大鼠肝移植免疫耐受的蛋白质组学初步研究
|
摘要
器官移植是众多终末期器官功能衰竭患者的唯一选择。虽然高效免疫抑制剂的应用明显提高了肝移植患者的生存率,但急性排斥反应(Acute Rejection, AR)仍然是肝移植术后最严重的并发症之一,其发生率仍然高达40%-60%,因此免疫耐受仍然是肝移植研究的重点之一。新兴的蛋白质组学是对基因组所编码的所有蛋白质进行研究的学科。通过蛋白质组学使我们对肝移植免疫耐受的发生、发展有了更详尽的理解。
为了观察两种不同品系即近交系Wistar和封闭群SD大鼠原位肝移植术后的急性排斥反应及F蛋白诱导肝移植免疫耐受的情况。我们将健康雄性清洁的近交系Wistar和封闭群SD大鼠随机分为3组,A：同基因对照组：SD→SD；B：急性排斥组：Wistar→SD;C:F蛋白干预组：术前一周胸腺内注射0.4mg。采用Kamada二袖套法,建立上述Wistar和SD组合之间的肝移植模型。A、B和C组受体分别于术后3d、6d、9d及100d,四个时间点各杀死三只大鼠,收集肝组织和血清分别做相应检测,另留取部分新鲜肝组织立即置入液氮中保存备用。剩余的大鼠用于观察肝移植术后长期生存率。实验过程中共完成108例次大鼠原位肝移植(rat orthotopic liver transplantation, ROLT)操作,总的手术成功率为62.96%(68/108),其中模型训练的前期完成45例,24h成活率仅24.44%(11/45)以后随着手术技巧成熟,手术成功率逐渐达到90%以上。大鼠肝移植手术死亡的主要原因为出血、麻醉意外、无肝期太长、血栓形成等。A组同基因对照组,术后组织学检查无排斤反应表现。B组急性排斥组自术后3d开始,出现Ⅰ级排斥反应,6d以后逐渐达到高峰,大多数死于术后12-21d。C组F蛋白干预组亦未见确切排斥反应证据。结果表明：(1)二袖套法操作简便,手术成功率高,可作为肝移植实验的有效手段。(2)成功建立大鼠原位肝移植模型的关键在于完善的手术技术,主要包括高质量的供肝获取、熟练的显微血管吻合技术和尽量短的无肝期。(3)近交系Wistar和封闭群SD大鼠之间建立的原位肝移植模型,能满足急性排斥反应或免疫耐受实验研究的需要。(4)F蛋白可以诱导大鼠肝移植免疫耐受。
应用超高效液相色谱串联质谱法(nanoUPLC-MS/MS)研究大鼠原位肝移植术后7天免疫耐受组和同基因对照组差异表达蛋白质的情况,对大鼠原位肝移植后的免疫耐受情况进行初步研究。建立免疫耐受组(Wistar→SD+F蛋白)n=5,建立同基因对照组(SD→SD)n=5.分别于术后7天处死5只受体,取肝组织,提取总蛋白,Trypsin酶解后,使用Waters公司独家专利MSe无标记定量技术,通过利用nanoUPLC-MS/MS技术对酶解后的肽段进行分析,数据库检索鉴定蛋白质,生物信息学工具对所鉴定的蛋白质进行功能分类。结果显示免疫耐受组和同基因对照组相比,两样品中同时存在的大部分蛋白上调,其中9个蛋白点表达水平明显上调,比值变化在2.59倍以上。结合数据库搜索得到鉴定,这些明显上调的蛋白的分子功能主要与细胞骨架、离子结合、氧化应激反应、能量代谢等相关。结果表明本研究建立了利用nanoUPLC-MS/MS技术对大鼠原位肝移植进行蛋白质组学分析的方法,对F蛋白诱导大鼠原位肝移植免疫耐受进行了初步研究,为建立动物模型提供量化的指标,为发现潜在的生物标记物提供方向和数据支持。
Organ transplantation is the treatment of choice for patients with end-stage organ failure. The incidence of acute rejection (AR) of liver allograft is reported to be between 40% and 60%. Therefore, the immune tolerance of liver transplantation remains one of the keys. High throughput genomics and proteomics techniques have facilitated a better understanding of immune tolerance.
To observe the acute rejection and the immune tolerance after orthotopic liver transplantation in rats between inbred line Wistar and closed colony SD. We established models with Kamada's two cuff technique and then randomly divided into three groups:Group A:SD→SD;Group B:Wistar→SD; Group C:F protein intervention:intrathymic injection one week before surgery. Three recipients were killed after 3d、6d、9d and 100d. Representative liver allografts tissue and serum specimens were collected for corresponding detection. Remaining recipients were observed for long-term survival. During the experiment 108 models were established, the total success rate was 62.96%(68/108). The success rate was only 24.44%(11/45) during the protophase of model manufacture. Afterward the success rate gradually comes up to more than 90%.The causes of death were hemorrhage, anesthetic accident, long anhepatic phase, thrombus formation and so on. In recipients of A group, rejection was not founded. In recipients of B group, I grade rejection occurred at 3rd day and rejection gradually reached the peak 6 days after transplantation, and most died between 12 to 21 days after operation. In recipients of C group rejection was not founded either. Conclusions:(1) Two cuff technique may provide a practical and stable experimental model.(2)The keys of establishing successful models include high quality donor liver, practiced microsurgical techniques and short anhepatic phase.(3) The model can be used to study acute rejection or immunologic tolerance effectively. (4) F protein can induce immune tolerance in liver transplantation.
Application of nanoUPLC-MS/MS technology to research the differentially expressed proteins at 7 days after rat liver orthotopic transplantation between immune tolerance group and syngeneic control group and to have a preliminary study of the immune tolerance. Establish immune tolerance group(Wistar→SD+F protein) n=5, establish syngeneic control group (SD→SD) n=5. After 7 days kill five receptors, obtain liver tissues, extract total protein, after Trypsin hydrolysis, use Waters exclusive patented MSe unmarked quantitative techniques, through the technology of nanoUPLC-MS/MS analyse the peptide hydrolysis, database search identify the protein, bio-informatics tools for the identification of protein functional classification. Comparing the two groups, the two samples simultaneously exist in most of the proteins increases, of which 9 protein spots were significantly up-regulated expression levels, changes in the ratio of 2.59 times. Combination of database search to be identified, these were significantly increased in the main function of the protein molecules with the cytoskeleton, ion binding, oxidative stress response, energy metabolism and other related. This study established a proteomic analysis methods for rat orthotopic liver transplantation with nanoUPLC-MS/MS, carried out a preliminary study for F protein induced immune tolerance after rat liver transplantation, provided quantitative indicators for the establishment of animal models and provided direction and data support for identifying potential biological markers.
为了观察两种不同品系即近交系Wistar和封闭群SD大鼠原位肝移植术后的急性排斥反应及F蛋白诱导肝移植免疫耐受的情况。我们将健康雄性清洁的近交系Wistar和封闭群SD大鼠随机分为3组,A：同基因对照组：SD→SD；B：急性排斥组：Wistar→SD;C:F蛋白干预组：术前一周胸腺内注射0.4mg。采用Kamada二袖套法,建立上述Wistar和SD组合之间的肝移植模型。A、B和C组受体分别于术后3d、6d、9d及100d,四个时间点各杀死三只大鼠,收集肝组织和血清分别做相应检测,另留取部分新鲜肝组织立即置入液氮中保存备用。剩余的大鼠用于观察肝移植术后长期生存率。实验过程中共完成108例次大鼠原位肝移植(rat orthotopic liver transplantation, ROLT)操作,总的手术成功率为62.96%(68/108),其中模型训练的前期完成45例,24h成活率仅24.44%(11/45)以后随着手术技巧成熟,手术成功率逐渐达到90%以上。大鼠肝移植手术死亡的主要原因为出血、麻醉意外、无肝期太长、血栓形成等。A组同基因对照组,术后组织学检查无排斤反应表现。B组急性排斥组自术后3d开始,出现Ⅰ级排斥反应,6d以后逐渐达到高峰,大多数死于术后12-21d。C组F蛋白干预组亦未见确切排斥反应证据。结果表明：(1)二袖套法操作简便,手术成功率高,可作为肝移植实验的有效手段。(2)成功建立大鼠原位肝移植模型的关键在于完善的手术技术,主要包括高质量的供肝获取、熟练的显微血管吻合技术和尽量短的无肝期。(3)近交系Wistar和封闭群SD大鼠之间建立的原位肝移植模型,能满足急性排斥反应或免疫耐受实验研究的需要。(4)F蛋白可以诱导大鼠肝移植免疫耐受。
应用超高效液相色谱串联质谱法(nanoUPLC-MS/MS)研究大鼠原位肝移植术后7天免疫耐受组和同基因对照组差异表达蛋白质的情况,对大鼠原位肝移植后的免疫耐受情况进行初步研究。建立免疫耐受组(Wistar→SD+F蛋白)n=5,建立同基因对照组(SD→SD)n=5.分别于术后7天处死5只受体,取肝组织,提取总蛋白,Trypsin酶解后,使用Waters公司独家专利MSe无标记定量技术,通过利用nanoUPLC-MS/MS技术对酶解后的肽段进行分析,数据库检索鉴定蛋白质,生物信息学工具对所鉴定的蛋白质进行功能分类。结果显示免疫耐受组和同基因对照组相比,两样品中同时存在的大部分蛋白上调,其中9个蛋白点表达水平明显上调,比值变化在2.59倍以上。结合数据库搜索得到鉴定,这些明显上调的蛋白的分子功能主要与细胞骨架、离子结合、氧化应激反应、能量代谢等相关。结果表明本研究建立了利用nanoUPLC-MS/MS技术对大鼠原位肝移植进行蛋白质组学分析的方法,对F蛋白诱导大鼠原位肝移植免疫耐受进行了初步研究,为建立动物模型提供量化的指标,为发现潜在的生物标记物提供方向和数据支持。
Organ transplantation is the treatment of choice for patients with end-stage organ failure. The incidence of acute rejection (AR) of liver allograft is reported to be between 40% and 60%. Therefore, the immune tolerance of liver transplantation remains one of the keys. High throughput genomics and proteomics techniques have facilitated a better understanding of immune tolerance.
To observe the acute rejection and the immune tolerance after orthotopic liver transplantation in rats between inbred line Wistar and closed colony SD. We established models with Kamada's two cuff technique and then randomly divided into three groups:Group A:SD→SD;Group B:Wistar→SD; Group C:F protein intervention:intrathymic injection one week before surgery. Three recipients were killed after 3d、6d、9d and 100d. Representative liver allografts tissue and serum specimens were collected for corresponding detection. Remaining recipients were observed for long-term survival. During the experiment 108 models were established, the total success rate was 62.96%(68/108). The success rate was only 24.44%(11/45) during the protophase of model manufacture. Afterward the success rate gradually comes up to more than 90%.The causes of death were hemorrhage, anesthetic accident, long anhepatic phase, thrombus formation and so on. In recipients of A group, rejection was not founded. In recipients of B group, I grade rejection occurred at 3rd day and rejection gradually reached the peak 6 days after transplantation, and most died between 12 to 21 days after operation. In recipients of C group rejection was not founded either. Conclusions:(1) Two cuff technique may provide a practical and stable experimental model.(2)The keys of establishing successful models include high quality donor liver, practiced microsurgical techniques and short anhepatic phase.(3) The model can be used to study acute rejection or immunologic tolerance effectively. (4) F protein can induce immune tolerance in liver transplantation.
Application of nanoUPLC-MS/MS technology to research the differentially expressed proteins at 7 days after rat liver orthotopic transplantation between immune tolerance group and syngeneic control group and to have a preliminary study of the immune tolerance. Establish immune tolerance group(Wistar→SD+F protein) n=5, establish syngeneic control group (SD→SD) n=5. After 7 days kill five receptors, obtain liver tissues, extract total protein, after Trypsin hydrolysis, use Waters exclusive patented MSe unmarked quantitative techniques, through the technology of nanoUPLC-MS/MS analyse the peptide hydrolysis, database search identify the protein, bio-informatics tools for the identification of protein functional classification. Comparing the two groups, the two samples simultaneously exist in most of the proteins increases, of which 9 protein spots were significantly up-regulated expression levels, changes in the ratio of 2.59 times. Combination of database search to be identified, these were significantly increased in the main function of the protein molecules with the cytoskeleton, ion binding, oxidative stress response, energy metabolism and other related. This study established a proteomic analysis methods for rat orthotopic liver transplantation with nanoUPLC-MS/MS, carried out a preliminary study for F protein induced immune tolerance after rat liver transplantation, provided quantitative indicators for the establishment of animal models and provided direction and data support for identifying potential biological markers.
引文
[1]WASINGER VC, CORDWELL SJ, CERPA-POLJAK A, et al. Progress with gene-product mapping of the mollicutes:mycoplasmagenitalium[J]. Electrophoresis,1995,16(7):1090-1094.
[2]Tyers M, Mann M. From genomics to proteomics. Nature 2003; 422:193-197.
[3]Gharbi S, Gaffiney P, Yang A, et al. Evaluation of two-dimensional differential gel electrophoresis for p roteomic exp ression analysis of a model breast cancer cell system [J]. Mol Cell Proteomics,2002,1 (2):91-98.
[4]Diamandis EP. Mass spectrometry as a diagnostic and a cancer biomarker discovery tool:opportunities and potential limitations [J]. Mol Cell Proteomics, 2004,3:367-378.
[5]Fravi.G, Lindenmann.J. Induction by Allogenic extracts of liver-specific precipitating autoantibodies in the mouse [J]. Nature,1968,218:141-143.
[6]Arakawa Y, Bull D M, Schoff C F, et al. F antigen:nature, liver-specificity, and release in experimental liver injury [J]. Gastraenterology.1976,71:118.
[7]Espinosa E, Caple S. Extrahepatic immunofluorescent reactivity of antisera detecting liver F antigen [J]. Fed.Proc,1981,40:764.
[8]Foster M, Celio M, Winterhalter R H, et al. Cellular location of liver specific antigen [J]. Immunol,1986,24:745.
[9]Oliveira DB. Bulk immunopurification and development of a radioimmunoassay for human and murine F liver protein [J]. J Immunol Methods,1986, 91(1):99-105.
[10]Griffiths JA, Mintchison N A, Nardi N, et al. The organ distribution of F protein in the mouse [J]. Scand J Immunol,1986,24:745.
[11]Griffiths JA, Oliveira DB. The organ distribution of F protein in the mouse [J]. Scand J Immunol,1988,27(3):357-60.
[12]郭海周,张春芳.肺癌蛋白质组学研究进展[J].中国医学工程,2004,12(3)：46-49.
[13]Vascotto C, Cesaratto L, DAmbrosio C, et al. Proteomic analysis of liver tissues subjected to early ischemia/reperfusion injury during human orthotopic liver transplantation. Proteomics,2006;6:3455-3465.
[14]张春潮,朱峰,尉建锋,等.大鼠同种异体肝移植急性排异反应的蛋白质组学研究[J].中国科学C辑：生命科学,2007,37(1)：88-95.
[15]PAN TL, GOTO S, LORD R, et al. Proteome Analysis in Liver Transplantation[J]. Transplant Proc,2001,33(1-2):156.
[16]Lee S, Charters AC, Chandler JG, et al. A technique for orthotopic liver transplantation in the rat [J]. Transplantation,1973,16(6):664-669.
[17]Kamada N,Calne RY. Orthotopic liver transplantation in the rat. Techniques using cuff for portal vein anastomosis and biliary drainage[J].Transplantation,1979,28(1):47-50.
[18]Inoue S, Tahara K, Shimizu H, et al. Rat liver transplantation for total vascular reconstruction, using a suture method [J]. Microsurgery,2003,23(5):470-475.
[19]Tan F, Chen Z, Zhao Y, et al. Novel technique for suprahepatic vena cava reconstruction in rat orthotopic liver transplantation [J]. Microsurgery, 2005,25(7):556-560.
[20]Kluas PP, Mueller AR, Rossaint R, et al.Cytokine Pattern during rejection and infection after liver transplantation-improvements in postoperative monitoring.Transplantation,1994,62:1441-50.
[21]Miyata M, Fischer JH, Fuhs M,et al.A simple method for orthotopic liver transplantation in the rat. Transplantation 1980,30:335.
[22]Jay A,Rubin RH, Infection in organ transplantation recipients,N Engl J Med, 1998,338:1741-1749.
[23]Pirenne J,Moon C,Gruessner A,et al.Bone marrow augmentation of kidney allografts can cause graft-versus-host disease in immunosuppressed recipients.Transplant Proc,1996;28(2):941-942.
[24]Starzl TE,Demetris AJ,Murase N,et al.Cell migration,chimerism,and acceptance[J]. Lancet,1992,339(27):1579-1582.
[25]傅宇阳,何晓顺,陈剑琳,等。检测胆汁中sIC AM-1诊断大鼠肝移植急性排斥反应。肝胆外科杂志,2001；9(1)：66-8.
[26]Miyata M,Fischer JH,Fuhs M,et al.A simple method for orthotopic liver transplantation in the rat. Cuf technique for three vascular anastomoses. Transplantation,1980,30(5):335-338.
[27]孙倍成,王学浩,胡建平,等.大鼠肝移植技术改进及免疫排斥初步观察.南京医科大学学报,2001,7：281-3.
[28]李相成,王学浩,俞悦,等.大鼠部分肝移植的实验研究.中华器官移植杂志,2002,23(3)：167-9.
[29]Peng Y,Gong JA,et al.Selection of anesthetic method in setting up the animal model of orthotopic liver transplantation in rats.Chinese Journal of General surgery,2003,12(9):673-676.
[30], Man,-K;Lo,-C-M.;Ng,-I-O;Wong,-Y-C;Qin,-L-F;Fan,-S-T; Wong,-J. liver transplantation in rats using small-for-size grafts:a study of hemodynamic and morphological changes.Arch-Surg.2001 Mar 136(3):280-5.
[31]邵堂雷,蔡伟耀,张明钧,杨卫平,李宏为,影响肝移植鼠近期存活的术中因素分析。上海第二医科大学学报,2001,3(21,)223.
[32]Gershwin ME, Coppel RL, Bearer E, et al. Molecular cloning of the liver-specific rat F antigen [J]. Immunol,1987,139:3828-33.
[33]Hummel R, Nergaard P, Andreasen PH, et al. Tetrahymena gene encode a prortein that is homologous of the Golgi apparatus and trasport vesicols [J]. Mol.Biol,1992,228:850-861.
[34]Yang C, shi YP, Udhayakumar V, et al. Sequence variations in the non-repetitive regions of the liver stage-specific antigen-1 (LSA-1) of plasmodium falciparum from field isolates[J]. Mol. Biochem Parasitol, 1995,71 (2):291-4.
[35]C K Jia, S S Zheng, Y F Zhu. Intrathymic inoculation of liver specific antigen alleviates liver transplant rejection [J]. Chinese Medical Sciences Journal,2004, 19(1):38-43.
[36]He JK, Tam AW, Yarbough PO, et al. Expression and diagnostic utility of hepatitis virus potative structural proteins expressed in insect cell [J]. Clin Microbiol,1993,31:2167-2173.
[37]Silver DM, Lane DP.Dominant nonresponsiveness in the induction of autoimmunity to liver-specific Fantigen, J Exp Med.1975,142(6):1455-61.
[38]Long GW, Bernard CC, Mackay IR, et al. Localization of genetic control in mice over response to liver-specific F antigen. Tissue Antigens.1978,11(1): 45-9.
[39]Clark E, Lake P, Mitchison NA, et al. New ideas about self-tolerance and auto-immunity.Haematologia.1978-1979; 12(1-4):7-13.
[40]Sunshine GH, Cyrus M, Winchester G. In vitro responses to the liver antigen F.lmmunology.1982,45(2):357-63.
[41]Nardi NB. Absence of suppression in natural and induced tolerance to F antigen. Immunogenetics.1984,20(4):357-95.
[42]Lukie ML, Mitchison NA. Self-and allo-specific suppressor T cells evoked by intravenous injection of F protein. Eur J Immunol.1984,14(8):766-8.
[43]Teuber SS, Coppel RL, Ansari AA, et al. The identification and cloning of the murine genes encoding the liver specific F alloantigens.J Autoimmun.1991, 4(6):857-70.
[44]Mitchison NA, Simon K. Dominant reduced responsiveness controlled by H-2(Kb) Ab. A new pattern evoked by Thy-1 antigen and F liver antigen. Immunogenetics.1990,32(2):104-9.
[45]Yamamoto Y, Gaynor RB.Therapeutic potential of inhibition of the NF-kappaB pathway in the treatment of inflammation and cancer.J Clin Invest.2001; 107(2):135-42.
[46]Bishop GA, Sun J, Sheil AG, et al. High dose/activation-associated tolerance: a mechanism for allograft tolerance.Transplantation.1997; 64(10):1377-82.
[47]Oluwole SF, Chowdhury NC, Jin MX.The relative contribution of intrathymic Inoculation of donor leukocyte subpopulations in the induction of specific tolerance.Cell Immunol.1994; 153(1):163-70.
[48]Oluwole SF, Jin MX, Chowdhury NC, et al. Effectiveness of intrathymic Inoculation of soluble antigens in the induction of specific unresponsiveness to rat islet allografts without transient recipient immunosuppression. Transplantation.1994; 58(10):1077-81.
[49]Donaldson P, Underhill J, Doherty D, et al. Influence of human leukocyte antigen matching on liver allograft survival and rejection:"the dualistic effect".Hepatology.1993; 17(6):1008-15.
[50]Doran TJ, Derley L, Chapman J, McCaughan G, et al. Severity of liver Transplantation rejection is associated with recipient HLA type. Transplant Proc.1992;24(1):192-3.
[51]Markus BH, Duquesnoy RJ, Gordon RD, et al. Histocompatibility and liver transplant outcome. Does HLA exert a dualistic effect? Transplantation.1988; 46(3):372-7.
[52]Fung JJ,leevi A, Starzl TE,et al.Functional characterization of infiltrating T lymphocytesin human hepatic allograft.Hum Immunol.1990;16:182-5.
[53]Martinez OM,Krams SM,Sterneck M, et al.Intragraft cytokine profile during human liver allograft rejection. Transplantation.1992; 53(2):449-53.
[54]Kita Y,Iwaki Y,Noguchi K, et al.Daily serum inflammatory cytokine(TNF-α, IL-6) Monitoring in liver transplantation focusing on allograft rejection:A five-case report. Transplant Proc.1996; 28(3):1237-42.
[55]Neuberger J. Liver allograft rejection--current concepts on diagnosis and treatment. J Hepatol,1995,23 Suppl 154-61.
[56]Alain Le Moine,Michel Goldman,and Daniel Abramowicz.Multiple pathways to allograft rejection, Transplantation,2002;73:1373-1381.
[57]Sikorski M, Bobek M, Zrubek H, Marcinkiewicz J. Dynamics of selected MHC classⅠand Ⅱ molecule expression in the course of HPV positive CIN treatment with the use of human recombinant IFN-gamma.Acta Obstet G ynecol Scand.2004;83(3):299-307.
[58]Dong D, Ni M, Li J, et al. Critical role of the stress chaperone GRP78/BiP in tumor proliferation, survival, and tumor angiogenesis in transgene-induced mammary tumor development [J]. Cancer Res,2008,68(2):498-505.
[59]Park HR, Tomida A, Sato S, et al. Effect on tumor cells of blocking survival response to glucose deprivation [J]. J Natl Cancer Inst, 2004,96(17): 1300-1310.
[60]Li J, Lee AS. Stress induction of GRP78/BiP and its role in cancer [J]. Curr Mol Med,2006,6(1):45-54.
[61]Yang GH, Li S, Pestka JJ. Down-regulation of the endoplasmic reticulum chaperone GRP78/BiP by vomitoxin (Deoxynivalenol) [J]. Toxicol Appl Pharmacol,2000,162(3):207-217.
[62]Yang Y, Turner RS, Gaut JR. The chaperone BiP/GRP78 binds to amyloid precursor protein and decreases Abeta40 and Abeta42 secretion [J]. J Biol Chem,1998,273(40):25552-25555.
[63]Arap MA, Lahdenranta J, Mintz PJ,et al. Cell surface expression of the stress response chaperone GRP78 enables tumor targeting by circulating ligands[J]. Cancer Cell,2004,6(3):275-284. 及氧化应激过程中发挥作用。张春潮等利用荧光差异显示双向凝胶电泳并整合内标法与正、反相荧光标记,对急性排异组和对照组大鼠肝移植后的肝组织蛋白质表达谱进行了定量蛋白质组学研究。结果表明,有27个蛋白点的表达水平在急性排异组有显著差异,其中19个差异表达蛋白经胶内酶切后,利用基质辅助激光解析电离飞行时间质谱获得相应的肽指纹图谱并结合数据库搜索得到鉴定。这些差异蛋白的分子功能主要表现为氧化还原活性及离子结合活性,实验结果将有助于进一步了解器官移植排异反应的分子机制。此外,蛋白质组学在肝移植免疫耐受和肝移植术后并发症的研究也在逐渐开展之中。
综上所述,随着蛋白质组技术的不断更新发展,蛋白质组学的研究将更为全面深入。肝脏疾病蛋白质组学的研究对于肝病的预防、诊断、治疗都有重大意义。相信随着研究的深入将会不断产生新的和更权威的具有诊断潜能的生物分子靶标。
[1]Wasinger VC, Cordwell SJ, Cerepa-Poljak A, et al. Progress with gene-product mapping of the Mollicutes:Mycoplasma genitalium. Electrophoresis 1995;16:1090-1094.
[2]夏其昌,主编.蛋白质化学与蛋白质组学[M].北京：科学出版社,2004：510-513.
[3]Feng B, Smith RD. A simple nanoelectrospray arrangement with controllable flow rate for mass analysis of submicroliter protein samples[J]. J Am Soc Mass Spectrom,2000,11(1):94-99.
[4]He QY, Lau GK, Zhou Y, et al. Serum biomarkers of hepatitis B virus infected liver inflammation:a proteomic study. Proteomics,2003;3:666-674.
[5]Fukuda Y, Yotsuyanagi H, Ooka S, et al. Identification of a new autoantibody in patients with chronic hepatitis. Hum Immunol,2004,65:1530-1538.
[6]Paradis V, Asselah T, Dargere D,et al. Serumproteome to predict virologic response in patients with hepatitis C treated by pegylated interferon plus ribavirin. Gastroenterology,2006,130:2189-2197.
[7]Terence CP, Alex YH, Henry LC, et al. Prediction of liver fibrosis and irrhosis in chronic hepatitis B infection by serum proteomic fingerprinting:A pilot study. Clin Chem,2005,51:2328-2335.
[8]Poon TC, Hui AY, Chan HL, et al. Prediction of liver fibrosis and cirrhosis in chronic Hepatitis B infection by serum proteomic fingerprinting:a pilot study. Clin Chem 2005;51:328-335.
[9]LI C, TAN YX, ZHOU H, et al. Proteomic analysis of hepatitis B virus-associated hepatocellular carcinoma:identification of potential tumor markers[J]. Proteomics,2005,5(4):1125-39.
[10]FEITEI SON MA, SUN B. SATIROGLU TUFAN NL, et al. Genetic mechanisms of hepatocarcinogenesis[J]. Oncogene,2002,21(16):2593-604.
[11]SEOW TK, LIANG RC, LEOW CK, et al. Hepatocellular carcinoma:from bedside to proteomics[J]. Proteomics,2001,1(11):1249-63.
[12]Seow TK, Ong SE, Liang RC, et al. Two-dimensional electrophoresismap of the human hepatocellular carcinoma cell line, HCC-M, and identification of the separated p roteins by mass spectrometry. Electrophoresis,2000,21: 1787-1813.
[13]Ou K, Seow TK, Liang RC, et al. Proteome analysis of a human hepatocellular carcinoma cell line, HCC-M:an update. Electrophoresis,2001,22:2804-2811.
[14]Steel LF, Shumpert D, TrotterM, et al. A strategy for the com-parative analysis of serum proteomes for the discovery of biomarkers for hepatocellular carcinoma[J]. Proteomics,2003,3:601-609.
[15]Wang Z, Ruan YB, Guan Y. Analysis of proteomic components of sera from patients with hepatocellular carcinomas by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time of flyingmass spectrometry[J]. Zhonghua Bing Li Xue Za Zhi,2003,32 (4):333-336.
[16]Liang CR, Leow CK, Neo JC, et al. Proteome analysis of human hepatocellular carcinoma tissues by two-dimensional difference gel electrophoresis and mass spectrometry. Proteomics,2005,5:2258-2271.
[17]PAN TL, WANG PW, HUANG CC, et al. Expression, by functional proteomics, of spontaneous tolerance in rat orthotopic liver transplantation[J]. Immunology,2004,113(1):57-64.
[18]ARREDOUANI M, MATTHIJS P, VAN HOEYVELD E, et al. Haptoglobin directly affects T cells and suppresses T helper cell type 2 cytokine release [J]. Immunology,2003,108 (2):144-151.
[19]Vascotto C, Cesaratto L, DAmbrosio C, et al. Proteomic analysis of liver tissues subjected to early ischemia/reperfusion injury during human orthotopic liver transplantation. Proteomics,2006;6:3455-3465.
[20]张春潮,朱峰,尉建锋,等.大鼠同种异体肝移植急性排异反应的蛋白质组学研究[J].中国科学C辑：生命科学,2007,37(1)：88-95.
[2]Tyers M, Mann M. From genomics to proteomics. Nature 2003; 422:193-197.
[3]Gharbi S, Gaffiney P, Yang A, et al. Evaluation of two-dimensional differential gel electrophoresis for p roteomic exp ression analysis of a model breast cancer cell system [J]. Mol Cell Proteomics,2002,1 (2):91-98.
[4]Diamandis EP. Mass spectrometry as a diagnostic and a cancer biomarker discovery tool:opportunities and potential limitations [J]. Mol Cell Proteomics, 2004,3:367-378.
[5]Fravi.G, Lindenmann.J. Induction by Allogenic extracts of liver-specific precipitating autoantibodies in the mouse [J]. Nature,1968,218:141-143.
[6]Arakawa Y, Bull D M, Schoff C F, et al. F antigen:nature, liver-specificity, and release in experimental liver injury [J]. Gastraenterology.1976,71:118.
[7]Espinosa E, Caple S. Extrahepatic immunofluorescent reactivity of antisera detecting liver F antigen [J]. Fed.Proc,1981,40:764.
[8]Foster M, Celio M, Winterhalter R H, et al. Cellular location of liver specific antigen [J]. Immunol,1986,24:745.
[9]Oliveira DB. Bulk immunopurification and development of a radioimmunoassay for human and murine F liver protein [J]. J Immunol Methods,1986, 91(1):99-105.
[10]Griffiths JA, Mintchison N A, Nardi N, et al. The organ distribution of F protein in the mouse [J]. Scand J Immunol,1986,24:745.
[11]Griffiths JA, Oliveira DB. The organ distribution of F protein in the mouse [J]. Scand J Immunol,1988,27(3):357-60.
[12]郭海周,张春芳.肺癌蛋白质组学研究进展[J].中国医学工程,2004,12(3)：46-49.
[13]Vascotto C, Cesaratto L, DAmbrosio C, et al. Proteomic analysis of liver tissues subjected to early ischemia/reperfusion injury during human orthotopic liver transplantation. Proteomics,2006;6:3455-3465.
[14]张春潮,朱峰,尉建锋,等.大鼠同种异体肝移植急性排异反应的蛋白质组学研究[J].中国科学C辑：生命科学,2007,37(1)：88-95.
[15]PAN TL, GOTO S, LORD R, et al. Proteome Analysis in Liver Transplantation[J]. Transplant Proc,2001,33(1-2):156.
[16]Lee S, Charters AC, Chandler JG, et al. A technique for orthotopic liver transplantation in the rat [J]. Transplantation,1973,16(6):664-669.
[17]Kamada N,Calne RY. Orthotopic liver transplantation in the rat. Techniques using cuff for portal vein anastomosis and biliary drainage[J].Transplantation,1979,28(1):47-50.
[18]Inoue S, Tahara K, Shimizu H, et al. Rat liver transplantation for total vascular reconstruction, using a suture method [J]. Microsurgery,2003,23(5):470-475.
[19]Tan F, Chen Z, Zhao Y, et al. Novel technique for suprahepatic vena cava reconstruction in rat orthotopic liver transplantation [J]. Microsurgery, 2005,25(7):556-560.
[20]Kluas PP, Mueller AR, Rossaint R, et al.Cytokine Pattern during rejection and infection after liver transplantation-improvements in postoperative monitoring.Transplantation,1994,62:1441-50.
[21]Miyata M, Fischer JH, Fuhs M,et al.A simple method for orthotopic liver transplantation in the rat. Transplantation 1980,30:335.
[22]Jay A,Rubin RH, Infection in organ transplantation recipients,N Engl J Med, 1998,338:1741-1749.
[23]Pirenne J,Moon C,Gruessner A,et al.Bone marrow augmentation of kidney allografts can cause graft-versus-host disease in immunosuppressed recipients.Transplant Proc,1996;28(2):941-942.
[24]Starzl TE,Demetris AJ,Murase N,et al.Cell migration,chimerism,and acceptance[J]. Lancet,1992,339(27):1579-1582.
[25]傅宇阳,何晓顺,陈剑琳,等。检测胆汁中sIC AM-1诊断大鼠肝移植急性排斥反应。肝胆外科杂志,2001；9(1)：66-8.
[26]Miyata M,Fischer JH,Fuhs M,et al.A simple method for orthotopic liver transplantation in the rat. Cuf technique for three vascular anastomoses. Transplantation,1980,30(5):335-338.
[27]孙倍成,王学浩,胡建平,等.大鼠肝移植技术改进及免疫排斥初步观察.南京医科大学学报,2001,7：281-3.
[28]李相成,王学浩,俞悦,等.大鼠部分肝移植的实验研究.中华器官移植杂志,2002,23(3)：167-9.
[29]Peng Y,Gong JA,et al.Selection of anesthetic method in setting up the animal model of orthotopic liver transplantation in rats.Chinese Journal of General surgery,2003,12(9):673-676.
[30], Man,-K;Lo,-C-M.;Ng,-I-O;Wong,-Y-C;Qin,-L-F;Fan,-S-T; Wong,-J. liver transplantation in rats using small-for-size grafts:a study of hemodynamic and morphological changes.Arch-Surg.2001 Mar 136(3):280-5.
[31]邵堂雷,蔡伟耀,张明钧,杨卫平,李宏为,影响肝移植鼠近期存活的术中因素分析。上海第二医科大学学报,2001,3(21,)223.
[32]Gershwin ME, Coppel RL, Bearer E, et al. Molecular cloning of the liver-specific rat F antigen [J]. Immunol,1987,139:3828-33.
[33]Hummel R, Nergaard P, Andreasen PH, et al. Tetrahymena gene encode a prortein that is homologous of the Golgi apparatus and trasport vesicols [J]. Mol.Biol,1992,228:850-861.
[34]Yang C, shi YP, Udhayakumar V, et al. Sequence variations in the non-repetitive regions of the liver stage-specific antigen-1 (LSA-1) of plasmodium falciparum from field isolates[J]. Mol. Biochem Parasitol, 1995,71 (2):291-4.
[35]C K Jia, S S Zheng, Y F Zhu. Intrathymic inoculation of liver specific antigen alleviates liver transplant rejection [J]. Chinese Medical Sciences Journal,2004, 19(1):38-43.
[36]He JK, Tam AW, Yarbough PO, et al. Expression and diagnostic utility of hepatitis virus potative structural proteins expressed in insect cell [J]. Clin Microbiol,1993,31:2167-2173.
[37]Silver DM, Lane DP.Dominant nonresponsiveness in the induction of autoimmunity to liver-specific Fantigen, J Exp Med.1975,142(6):1455-61.
[38]Long GW, Bernard CC, Mackay IR, et al. Localization of genetic control in mice over response to liver-specific F antigen. Tissue Antigens.1978,11(1): 45-9.
[39]Clark E, Lake P, Mitchison NA, et al. New ideas about self-tolerance and auto-immunity.Haematologia.1978-1979; 12(1-4):7-13.
[40]Sunshine GH, Cyrus M, Winchester G. In vitro responses to the liver antigen F.lmmunology.1982,45(2):357-63.
[41]Nardi NB. Absence of suppression in natural and induced tolerance to F antigen. Immunogenetics.1984,20(4):357-95.
[42]Lukie ML, Mitchison NA. Self-and allo-specific suppressor T cells evoked by intravenous injection of F protein. Eur J Immunol.1984,14(8):766-8.
[43]Teuber SS, Coppel RL, Ansari AA, et al. The identification and cloning of the murine genes encoding the liver specific F alloantigens.J Autoimmun.1991, 4(6):857-70.
[44]Mitchison NA, Simon K. Dominant reduced responsiveness controlled by H-2(Kb) Ab. A new pattern evoked by Thy-1 antigen and F liver antigen. Immunogenetics.1990,32(2):104-9.
[45]Yamamoto Y, Gaynor RB.Therapeutic potential of inhibition of the NF-kappaB pathway in the treatment of inflammation and cancer.J Clin Invest.2001; 107(2):135-42.
[46]Bishop GA, Sun J, Sheil AG, et al. High dose/activation-associated tolerance: a mechanism for allograft tolerance.Transplantation.1997; 64(10):1377-82.
[47]Oluwole SF, Chowdhury NC, Jin MX.The relative contribution of intrathymic Inoculation of donor leukocyte subpopulations in the induction of specific tolerance.Cell Immunol.1994; 153(1):163-70.
[48]Oluwole SF, Jin MX, Chowdhury NC, et al. Effectiveness of intrathymic Inoculation of soluble antigens in the induction of specific unresponsiveness to rat islet allografts without transient recipient immunosuppression. Transplantation.1994; 58(10):1077-81.
[49]Donaldson P, Underhill J, Doherty D, et al. Influence of human leukocyte antigen matching on liver allograft survival and rejection:"the dualistic effect".Hepatology.1993; 17(6):1008-15.
[50]Doran TJ, Derley L, Chapman J, McCaughan G, et al. Severity of liver Transplantation rejection is associated with recipient HLA type. Transplant Proc.1992;24(1):192-3.
[51]Markus BH, Duquesnoy RJ, Gordon RD, et al. Histocompatibility and liver transplant outcome. Does HLA exert a dualistic effect? Transplantation.1988; 46(3):372-7.
[52]Fung JJ,leevi A, Starzl TE,et al.Functional characterization of infiltrating T lymphocytesin human hepatic allograft.Hum Immunol.1990;16:182-5.
[53]Martinez OM,Krams SM,Sterneck M, et al.Intragraft cytokine profile during human liver allograft rejection. Transplantation.1992; 53(2):449-53.
[54]Kita Y,Iwaki Y,Noguchi K, et al.Daily serum inflammatory cytokine(TNF-α, IL-6) Monitoring in liver transplantation focusing on allograft rejection:A five-case report. Transplant Proc.1996; 28(3):1237-42.
[55]Neuberger J. Liver allograft rejection--current concepts on diagnosis and treatment. J Hepatol,1995,23 Suppl 154-61.
[56]Alain Le Moine,Michel Goldman,and Daniel Abramowicz.Multiple pathways to allograft rejection, Transplantation,2002;73:1373-1381.
[57]Sikorski M, Bobek M, Zrubek H, Marcinkiewicz J. Dynamics of selected MHC classⅠand Ⅱ molecule expression in the course of HPV positive CIN treatment with the use of human recombinant IFN-gamma.Acta Obstet G ynecol Scand.2004;83(3):299-307.
[58]Dong D, Ni M, Li J, et al. Critical role of the stress chaperone GRP78/BiP in tumor proliferation, survival, and tumor angiogenesis in transgene-induced mammary tumor development [J]. Cancer Res,2008,68(2):498-505.
[59]Park HR, Tomida A, Sato S, et al. Effect on tumor cells of blocking survival response to glucose deprivation [J]. J Natl Cancer Inst, 2004,96(17): 1300-1310.
[60]Li J, Lee AS. Stress induction of GRP78/BiP and its role in cancer [J]. Curr Mol Med,2006,6(1):45-54.
[61]Yang GH, Li S, Pestka JJ. Down-regulation of the endoplasmic reticulum chaperone GRP78/BiP by vomitoxin (Deoxynivalenol) [J]. Toxicol Appl Pharmacol,2000,162(3):207-217.
[62]Yang Y, Turner RS, Gaut JR. The chaperone BiP/GRP78 binds to amyloid precursor protein and decreases Abeta40 and Abeta42 secretion [J]. J Biol Chem,1998,273(40):25552-25555.
[63]Arap MA, Lahdenranta J, Mintz PJ,et al. Cell surface expression of the stress response chaperone GRP78 enables tumor targeting by circulating ligands[J]. Cancer Cell,2004,6(3):275-284. 及氧化应激过程中发挥作用。张春潮等利用荧光差异显示双向凝胶电泳并整合内标法与正、反相荧光标记,对急性排异组和对照组大鼠肝移植后的肝组织蛋白质表达谱进行了定量蛋白质组学研究。结果表明,有27个蛋白点的表达水平在急性排异组有显著差异,其中19个差异表达蛋白经胶内酶切后,利用基质辅助激光解析电离飞行时间质谱获得相应的肽指纹图谱并结合数据库搜索得到鉴定。这些差异蛋白的分子功能主要表现为氧化还原活性及离子结合活性,实验结果将有助于进一步了解器官移植排异反应的分子机制。此外,蛋白质组学在肝移植免疫耐受和肝移植术后并发症的研究也在逐渐开展之中。
综上所述,随着蛋白质组技术的不断更新发展,蛋白质组学的研究将更为全面深入。肝脏疾病蛋白质组学的研究对于肝病的预防、诊断、治疗都有重大意义。相信随着研究的深入将会不断产生新的和更权威的具有诊断潜能的生物分子靶标。
[1]Wasinger VC, Cordwell SJ, Cerepa-Poljak A, et al. Progress with gene-product mapping of the Mollicutes:Mycoplasma genitalium. Electrophoresis 1995;16:1090-1094.
[2]夏其昌,主编.蛋白质化学与蛋白质组学[M].北京：科学出版社,2004：510-513.
[3]Feng B, Smith RD. A simple nanoelectrospray arrangement with controllable flow rate for mass analysis of submicroliter protein samples[J]. J Am Soc Mass Spectrom,2000,11(1):94-99.
[4]He QY, Lau GK, Zhou Y, et al. Serum biomarkers of hepatitis B virus infected liver inflammation:a proteomic study. Proteomics,2003;3:666-674.
[5]Fukuda Y, Yotsuyanagi H, Ooka S, et al. Identification of a new autoantibody in patients with chronic hepatitis. Hum Immunol,2004,65:1530-1538.
[6]Paradis V, Asselah T, Dargere D,et al. Serumproteome to predict virologic response in patients with hepatitis C treated by pegylated interferon plus ribavirin. Gastroenterology,2006,130:2189-2197.
[7]Terence CP, Alex YH, Henry LC, et al. Prediction of liver fibrosis and irrhosis in chronic hepatitis B infection by serum proteomic fingerprinting:A pilot study. Clin Chem,2005,51:2328-2335.
[8]Poon TC, Hui AY, Chan HL, et al. Prediction of liver fibrosis and cirrhosis in chronic Hepatitis B infection by serum proteomic fingerprinting:a pilot study. Clin Chem 2005;51:328-335.
[9]LI C, TAN YX, ZHOU H, et al. Proteomic analysis of hepatitis B virus-associated hepatocellular carcinoma:identification of potential tumor markers[J]. Proteomics,2005,5(4):1125-39.
[10]FEITEI SON MA, SUN B. SATIROGLU TUFAN NL, et al. Genetic mechanisms of hepatocarcinogenesis[J]. Oncogene,2002,21(16):2593-604.
[11]SEOW TK, LIANG RC, LEOW CK, et al. Hepatocellular carcinoma:from bedside to proteomics[J]. Proteomics,2001,1(11):1249-63.
[12]Seow TK, Ong SE, Liang RC, et al. Two-dimensional electrophoresismap of the human hepatocellular carcinoma cell line, HCC-M, and identification of the separated p roteins by mass spectrometry. Electrophoresis,2000,21: 1787-1813.
[13]Ou K, Seow TK, Liang RC, et al. Proteome analysis of a human hepatocellular carcinoma cell line, HCC-M:an update. Electrophoresis,2001,22:2804-2811.
[14]Steel LF, Shumpert D, TrotterM, et al. A strategy for the com-parative analysis of serum proteomes for the discovery of biomarkers for hepatocellular carcinoma[J]. Proteomics,2003,3:601-609.
[15]Wang Z, Ruan YB, Guan Y. Analysis of proteomic components of sera from patients with hepatocellular carcinomas by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time of flyingmass spectrometry[J]. Zhonghua Bing Li Xue Za Zhi,2003,32 (4):333-336.
[16]Liang CR, Leow CK, Neo JC, et al. Proteome analysis of human hepatocellular carcinoma tissues by two-dimensional difference gel electrophoresis and mass spectrometry. Proteomics,2005,5:2258-2271.
[17]PAN TL, WANG PW, HUANG CC, et al. Expression, by functional proteomics, of spontaneous tolerance in rat orthotopic liver transplantation[J]. Immunology,2004,113(1):57-64.
[18]ARREDOUANI M, MATTHIJS P, VAN HOEYVELD E, et al. Haptoglobin directly affects T cells and suppresses T helper cell type 2 cytokine release [J]. Immunology,2003,108 (2):144-151.
[19]Vascotto C, Cesaratto L, DAmbrosio C, et al. Proteomic analysis of liver tissues subjected to early ischemia/reperfusion injury during human orthotopic liver transplantation. Proteomics,2006;6:3455-3465.
[20]张春潮,朱峰,尉建锋,等.大鼠同种异体肝移植急性排异反应的蛋白质组学研究[J].中国科学C辑：生命科学,2007,37(1)：88-95.