NF-kB诱饵双链核苷酸在大鼠心脏移植中作用的实验研究
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摘要
目的：利用大鼠异位心脏移植模型，探讨转染入心肌细胞中的NF-decoy诱饵双链核苷酸在心脏移植中的作用，并对其可能的作用机制作一探讨。
方法：在建立Wistar大鼠→SD大鼠颈部异位心脏移植模型基础上，用不同浓度的Lipofectmine2000与NF-decoy诱饵ds ODN混合物处理供心，比较其在心肌细胞中存活时间的长短和移植后供心在Langendorff-Neeley离体心脏顺灌左心做功模型在功能表现，测定心脏组织损伤生化学指标；半定量RT-PCR法测定ICAM-1、VCAM、iNOS mRNA的表达。用10OD诱饵ds ODN脂质体混合物处理供心，观察移植术后供心存活时间，心室组织病理切片排斥反应分级的差别和抗ICAM-1免疫组织化学染色情况。
结果：Lipofectamine2000可将ds ODN转染入心肌细胞内，存在时间与ds ODN的浓度有关。乱序ds ODN不能对供心提供缺血再灌注的保护作用，而用5 OD及10 OD的ds ODN处理的供心表现出较佳的心脏功能，减轻心肌组织中损伤的生化学指标以及心肌mRNA中ICAM-1、VCAM、iNOS的表达。10 OD ds ODN能延长供心的存活时间与亚治疗剂量(2.5mg／kg)免疫抑制剂CsA所获得的效果类似，而与亚治疗剂量CsA合用可进一步延长供心的存活时间，减轻病理切片中排斥反应的分级和免疫组化染色ICAM—1的表达。
结论：1)转染入心肌细胞的NF-kB诱饵双链核苷酸可有效减轻移植术后的供心的缺血再灌注损伤，其机理与抑制ICAM-1、VCAM、iNOS的表达有关。2)NF-kB ds ODN还能减轻移植术后的急性排斥反应，延长供心的存活时间，减少了心肌细胞中ICAM-1的表达可能是其机理之一。
Objectives: By using a rat heterotopical heart transplantation model, we investigate the effect of the lipofectin-mediated delivery double-strand oligonuide(ds ODN) decoy to NF-kB binding site in the heart transplantation.
Methods: First, we developed a new heterotopic heart transplantation model by using cuff technique. Then, the donor hearts were treated with 1 OD decoy, 1OD decoy Iipofectamine2000 compound or 5 OD decoy Iipofectamine2000 compound. Transfection efficiency was determined with FITC-labeled ODN and fluorescent microscopy. Reperfusion injury was assessed by Langendorff-Neeley reperfusion system and the content of MDA, MPO in heart tissure and NO in right atrial. Reverse transcription-PCR was used to analyze intercellular adhesion molecule 1, vascular celluar adhesion molecule and inducible NOS mRNA expression. Acute rejection was determined by daily palpation. Histopathological and immunohistochemical study for measurment ICAM-1 were examined after 5 days of transplantation.
Results: The heterotopic heart transplantation using cuff technique got the similar results as classical heterotopic heart transplantation. Without Lipofectamine2000, the ds ODN can not transfected into the donor heart cell. The transfected 5 OD ds ODN can lasted 14 days after heart transplantation. In the reperfusion experiment, the 5 OD ds ODN treated group had better donor heart function, less tissure MDA, myeloperoxidase(MPO) content and blood nitric oxide (NO) concentration (P<0.01 vs the control group). And the 10 OD ds ODN treated group had more efficient heart preservation effect when compared to the 5 OD treated group( P<0.01 ). The NF-kB decoy(5 OD and 10 OD), not the scramble ds ODN inhibited the donor heart ICAM-1, VCAM, iNOS mRNA expression in vivo after transplantation as assessed by semi-quantitative RT-PCR analysis(P< 0.01 vs controls). The 10 OD ds ODN treatment also prolonged donor heart survival over the controls (10.88 ± 1.55 vs. 6.75 ± 089 08
0.01), and had the similar results as the low-dose cyclosporine A (2.5 mg/kg) treatment.
When decoy employment combined with the the low-dose cyclosporine A treatment can further prolonged the allografts survival. The histopathology showed less rejection scores after 5 days of transplantation while treated with the NF-kB decoy. The immunohistochemical study also showed decoy ds ODN can alleviated the expression of ICAM-1 in allograft rejection tissure .
Conclusion: Our results suggested that Lipofectamine2000 could effectively deliver the ds ODN into the donor heart during preservation period. The NF-kB decoy ds ODN treatment can reduce the reperfusion injury and block the donor heart ICAM-1, VCAM, iNOS expression after transplantation. And the NF-kB decoy can also attenuate the acute rejection of the donor heart, the possible mechanism may contribute to the suppression of ICAM-1 expression. This highly targeted therapy may be a clinically viable strategy for the heart transplantation.
方法：在建立Wistar大鼠→SD大鼠颈部异位心脏移植模型基础上，用不同浓度的Lipofectmine2000与NF-decoy诱饵ds ODN混合物处理供心，比较其在心肌细胞中存活时间的长短和移植后供心在Langendorff-Neeley离体心脏顺灌左心做功模型在功能表现，测定心脏组织损伤生化学指标；半定量RT-PCR法测定ICAM-1、VCAM、iNOS mRNA的表达。用10OD诱饵ds ODN脂质体混合物处理供心，观察移植术后供心存活时间，心室组织病理切片排斥反应分级的差别和抗ICAM-1免疫组织化学染色情况。
结果：Lipofectamine2000可将ds ODN转染入心肌细胞内，存在时间与ds ODN的浓度有关。乱序ds ODN不能对供心提供缺血再灌注的保护作用，而用5 OD及10 OD的ds ODN处理的供心表现出较佳的心脏功能，减轻心肌组织中损伤的生化学指标以及心肌mRNA中ICAM-1、VCAM、iNOS的表达。10 OD ds ODN能延长供心的存活时间与亚治疗剂量(2.5mg／kg)免疫抑制剂CsA所获得的效果类似，而与亚治疗剂量CsA合用可进一步延长供心的存活时间，减轻病理切片中排斥反应的分级和免疫组化染色ICAM—1的表达。
结论：1)转染入心肌细胞的NF-kB诱饵双链核苷酸可有效减轻移植术后的供心的缺血再灌注损伤，其机理与抑制ICAM-1、VCAM、iNOS的表达有关。2)NF-kB ds ODN还能减轻移植术后的急性排斥反应，延长供心的存活时间，减少了心肌细胞中ICAM-1的表达可能是其机理之一。
Objectives: By using a rat heterotopical heart transplantation model, we investigate the effect of the lipofectin-mediated delivery double-strand oligonuide(ds ODN) decoy to NF-kB binding site in the heart transplantation.
Methods: First, we developed a new heterotopic heart transplantation model by using cuff technique. Then, the donor hearts were treated with 1 OD decoy, 1OD decoy Iipofectamine2000 compound or 5 OD decoy Iipofectamine2000 compound. Transfection efficiency was determined with FITC-labeled ODN and fluorescent microscopy. Reperfusion injury was assessed by Langendorff-Neeley reperfusion system and the content of MDA, MPO in heart tissure and NO in right atrial. Reverse transcription-PCR was used to analyze intercellular adhesion molecule 1, vascular celluar adhesion molecule and inducible NOS mRNA expression. Acute rejection was determined by daily palpation. Histopathological and immunohistochemical study for measurment ICAM-1 were examined after 5 days of transplantation.
Results: The heterotopic heart transplantation using cuff technique got the similar results as classical heterotopic heart transplantation. Without Lipofectamine2000, the ds ODN can not transfected into the donor heart cell. The transfected 5 OD ds ODN can lasted 14 days after heart transplantation. In the reperfusion experiment, the 5 OD ds ODN treated group had better donor heart function, less tissure MDA, myeloperoxidase(MPO) content and blood nitric oxide (NO) concentration (P<0.01 vs the control group). And the 10 OD ds ODN treated group had more efficient heart preservation effect when compared to the 5 OD treated group( P<0.01 ). The NF-kB decoy(5 OD and 10 OD), not the scramble ds ODN inhibited the donor heart ICAM-1, VCAM, iNOS mRNA expression in vivo after transplantation as assessed by semi-quantitative RT-PCR analysis(P< 0.01 vs controls). The 10 OD ds ODN treatment also prolonged donor heart survival over the controls (10.88 ± 1.55 vs. 6.75 ± 089 08
0.01), and had the similar results as the low-dose cyclosporine A (2.5 mg/kg) treatment.
When decoy employment combined with the the low-dose cyclosporine A treatment can further prolonged the allografts survival. The histopathology showed less rejection scores after 5 days of transplantation while treated with the NF-kB decoy. The immunohistochemical study also showed decoy ds ODN can alleviated the expression of ICAM-1 in allograft rejection tissure .
Conclusion: Our results suggested that Lipofectamine2000 could effectively deliver the ds ODN into the donor heart during preservation period. The NF-kB decoy ds ODN treatment can reduce the reperfusion injury and block the donor heart ICAM-1, VCAM, iNOS expression after transplantation. And the NF-kB decoy can also attenuate the acute rejection of the donor heart, the possible mechanism may contribute to the suppression of ICAM-1 expression. This highly targeted therapy may be a clinically viable strategy for the heart transplantation.
引文
1． Hosenpud JD, Bennett LE, Keck BM, et al. The registry of the international society for heart and lung tranaplantation: eighteenth official report-2001． J Heart Lung Transplant, 2001; 20: 805-815．
2． Siebenlist U, Franzoso G, Brown K, et al. Structure, regulation and function of NF-K B. Ann Rev Cell Biol 1994, 10: 405-455．
3． Li C, Browder W, Kao RL. Early activation of transcription factor NF-kappaB during ischemia in perfused rat heart. Am J Physiol, 1999,276: H543-552．
4． Cooper M, Lindholm P, Pieper G, et al. Myocardial nuclear factor-K B activity and nitric oxide production in rejecting cardiac allograts. Transplantation 1998; 66:838-844．
5． Guzman RJ, Lemarchand P, Crystal RG, et al. Efficient gene transfer into myocardium by direct injection of adenovirus vector. Cir Res 1993; 73: 1202-7．
6． Lee J, Laks H, Drinkwater DC, et al. Cardiac gene transfer by intracoronary infusion of adenovirus vector-mediatd reporter gene in the transplanted mouse heart. J Thorac Cardiovasc Surg 1996; 111: 246-52．
7． Bielinska A, Shivdasani RA, Zhang L, et al. Regulation of gene expression with double-stranded phosphotothioate oligonucleotides. Science 1990; 250: 997-1000
8． Lee J, Laks H, Drinkwater DC, etal. Cardiac gene transfer by intracoronary infusion of adenovirus vector-mediated reporter gene in the transplanted mouse heart. J Thorac Cardiovasc Surg 1996; 111:246-52．
9． Billinham ME, Gary NRB, Hammond ME, et al. A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: Heart rejection study group. J Heart Transplant, 1990; 9: 587-592．
10． Ono K , Lindsey ES . Improved technique of heart transplantation in rats . J Thorac Cardiovasc Surg 1969 ; 57: 225-229．
11． Sano I, Takahashi T, Koji Takehiko, et al. Prolonged survival of rat cardiac allograft with proinflammatory cytokine inhibitor. J Heart Lung Transplant 2001; 20: 583-89．
12． Chen, F. E., Huang, D. B., Chen, Y. Q., Ghosh, G. (Crystal structure of p50/p65 heterodimer of transcription factor NF-B bound to DNA. Nature 1998; 391:410-413．
13． Buchwald AB,Wagner AH, Webel C, et al. Decoy oligodeoxynucleotide against activator
protein-1 reduces neointimal proliferation after coronary angioplasty in hypercholesterolemic minipigs. J Am Coll Cardiol 2002; 39: 732-8．
14． Brauner R, Wu lily, Laks H et al. Intracoronary gene transfer of immunosuppressive cytokines to cardiac allografts method and efficacy of adenovirus-mediated transduction. J Thorac Cardiovasc Surg 1997; 113:1059-67．
15． Poston RS, Billingham M, Hoyt EG, et al. Rapamycin reverses chronic graft vascular disease in a novel cardiac allograft model. Circulation 1999; 100: 67-74．
16． Tatjana I; Fulong T; Viktor B; et af. Novel mode of action of angiotensin I converting enzyme inhibitors: direct activation of bradykinin Bl receptor. J Biol Chem 2002;277(19) :16847-52．
17． 李国新，董为人。反义寡核苷酸防治自体静脉移植物狭窄的策略 国外医学外科学分册， 1998， 25： 71-74。
18． Morishita, R, Sugimoto, T., Aoki, M., Kida, I., Tomita, N., Moriguchi, A., Maeda, K., Sawa, Y., Kaneda, Y., Higaki, J., Ogihara, T.) In vivo transfection of cis element 'decoy' against nuclear factor-B binding site prevents myocardial infarction. Nat. Med. (1997;8: 894-899．
19． Neely J R,Liebermeister H,Battersby E J,et al.Effect of pressure development on oxygen comsumption by isolated rat heart. Am J Physiol,1967,212(4) :804-814．
20． Liu P, Xu B, John Quilley,et al. Peroxynitrite attenuates hepatic ischemia-reperfusion injury. Am J Physiol Cell Physiol 2000; 279: c 1970-1977．
21． Freyer D, Manz R, Ziegenhorn A et al. Cerebral Endothelial Cells Release TNF-α After Stimulation with Cell Walls of Streptococcus pneumoniae and Regulate Inducible Nitric Oxide Synthase and ICAM-1 Expression Via Autocrine Loops J. Immun., 1999; 163:4308-4314
22． Nadaud S, Philippe M, Arnal JF, Michel JB, Soubrier F. Sustained increase in aortic endothelial nitric oxide synthase expression in vivo in a model of chronic high blood flow. Circ Res. 1996;79:857-863．
23． Piette J, Piret B. Multiple redox regulation in NF-kB transcription factor activation. Biol Chem, 1997; 378: 1237-1245．
24． Brand K , Page S, Rogler G, etal. Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion. J Clin Invest, 1996; 97: 1715-1722．
25． Blackwell TS, Chrisman JW. The role of nuclear factor-kappa B in cytokine gene regulation. Am J Respir Cell Mol Biol, 1997;17: 3-9．
26． Dpie LH. Reperfusion injury and its pharmacologic modification. Circulation, 1989, 80: 1049-57
27． Byrne JG, Martin WH, Hille B, et al. Complete prevention of myocardial stunning, contracture, low-reflow and edema after heart transplantation by blocking neutrophil adhesion molecules during reperfusion. J Thorac Cardiovasc Surg 1992; 104: 1589-95．
28． Chandrasekar B, Freeman GL. Induction of nuclear factor kappaB and activation protein 1 in postischemic myocardium. FEBS Lett, 1997; 401:30-35．
29． Cooper M, Lindholm P, Pieper G, etal. Myocardial nuclear factor-kB activity and nitric oxide production in rejecting cardiac allografts. Transplant, 1998; 66: 838-844．
30． Ma XL, Gao F, Lopez BL, et al. A two-edged sword in post-ischemic myocardial injury-dichotomy of action in crystalloid-versus blood-perfused hearts. J Pharmacol Exp Ther, 2000, 292: 912-920
31． Jacobs MD, Harrison SC. Structure of an IkBa/NF-kB complex. Cell 1998; 95: 749-758．
32． Mery PF, Lohmann SM, Walter U, et al. Ca2+ current is regulated by cyclic GMP-dependent protein kinase in mammalian cardiac myocytes. Proc Natl Acad Sci, 1991,88: 1197-1204． .
33． Suzuki J, Morishita R, Amano J, et al. Decoy against nuclear factor-kappa B attenuates myocardial cell infiltration and arterial neointimal formation in murine cardiac allografts. Gene Therapy 2000,7:1847-1852．
34． Hoang K, Chen YD, Reaven G, et al. Diabetes and dyslipidemia. A new model for transplant coronary artery disease. Circulation. 1998; 97: 2160-8．
35． Halloran PF. The immune response to an organ transplant. The handbook of transplant immunology. Bucks, UK: MedSci Publication, 1995:223．
36． Nadeau K C, Azuma H, Tibney NL. Cytokines in the pathophysiology of acute and chronic allograft rejection. Transplantation Review, 1996; 10: 99-107．
37． Kirby JA, Wilson JL. Allograft rejection: the role played by adhesion molecules. Transplantation Reviews, 1994; 8: 114-126．
38． Poston RS, Ennen M, Pollard J, et al. Ex vivo gene therapy prevents chronic graft vascular disease in cardiac allografts. J Thorac Cardiovasc Surg 1998; 116: 386-96
39． Stepkowski SM, Wang ME, Condon TP , et al. Protection against allograft rejection with intercellular adhesion molecule-1 antisense oligodeoxynucleotides. Transplantation 1998 ; 66:
699-707
40． Baeuerle PA, Henkel T. Function and activation of NF-kappa B in the immune system. Annu Revlmmunol 1994,12: 141-179
41． Lee JI, Burckart GJ. Nuclear factor kappa B: important transcription factor and therapeutic target. J Clin Pharmacol, 1998; 38: 981-993．
2． Siebenlist U, Franzoso G, Brown K, et al. Structure, regulation and function of NF-K B. Ann Rev Cell Biol 1994, 10: 405-455．
3． Li C, Browder W, Kao RL. Early activation of transcription factor NF-kappaB during ischemia in perfused rat heart. Am J Physiol, 1999,276: H543-552．
4． Cooper M, Lindholm P, Pieper G, et al. Myocardial nuclear factor-K B activity and nitric oxide production in rejecting cardiac allograts. Transplantation 1998; 66:838-844．
5． Guzman RJ, Lemarchand P, Crystal RG, et al. Efficient gene transfer into myocardium by direct injection of adenovirus vector. Cir Res 1993; 73: 1202-7．
6． Lee J, Laks H, Drinkwater DC, et al. Cardiac gene transfer by intracoronary infusion of adenovirus vector-mediatd reporter gene in the transplanted mouse heart. J Thorac Cardiovasc Surg 1996; 111: 246-52．
7． Bielinska A, Shivdasani RA, Zhang L, et al. Regulation of gene expression with double-stranded phosphotothioate oligonucleotides. Science 1990; 250: 997-1000
8． Lee J, Laks H, Drinkwater DC, etal. Cardiac gene transfer by intracoronary infusion of adenovirus vector-mediated reporter gene in the transplanted mouse heart. J Thorac Cardiovasc Surg 1996; 111:246-52．
9． Billinham ME, Gary NRB, Hammond ME, et al. A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: Heart rejection study group. J Heart Transplant, 1990; 9: 587-592．
10． Ono K , Lindsey ES . Improved technique of heart transplantation in rats . J Thorac Cardiovasc Surg 1969 ; 57: 225-229．
11． Sano I, Takahashi T, Koji Takehiko, et al. Prolonged survival of rat cardiac allograft with proinflammatory cytokine inhibitor. J Heart Lung Transplant 2001; 20: 583-89．
12． Chen, F. E., Huang, D. B., Chen, Y. Q., Ghosh, G. (Crystal structure of p50/p65 heterodimer of transcription factor NF-B bound to DNA. Nature 1998; 391:410-413．
13． Buchwald AB,Wagner AH, Webel C, et al. Decoy oligodeoxynucleotide against activator
protein-1 reduces neointimal proliferation after coronary angioplasty in hypercholesterolemic minipigs. J Am Coll Cardiol 2002; 39: 732-8．
14． Brauner R, Wu lily, Laks H et al. Intracoronary gene transfer of immunosuppressive cytokines to cardiac allografts method and efficacy of adenovirus-mediated transduction. J Thorac Cardiovasc Surg 1997; 113:1059-67．
15． Poston RS, Billingham M, Hoyt EG, et al. Rapamycin reverses chronic graft vascular disease in a novel cardiac allograft model. Circulation 1999; 100: 67-74．
16． Tatjana I; Fulong T; Viktor B; et af. Novel mode of action of angiotensin I converting enzyme inhibitors: direct activation of bradykinin Bl receptor. J Biol Chem 2002;277(19) :16847-52．
17． 李国新，董为人。反义寡核苷酸防治自体静脉移植物狭窄的策略 国外医学外科学分册， 1998， 25： 71-74。
18． Morishita, R, Sugimoto, T., Aoki, M., Kida, I., Tomita, N., Moriguchi, A., Maeda, K., Sawa, Y., Kaneda, Y., Higaki, J., Ogihara, T.) In vivo transfection of cis element 'decoy' against nuclear factor-B binding site prevents myocardial infarction. Nat. Med. (1997;8: 894-899．
19． Neely J R,Liebermeister H,Battersby E J,et al.Effect of pressure development on oxygen comsumption by isolated rat heart. Am J Physiol,1967,212(4) :804-814．
20． Liu P, Xu B, John Quilley,et al. Peroxynitrite attenuates hepatic ischemia-reperfusion injury. Am J Physiol Cell Physiol 2000; 279: c 1970-1977．
21． Freyer D, Manz R, Ziegenhorn A et al. Cerebral Endothelial Cells Release TNF-α After Stimulation with Cell Walls of Streptococcus pneumoniae and Regulate Inducible Nitric Oxide Synthase and ICAM-1 Expression Via Autocrine Loops J. Immun., 1999; 163:4308-4314
22． Nadaud S, Philippe M, Arnal JF, Michel JB, Soubrier F. Sustained increase in aortic endothelial nitric oxide synthase expression in vivo in a model of chronic high blood flow. Circ Res. 1996;79:857-863．
23． Piette J, Piret B. Multiple redox regulation in NF-kB transcription factor activation. Biol Chem, 1997; 378: 1237-1245．
24． Brand K , Page S, Rogler G, etal. Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion. J Clin Invest, 1996; 97: 1715-1722．
25． Blackwell TS, Chrisman JW. The role of nuclear factor-kappa B in cytokine gene regulation. Am J Respir Cell Mol Biol, 1997;17: 3-9．
26． Dpie LH. Reperfusion injury and its pharmacologic modification. Circulation, 1989, 80: 1049-57
27． Byrne JG, Martin WH, Hille B, et al. Complete prevention of myocardial stunning, contracture, low-reflow and edema after heart transplantation by blocking neutrophil adhesion molecules during reperfusion. J Thorac Cardiovasc Surg 1992; 104: 1589-95．
28． Chandrasekar B, Freeman GL. Induction of nuclear factor kappaB and activation protein 1 in postischemic myocardium. FEBS Lett, 1997; 401:30-35．
29． Cooper M, Lindholm P, Pieper G, etal. Myocardial nuclear factor-kB activity and nitric oxide production in rejecting cardiac allografts. Transplant, 1998; 66: 838-844．
30． Ma XL, Gao F, Lopez BL, et al. A two-edged sword in post-ischemic myocardial injury-dichotomy of action in crystalloid-versus blood-perfused hearts. J Pharmacol Exp Ther, 2000, 292: 912-920
31． Jacobs MD, Harrison SC. Structure of an IkBa/NF-kB complex. Cell 1998; 95: 749-758．
32． Mery PF, Lohmann SM, Walter U, et al. Ca2+ current is regulated by cyclic GMP-dependent protein kinase in mammalian cardiac myocytes. Proc Natl Acad Sci, 1991,88: 1197-1204． .
33． Suzuki J, Morishita R, Amano J, et al. Decoy against nuclear factor-kappa B attenuates myocardial cell infiltration and arterial neointimal formation in murine cardiac allografts. Gene Therapy 2000,7:1847-1852．
34． Hoang K, Chen YD, Reaven G, et al. Diabetes and dyslipidemia. A new model for transplant coronary artery disease. Circulation. 1998; 97: 2160-8．
35． Halloran PF. The immune response to an organ transplant. The handbook of transplant immunology. Bucks, UK: MedSci Publication, 1995:223．
36． Nadeau K C, Azuma H, Tibney NL. Cytokines in the pathophysiology of acute and chronic allograft rejection. Transplantation Review, 1996; 10: 99-107．
37． Kirby JA, Wilson JL. Allograft rejection: the role played by adhesion molecules. Transplantation Reviews, 1994; 8: 114-126．
38． Poston RS, Ennen M, Pollard J, et al. Ex vivo gene therapy prevents chronic graft vascular disease in cardiac allografts. J Thorac Cardiovasc Surg 1998; 116: 386-96
39． Stepkowski SM, Wang ME, Condon TP , et al. Protection against allograft rejection with intercellular adhesion molecule-1 antisense oligodeoxynucleotides. Transplantation 1998 ; 66:
699-707
40． Baeuerle PA, Henkel T. Function and activation of NF-kappa B in the immune system. Annu Revlmmunol 1994,12: 141-179
41． Lee JI, Burckart GJ. Nuclear factor kappa B: important transcription factor and therapeutic target. J Clin Pharmacol, 1998; 38: 981-993．