基础研究 Open Access
Copyright ©The Author(s) 2004. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2004-06-15; 12(6): 1333-1336
在线出版日期: 2004-06-15. doi: 10.11569/wcjd.v12.i6.1333
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
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.
基金项目: 国家自然科学基金资助项目, No. 30300337; 30200278; 30170919.
通讯作者: 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
修回日期: 2004-02-09
接受日期: 2004-02-24
在线出版日期: 2004-06-15

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-α、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(mRNA 1.28±0.12 vs 0.42±0.02; 膜蛋白23.7±2.36 vs 6.3±1.27, P<0.01); 再灌注后核转录因子kB活性、培养上清TNF-α、IL-1表达量明显高于对照组(NF-κB 2.79±0.48 vs 0.27±0.01; TNF-α 205.9±12.04 ng/L vs 57.4±4.35 ng/L; IL-1 176.8±8.94 ng/L vs 37.6±3.47 ng/L, P<0.01); 用抗CD14抗体后NF-κB活性、TNF-α、IL-1表达量与再灌注相比明显下降(NF-κB 1.34±0.24 vs 2.79±0.48; TNF-α 129.6±6.48 ng/L vs 205.9±12.04 ng/L; IL-1 103.4±5.74 ng/L vs 176.8±8.94 ng/L; P<0.05), 但仍然高于对照组(NF-κB 1.34±0.24 vs 0.27±0.01; TNF-α 129.6±6.48 ng/L vs 57.4±4.35 ng/L; IL-1 103.4±5.74 ng/L vs 37.6±3.47 ng/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-κB, 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-κB的激活和缺血再灌注损伤.

关键词: N/A
引文著录: 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
CD14 expression in Kupffer cells of ischemia-reperfusion injury after rat 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 of Medical Sciences, Chongqing 400010, China
Supported by: the National Natural Science Foundation of China, No. 30300337; 30200278; 30170919.
Correspondence to: Dr. Yong Peng, Department of Hepatobiliary Surgery, the Second Affiliated Hospital, Chongqing University of Medical Sciences, 74 Linjiang Road, Chongqing 400010, China. pengyong725@sina.com
Received: January 15, 2004
Revised: February 9, 2004
Accepted: February 24, 2004
Published online: June 15, 2004

AIM: To study the expression of lipopolysaccharide receptor CD14 mRNA and 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-κB) activity, and TNF-α and IL-1 level in the culture supernatant were measured.

RESULTS: The CD14 mRNA, and protein in IR group were significantly higher than those in control group (mRNA 1.28±0.12 vs 0.42±0.02, protein 23.7±2.36 vs 6.3±1.27, P < 0.01). The NF-κB activity, TNF-α and IL-1 level in IR group were significantly higher than those in control group (NF-κB 2.79±0.48 vs 0.27±0.01, TNF-α 205.9±12.04 ng/L vs 57.4±4.35 ng/L, IL-1 176.8±8.94 ng/L vs 37.6±3.47 ng/L, P < 0.01), and they greatly decreased after anti-CD14 antibody treatment compared with IR group (NF-κB 1.34±0.24 vs 2.79±0.48, TNF-α 129.6±6.48 ng/L vs 205.9±12.04 ng/L, IL-1 103.4±5.74 ng/L vs 176.8±8.94 ng/L, P < 0.05), but still significantly higher than those in control group (NF-κB 1.34±0.24 vs 0.27±0.01, TNF-α 129.6±6.48 ng/L vs 57.4±4.35 ng/L, IL-1 103.4±5.74 ng/L vs 37.6±3.47 ng/L, P < 0.01).

CONCLUSION: LPS following IR can up-regulate the expression of CD14 mRNA and protein in Kupffer cells, and subsequently activate NF-κB to produce cytokines. But other signal transduction pathways might also participate in the NF-κB activation and IRI.

Key Words: N/A
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-reperfusion injury, 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-κB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1 h Kupffer细胞为研究对象; 抗CD14抗体组, 取再灌注后1 h Kupffer细胞, 并在培养液中加入CD14抗体0.2 mL共同培养12 h. 健康♂Wistar大鼠, 质量210-250 g (重庆医科大学实验动物中心提供). 参照Peng et al[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Gong et 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, 50 g/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100 mL/L小牛血清的RPMI1640培养液, 37 ℃, 50 mL/L CO2培养6 h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CD14 mRNA检测: 采用Trizol试剂盒(life Technologies)提取Kupffer细胞总RNA, 取0.5 mL RNA产物, 用RT-PCR试剂盒(Roche)将其逆转录为互补DNA(cDNA), -70 ℃保存待用. 以β-actin作为内参对照进行PCR反应(表1), CD14以及β-actin引物由上海生工合成. PCR循环条件为: 94 ℃ 1 min, 58 ℃ 1 min, 72 ℃ 1 min, 30个循环, 72 ℃延长7 min. 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/β-actin的灰度比值表示.

表1 RT-PCR检测CD14引物设计.
引物序列长度(bp)
CD145'CTCAACCTAGAGCCGTTTCT 3'267
5'CAGGATTGTCAGACAGGTCT 3'
β-actin5'ATCATGTTTGAGACCTTCAACA 3'300
5'CATCTCTTGCTCGAAGTCCA 3'

1.2.2 Kupffer细胞 CD14蛋白的检测: 采用Western blot 检测Kupffer细胞 CD14蛋白的表达. Kupffer细胞蛋白提取物50 mg用100 g/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 ℃过夜, 20 g/L脱脂奶粉封闭1 h, 与抗CD14多克隆抗体(一抗, Santa Cruz)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2 h, 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.

1.2.3 Kupffer细胞 NF-κB活性检测: 应用凝胶迁移变动分析(EMSA)检测Kupffer细胞NF-κB活性. 提取KC细胞核蛋白, NF-κB寡核苷酸探针序列为5'GCC TCC 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-κB的相对活性.

1.2.4 细胞培养上清液TNF-α和IL-1检测: 采用ELISA检测试剂盒(Sigma)测定上清液TNF-α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CD14 mRNA表达

对照组有微量CD14 mRNA 表达, 再灌注后CD14 mRNA表达明显升高(CD14/β-actin 1.28±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±2.36 vs 6.3±1.27, P<0.01, 图1).

图1
图1 CD14蛋白Western blot检测结果.
2.3 Kupffer细胞 NF-κB活性改变

再灌注后NF-κB活性明显高于对照组(2.79±0.48 vs 0.27±0.01, P<0.01), 应用抗CD14抗体后, NF-κB活性与IR相比显著降低(1.34±0.24 vs 2.79±0.48, P<0.05), 但仍然高于对照组(1.34±0.24 vs 0.27±0.01, P<0.01, 图2).

图2
图2 NF-κB EMSA放射自显影图像. 1: 对照组; 2: IR组; 3: 抗CD14组.
2.4 TNF-α和IL-1表达量

IR组和抗CD14治疗组TNF-α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3
图3 Kupffer细胞培养上清中TNF-α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-α、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-κB的活性变化, 阐明了他们之间的联系. 再灌注后NF-κB活性明显高于对照, TNF-α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-κB-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-κB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-κB惟一上游信号, 通过用抗CD14抗体阻断CD14功能后, 发现NF-κB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-κB.

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-κB, 启动TNF-α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-κB之间的准确信号传导过程, 以及除CD14以外的其他激活NF-κB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!

1.  Heumann D, Lauener R, Ryffel B. The dual role of LBP and CD14 in response to Gram-negative bacteria or Gram-negative compounds. J Endotoxin Res. 2003;9:381-384.  [PubMed]  [DOI]
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.  [PubMed]  [DOI]
3.  Lin M, Rikihisa Y. Ehrlichia chaffeensis downregulates surface Toll-like receptors 2/4, CD14 and transcription factors PU.1 and inhibits lipopolysaccharide activation of NF-kappa B, ERK 1/2 and p38 MAPK in host monocytes. Cell Microbiol. 2004;6:175-186.  [PubMed]  [DOI]
4.  Gong JP, Wu CX, Liu CA, Li SW, Shi YJ, Yang K, Li Y, Li XH. Intestinal damage mediated by Kupffer cells in rats with endotoxemia. World J Gastroenterol. 2002;8:923-927.  [PubMed]  [DOI]
5.  Olszyna DP, Verbon A, Pribble JP, Turner T, Axtelle T, van Deventer SJ, van der Poll T. Effect of IC14, an anti-CD14 antibody, on plasma and cell-associated chemokines during human endotoxemia. Eur Cytokine Netw. 2003;14:158-162.  [PubMed]  [DOI]
6.  Takeda Y, Arii S, Mori A, Imamura M. High expression of the CD14 gene and interleukin-1beta gene in the liver and lungs of cirrhotic rats after partial hepatectomy. J Surg Res. 2003;115:9-17.  [PubMed]  [DOI]
7.  Nagy LE. Recent insights into the role of the innate immune system in the development of alcoholic liver disease. Exp Biol Med (Maywood). 2003;228:882-890.  [PubMed]  [DOI]
8.  Dai LL, Gong JP, Zuo GQ, Wu CX, Shi YJ, Li XH, Peng Y, Deng W, Li SW, Liu CA. Synthesis of endotoxin receptor CD14 protein in Kupffer cells and its role in alcohol-induced liver disease. World J Gastroenterol. 2003;9:622-626.  [PubMed]  [DOI]
9.  Peng Y, Gong JP, Yan LN, Li SB, Li XH. Improved two-cuff technique for orthotopic liver transplantation in rat. Hepatobiliary Pancreat Dis Int. 2004;3:33-37.  [PubMed]  [DOI]
10.  Vives-Pi M, Somoza N, Fernández-Alvarez J, Vargas F, Caro P, Alba A, 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 and of sensitivity to endotoxin (LPS) in islet beta cells. Clin Exp Immunol. 2003;133:208-218.  [PubMed]  [DOI]
11.  Opal SM, Palardy JE, Parejo N, Jasman RL. Effect of anti-CD14 monoclonal antibody on clearance of Escherichia coli bacteremia and endotoxemia. Crit Care Med. 2003;31:929-932.  [PubMed]  [DOI]
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 local inflammatory responses to endotoxemia in mice. World J Surg. 2003;27:1-9.  [PubMed]  [DOI]
13.  Vajdová K, Heinrich S, Tian Y, Graf R, Clavien PA. Ischemic preconditioning and intermittent clamping improve murine hepatic microcirculation and Kupffer cell function after ischemic injury. Liver Transpl. 2004;10:520-528.  [PubMed]  [DOI]
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 reperfusion injury after prolonged warm ischemia. Ann Surg. 2004;239:220-231.  [PubMed]  [DOI]
15.  Cavalieri B, Perrelli MG, Aragno M, Ramadori P, Poli G, Cutrìn JC. Ischaemic preconditioning modulates the activity of Kupffer cells during in vivo reperfusion injury of rat liver. Cell Biochem Funct. 2003;21:299-305.  [PubMed]  [DOI]
16.  Giakoustidis DE, Iliadis S, Tsantilas D, Papageorgiou G, Kontos N, Kostopoulou E, Botsoglou NA, Gerasimidis T, Dimitriadou A. Blockade of Kupffer cells by gadolinium chloride reduces lipid peroxidation and protects liver from ischemia/reperfusion injury. Hepatogastroenterology. 2003;50:1587-1592.  [PubMed]  [DOI]
17.  Tsoulfas G, Takahashi Y, Ganster RW, Yagnik G, Guo Z, Fung JJ, Murase N, Geller DA. Activation of the lipopolysaccharide signaling pathway in hepatic transplantation preservation injury. Transplantation. 2002;74:7-13.  [PubMed]  [DOI]
18.  Manigold T, Böcker U, Hanck C, Gundt J, Traber P, Antoni C, Rossol S. Differential expression of toll-like receptors 2 and 4 in patients with liver cirrhosis. Eur J Gastroenterol Hepatol. 2003;15:275-282.  [PubMed]  [DOI]
19.  Paik YH, Schwabe RF, Bataller R, Russo MP, Jobin C, Brenner DA. Toll-like receptor 4 mediates inflammatory signaling by bacterial lipopolysaccharide in human hepatic stellate cells. Hepatology. 2003;37:1043-1055.  [PubMed]  [DOI]
20.  Su GL, Goyert SM, Fan MH, Aminlari A, Gong KQ, Klein RD, Myc A, Alarcon WH, Steinstraesser L, Remick DG. Activation of human and mouse Kupffer cells by lipopolysaccharide is mediated by CD14. Am J Physiol Gastrointest Liver Physiol. 2002;283:G640-G645.  [PubMed]  [DOI]
21.  Wheeler MD, Thurman RG. Up-regulation of CD14 in liver caused by acute ethanol involves oxidant-dependent AP-1 pathway. J Biol Chem. 2003;278:8435-8441.  [PubMed]  [DOI]
22.  Su GL. Lipopolysaccharides in liver injury: molecular mechanisms of Kupffer cell activation. Am J Physiol Gastrointest Liver Physiol. 2002;283:G256-G265.  [PubMed]  [DOI]
23.  Enomoto N, Takei Y, Hirose M, Ikejima K, Miwa H, Kitamura T, Sato N. Thalidomide prevents alcoholic liver injury in rats through suppression of Kupffer cell sensitization and TNF-αlpha production. Gastroenterology. 2002;123:291-300.  [PubMed]  [DOI]
24.  van Oosten M, van Amersfoort ES, van Berkel TJ, Kuiper J. Scavenger receptor-like receptors for the binding of lipopolysaccharide and lipoteichoic acid to liver endothelial and Kupffer cells. J Endotoxin Res. 2001;7:381-384.  [PubMed]  [DOI]
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.  [PubMed]  [DOI]
26.  Zipfel A, Schenk M, Metzdorf B, Bode C, Viebahn R. Release of TNF-αlpha from lipopolysaccharide (LPS)-stimulated Kupffer cells in serum- and nutrient-free medium. Inflammation. 2001;25:287-292.  [PubMed]  [DOI]
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 to lipopolysaccharide. Alcohol Clin Exp Res. 2001;25:1360-1367.  [PubMed]  [DOI]
28.  Kozar RA, Schultz SG, Bick RJ, Poindexter BJ, DeSoignie R, Moore FA. Enteral glutamine but not alanine maintains small bowel barrier function after ischemia/reperfusion injury in rats. Shock. 2004;21:433-437.  [PubMed]  [DOI]
29.  Arumugam TV, Shiels IA, Woodruff TM, Granger DN, Taylor SM. The role of the complement system in ischemia-reperfusion injury. Shock. 2004;21:401-409.  [PubMed]  [DOI]
30.  Que X, Debonera F, Xie J, Furth EE, Aldeguer X, Gelman AE, Olthoff KM. Pattern of ischemia reperfusion injury in a mouse orthotopic liver transplant model. J Surg Res. 2004;116:262-268.  [PubMed]  [DOI]
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 by suppressing anti-inflammatory cytokine expression in mice. Hepatology. 2004;39:699-710.  [PubMed]  [DOI]