起缺血再灌注损伤的机制意义重大[17-20].我们发现，正常处于静息状态下的Kupffer细胞有少量的CD14表达，这可能对于维持正常Kupffer细胞功能，使之处于应激状态是必需的[15,21-23]. 但再灌注后，大量内毒素由门静脉入肝，立即引起CD14mRNA以及蛋白的表达大量增多，启动Kupffer细胞的激活[24-27].
缺血再灌注损伤的最终形式表现为大量有害细胞因子(如TNF-a、IL-1等)对移植物的攻击和损伤[28-31].Kupffer细胞CD14表达增加是否与细胞因子分泌增多存在因果关系？我们通过研究CD14以后的胞内信号传导途径，特别是处于CD14与细胞因子之间的NF-kB的活性变化，阐明了他们之间的联系.再灌注后NF-kB活性明显高于对照，TNF-a等细胞因子的产生也较对照明显升高.上述一系列连续的变化过程表明，LPS-CD14-NF-kB-细胞因子是存在于再灌注损伤中的一条重要信号传导途径，CD14对其下游的NF-kB活化及细胞因子的产生存在必然关系.但我们同时也发现，CD14并非激活NF-kB惟一上游信号，通过用抗CD14抗体阻断CD14功能后，发现NF-kB活性以及细胞因子的产生有所降低，但仍然高于正常，证明除CD14途径外，尚有其他信号途径可以激活NF-kB.
总之，我们的研究表明Kupffer有CD14基因以及膜蛋白的表达，缺血再灌注后，CD14表达增强，内毒素通过与CD14的结合，启动Kupffer细胞激活的信号传导，信号通过多级酶联反应由胞外传到胞内，激活NF-kB，启动TNF-a等细胞因子的转录和分泌，最终造成对移植物的攻击和损害.但尚需进一步研究CD14至NF-kB之间的准确信号传导过程，以及除CD14以外的其他激活NF-kB的信号途径，才能完整阐明参与缺血再灌注损伤的整个信号传导通路.
致谢:本研究是在重庆市肝胆外科重点实验室完成，在此感谢!
4 参考文献1 Heumann D, Lauener R, Ryffel B.The dual role of LBP and CD14 inresponse to Gram-negative bacteria or Gram-negative
compounds. J Endotoxin Res 2003;9:381-384
2 Latz E, Visintin A, Lien E, Fitzgerald KA, Espevik T, Golenbock DT.The LPS receptor generates inflammatory signals from
the cell surface. J Endotoxin Res 2003;9:375-380
3 Lin M, Rikihisa Y.Ehrlichia chaffeensis downregulates surfaceToll-like receptors 2/4, CD14 and transcription factors PU.1
and inhibits lipopolysaccharide activationof NF-kappa B, ERK 1/2 and p38 MAPK in host monocytes. Cell Microbiol
2004;6:175-186
4 Gong JP, Wu CX, Liu CA, Li SW, Shi YJ, Yang K, Li Y, LiXH.Intestinal damage mediated by Kupffer cells in rats with
endotoxemia. World J Gastroenterol 2002;8:923-927
5 Olszyna DP, Verbon A, Pribble JP, Turner T, Axtelle T, van DeventerSJ, van der Poll T. Effect of IC14, an anti-CD14
antibody, on plasma and cell-associatedchemokines during human endotoxemia. Eur Cytokine Netw 2003;14:158-162
6 Takeda Y, Arii S, Mori A, Imamura M. High expression of the CD14gene and interleukin-1beta gene in the liver and lungs
of cirrhotic rats after partial hepatectomy.J Surg Res 2003;115:9-17
7 Nagy LE. Recent insights into the role of the innate immune systemin the development of alcoholic liver disease. Exp Biol
Med (Maywood) 2003;228:882-890
8 Dai LL, Gong JP, Zuo GQ, Wu CX, Shi YJ, Li XH, Peng Y, Deng W, LiSW, Liu CA. Synthesis of endotoxin receptor CD14
protein in Kupffer cells and its role inalcohol-induced liver disease. World J Gastroenterol 2003;9:622-626
9 Peng Y, Gong JP, Yan LN, Li SB, Li XH. Improved two-cuff techniquefor orthotopic liver transplantation in rat.
Hepatob-iliary Pancreat Dis Int 2004;3:33-37
10 Vives-Pi M, Somoza N, Fernandez-Alvarez J, Vargas F, Caro P, AlbaA, Gomis R, Labeta MO, Pujol-Borrell R. Evidence of
expression of endotoxin receptors CD14,toll-like receptors TLR4 and TLR2 and associated molecule MD-2 andof
sensitivity to endotoxin (LPS) in isletbeta cells. Clin Exp Immunol 2003;133:208-218
11 Opal SM, Palardy JE, Parejo N, Jasman RL. Effect of anti-CD14monoclonal antibody on clearance of Escherichia coli
bacteremia and endotoxemia. Crit Care Med 2003;31:929-932
12 Jiang JX, Chen YH, Xie GQ, Ji SH, Liu DW, Qiu J, Zhu PF, Wang ZG.Intrapulmonary expression of scavenger receptor and
CD14 and their relation to localinflammatory responses to endotoxemia in mice. World J Surg 2003;27:1-9
13 Vajdova K, Heinrich S, Tian Y, Graf R, Clavien PA. Ischemicpreconditioning and intermittent clamping improve murine
hepatic microcirculation and Kupffer cellfunction after ischemic injury. Liver Transpl 2004;10:520-528
14 Schauer RJ, Gerbes AL, Vonier D, Meissner H, Michl P, Leiderer R,Schildberg FW, Messmer K, Bilzer M. Glutathione
protects the rat liver against reperfusioninjury after prolonged warm ischemia. Ann Surg 2004;239:220-231
15 Cavalieri B, Perrelli MG, Aragno M, Ramadori P, Poli G, Cutrin JC.Ischaemic preconditioning modulates the activity of
Kupffer cells during in vivo reperfusioninjury of rat liver. Cell Biochem Funct 2003;21:299-305
16 Giakoustidis DE, Iliadis S, Tsantilas D, Papageorgiou G, Kontos N,Kostopoulou E, Botsoglou NA, Gerasimidis T, Dimitriadou
A. Blockade of Kupffer cells by gadoliniumchloride reduces lipid peroxidation and protects liver from ischemia/reperfusion
injury. Hepatogastroenterology 2003;50:1587-1592
17 Tsoulfas G, Takahashi Y, Ganster RW, Yagnik G, Guo Z, Fung JJ,Murase N, Geller DA. Activation of the lipopolysaccharide
signaling pathway in hepatictransplantation preservation injury. Transplantation 2002;74:7-13
18 Manigold T, Bocker U, Hanck C, Gundt J, Traber P, Antoni C, RossolS. Differential expression of toll-like receptors 2 and 4
in patients with liver cirrhosis. Eur JGastroenterol Hepatol 2003;15:275-282
19 Paik YH, Schwabe RF, Bataller R, Russo MP, Jobin C, Brenner DA.Toll-likereceptor 4 mediates inflammatory signaling by
bacterial lipopolysaccharide in humanhepatic stellate cells. Hepatology 2003;37:1043-1055
20 Su GL, Goyert SM, Fan MH, Aminlari A, Gong KQ, Klein RD, Myc A,Alarcon WH, Steinstraesser L, Remick DG, Wang
SC.Activation of human and mouse Kupffercells by lipopolysaccharide is mediated by CD14. Am J Physiol Gastrointest
Liver Physiol 2002;283:G640-645
21 Wheeler MD, Thurman RG. Up-regulation of CD14 in liver caused byacute ethanol involves oxidant-dependent AP-1
pathway. J Biol Chem 2003;278:8435-8441
22 Su GL. Lipopolysaccharides in liver injury: molecular mechanisms ofKupffer cell activation. Am J Physiol Gastrointest Liver
Physiol 2002;283:G256-265
23 Enomoto N, Takei Y, Hirose M, Ikejima K, Miwa H, Kitamura T, SatoN. Thalidomide prevents alcoholic liver injury in rats
through suppression of Kupffer cellsensitization and TNF-alpha production. Gastroenterology 2002;123:291-300
24 van Oosten M, van Amersfoort ES, van Berkel TJ, Kuiper J.Scavengerreceptor-like receptors for the binding of
lipopolysaccharide and lipoteichoic acid toliver endothelial and Kupffer cells. J Endotoxin Res 2001;7:381-384
25 Van Amersfoort ES, Van Berkel TJ, Kuiper J. Receptors, mediators,and mechanisms involved in bacterial sepsis and septic
shock. Clin Microbiol Rev 2003;16:379-414
26 Zipfel A, Schenk M, Metzdorf B, Bode C, Viebahn R. Release of TNF-alphafrom lipopolysaccharide (LPS)-stimulated Kupffer
cells in serum- and nutrient-free medium.Inflammation 2001;25:287-92
27 Enomoto N, Schemmer P, Ikejima K, Takei Y, Sato N, Brenner DA,Thurman RG. Long-term alcohol exposure changes
sensitivity of rat Kupffer cells tolipopolysaccharide. Alcohol Clin Exp Res 2001;25:1360-1367
28 Kozar RA, Schultz SG, Bick RJ, Poindexter BJ, DeSoignie R, MooreFA. Enteral glutamine but not alanine maintains small
bowel barrier function after ischemia/reperfusioninjury in rats. Shock 2004;21:433-437
29 Arumugam TV, Shiels IA, Woodruff TM, Granger DN, Taylor SM. Therole of the complement system in
ischemia-reperfusion injury. Shock 2004;21:401-409
30 Que X, Debonera F, Xie J, Furth EE, Aldeguer X, Gelman AE, OlthoffKM. Pattern of ischemia reperfusion injury in a mouse
orthotopic liver transplant model. J SurgRes 2004;116:262-268
31 Takeuchi D, Yoshidome H, Kato A, Ito H, Kimura F, Shimizu H,Ohtsuka M, Morita Y, Miyazaki M. Interleukin 18 causes
hepatic ischemia/reperfusion injury bysuppressing anti-inflammatory cytokine expression in mice. Hepatology
2004;39:699-710, 百拇医药( 彭 勇, 龚建平, 刘长安, 李生伟, 刘海忠, 李寿柏)
