肿瘤坏死因子—α对骨骼肌缺血—再灌注损伤作用的实验研究
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摘要
肿瘤坏死因子-α对骨骼肌缺血—再灌注损伤作用的实验研究
骨骼肌缺血-再灌注不仅引起再灌注局部的损伤，也会导致远处器官的损害，其机制十分复杂。近年来发现，除氧自由基和钙超载作用外，细胞因子在骨骼肌缺血—再灌注损伤(skeletal muscle ischemia reperfusion injury，SMIRI)中也起重要作用。本研究探讨肿瘤坏死因子-α(TNF-α)在SMIRI中的作用。这一研究有助于阐明SMIRI的发病机制，为寻找防治SMIRI的新方法奠定基础。
本研究以大鼠后肢缺血—再灌注建立SMIRI动物模型，以抗TNF-α单克隆抗体阻断TNF-α的功能来研究TNF-α的作用。
本研究的第一部分，运用逆转录—聚合酶链反应(RT-PCR)检测单核细胞TNF-αmRNA的表达，并测定血浆TNF-α浓度。结果表明：骨骼肌缺血—再灌注导致单核细胞TNF-αmRNA表达显著增加，血浆TNF-α水平明显升高，应用抗TNF-α单克隆抗体能有效阻断升高的TNF-α。本研究首先应用RT-PCR方法检测SMIRI时TNF-αmRNA的高表达，有助于进一步阐明SMIRI的发生机制。
本研究的第二部分，运用免疫组织化学及流式细胞计数分别检测血管内皮细胞细胞间粘附分子-1(1CAM-1)与中性粒细胞CD18的表达。结果表明：骨骼肌缺血—再灌注上调了骨骼肌和肺组织血管内皮细胞ICAM-1以及中性粒细胞CD18的表达，应用抗TNF-α单克隆抗体能有效阻断这些粘附分子的表达。
本研究的第三部分，测定血浆丙二醛(MDA)、一氧化氮(NO)、肌酸激酶(CK)、骨骼肌和肺组织髓过氧化物酶(MPO)水平，以湿重/干重比例反映骨骼肌水肿程度，光镜电镜观察骨骼肌、肺组织结
福建医科大学硕土研究生毕业论文
构变化，采用末端脱氧核糖转移酶介导的生物素化脱氧尿嗜暄缺刻
标记技术（TUNEL）观察骨骼肌细胞凋亡的情况。结果表明：骨骼肌
缺血－再灌注导致mA、NO、CK、MPO以及湿重／干重比率显著升高，
应用抗孤F－。单克隆抗体能明显降低MDA、NO、CK和MPO的水平，
但不能有效缓解水肿程度：桔抗W卜。能明显减轻骨骼肌和肺组织
形态学和超微结构的改变；再灌注4小时骨骼肌可见大量TUNEL阳
性细胞，阻断TNF－a 能明显降低阳性细胞的比例。骨骼肌缺血－再
灌注激发T＼F－a的生成，异常升高的TNF－a发挥广泛的生物学活性，
在介导骨骼肌和肺的损伤中起重要作用，应用抗TNF－a单克隆抗体
能减轻损伤程度，为寻找防治SMIRI的新方法奠定基础。
Experimental study of effect of tumor necrosis factor-a on skeletal muscle ischemia-reperfusion injury
Ischemia in skeletal muscle followed by reperfusion results in local as well as remote organ injury. The mechanisms is very complex. In the past decade, studies showed that aside from oxygen free radicals and calcium overload, cytokines also play an important role in skeletal muscle ischemia- reperfusion injury (SMIRI). This study investigated the effect of tumor necrosis factor-a on skeletal muscle ischemia-reperfusion injury. It not only would help for further expounding the pathogenesis of SMIRI, but also could be informative for new prevention and cure of SMIRI.
A model of rat hind limb ischemia and reperfusion was established in this study. Anti-TNF-a monoclonal antibody was used to block the function of TNF-a to investigate the role of TNF-oc in SMIRI.
In the first part of this study, monocyte mRNA transcription level of TNF-a was determined by reverse transcription-polymerase chain reaction (RT-PCR), standard assay was performed for plasma level of TNF-a. Results showed that skeletal muscle ischemia-reperfusion (SMIR) contributed to significant increase in the level of TNF-a mRNA in monocyte and the level of TNF-a in plasma, anti-TNF-a monoclonal antibody managed to block the increasing TNF-a in plasma. The hyperexpression of TNF-a mRNA was firstly determined by RT-PCR in this study, this will help for further understanding the pathogenesis of SMIRI.
In the second part of this study, intercellular adhesion molecule-1 (ICAM-1) protein translation level of endothelium and CD 18 of neutrophil were assessed by immunohistochemistry and flow cytometry respectively. Results showed that ICAM-1 of vascular endothelium in skeletal muscle and lung was markedly upregulated by SMIR, so was CD 18 of neutrophil. The expression of these adhesion molecules was effectively blocked by treatment with anti-TNF-a monoclonal antibody.
In the third part of this study, standard assays were performed for plasma levels of malondialdehyde (MDA), creatine kinase (CK) and nitric oxide (NO), skeletal muscle and lung myeloperoxidase (MPO) activity was scored as well. The oedema degree was quantified by calculating the wet/dry weight ratio of skeletal muscle. Skeletal muscle and lung were also observed histologically and ultrastructurally. Terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labeling (TUNEL) method was used to identify apoptosis in skeletal muscle. Results showed that skeletal muscle ischemia-reperfusion resulted in a significant increase in the level of MDA, NO, CK, MPO and wet/dry weight ratio. Treatment with anti-TNF-a monoclonal antibody significantly lowered the levels of MDA, NO, CK and MPO, but couldn't reduce the oedema degree of skeletal muscle. Intravenous administration of anti-TNF-a monoclonal antibody at the time of reperfusion provided significant protection against histological and ultrastructural injury in both skeletal muscle and lung. A great number of TUNEL-positive cells in skeletal muscle were detected at 4-hour reperfusion. Monoclonal antibody targeting TNF-a distinctly reduced the rate of apoptotic cells. Skeletal muscle ischemia-reperfusion initiates a systemic TNF-a response. Both local
(skeletal muscle) and remote organ (lung) injury after ischemia-reperfusion requires participation of TNF-a, and the injury can be effectively attenuated by treatment with anti-TNF-a monoclonal antibody. This will be informative for new prevention and cure of SMIRI.
骨骼肌缺血-再灌注不仅引起再灌注局部的损伤，也会导致远处器官的损害，其机制十分复杂。近年来发现，除氧自由基和钙超载作用外，细胞因子在骨骼肌缺血—再灌注损伤(skeletal muscle ischemia reperfusion injury，SMIRI)中也起重要作用。本研究探讨肿瘤坏死因子-α(TNF-α)在SMIRI中的作用。这一研究有助于阐明SMIRI的发病机制，为寻找防治SMIRI的新方法奠定基础。
本研究以大鼠后肢缺血—再灌注建立SMIRI动物模型，以抗TNF-α单克隆抗体阻断TNF-α的功能来研究TNF-α的作用。
本研究的第一部分，运用逆转录—聚合酶链反应(RT-PCR)检测单核细胞TNF-αmRNA的表达，并测定血浆TNF-α浓度。结果表明：骨骼肌缺血—再灌注导致单核细胞TNF-αmRNA表达显著增加，血浆TNF-α水平明显升高，应用抗TNF-α单克隆抗体能有效阻断升高的TNF-α。本研究首先应用RT-PCR方法检测SMIRI时TNF-αmRNA的高表达，有助于进一步阐明SMIRI的发生机制。
本研究的第二部分，运用免疫组织化学及流式细胞计数分别检测血管内皮细胞细胞间粘附分子-1(1CAM-1)与中性粒细胞CD18的表达。结果表明：骨骼肌缺血—再灌注上调了骨骼肌和肺组织血管内皮细胞ICAM-1以及中性粒细胞CD18的表达，应用抗TNF-α单克隆抗体能有效阻断这些粘附分子的表达。
本研究的第三部分，测定血浆丙二醛(MDA)、一氧化氮(NO)、肌酸激酶(CK)、骨骼肌和肺组织髓过氧化物酶(MPO)水平，以湿重/干重比例反映骨骼肌水肿程度，光镜电镜观察骨骼肌、肺组织结
福建医科大学硕土研究生毕业论文
构变化，采用末端脱氧核糖转移酶介导的生物素化脱氧尿嗜暄缺刻
标记技术（TUNEL）观察骨骼肌细胞凋亡的情况。结果表明：骨骼肌
缺血－再灌注导致mA、NO、CK、MPO以及湿重／干重比率显著升高，
应用抗孤F－。单克隆抗体能明显降低MDA、NO、CK和MPO的水平，
但不能有效缓解水肿程度：桔抗W卜。能明显减轻骨骼肌和肺组织
形态学和超微结构的改变；再灌注4小时骨骼肌可见大量TUNEL阳
性细胞，阻断TNF－a 能明显降低阳性细胞的比例。骨骼肌缺血－再
灌注激发T＼F－a的生成，异常升高的TNF－a发挥广泛的生物学活性，
在介导骨骼肌和肺的损伤中起重要作用，应用抗TNF－a单克隆抗体
能减轻损伤程度，为寻找防治SMIRI的新方法奠定基础。
Experimental study of effect of tumor necrosis factor-a on skeletal muscle ischemia-reperfusion injury
Ischemia in skeletal muscle followed by reperfusion results in local as well as remote organ injury. The mechanisms is very complex. In the past decade, studies showed that aside from oxygen free radicals and calcium overload, cytokines also play an important role in skeletal muscle ischemia- reperfusion injury (SMIRI). This study investigated the effect of tumor necrosis factor-a on skeletal muscle ischemia-reperfusion injury. It not only would help for further expounding the pathogenesis of SMIRI, but also could be informative for new prevention and cure of SMIRI.
A model of rat hind limb ischemia and reperfusion was established in this study. Anti-TNF-a monoclonal antibody was used to block the function of TNF-a to investigate the role of TNF-oc in SMIRI.
In the first part of this study, monocyte mRNA transcription level of TNF-a was determined by reverse transcription-polymerase chain reaction (RT-PCR), standard assay was performed for plasma level of TNF-a. Results showed that skeletal muscle ischemia-reperfusion (SMIR) contributed to significant increase in the level of TNF-a mRNA in monocyte and the level of TNF-a in plasma, anti-TNF-a monoclonal antibody managed to block the increasing TNF-a in plasma. The hyperexpression of TNF-a mRNA was firstly determined by RT-PCR in this study, this will help for further understanding the pathogenesis of SMIRI.
In the second part of this study, intercellular adhesion molecule-1 (ICAM-1) protein translation level of endothelium and CD 18 of neutrophil were assessed by immunohistochemistry and flow cytometry respectively. Results showed that ICAM-1 of vascular endothelium in skeletal muscle and lung was markedly upregulated by SMIR, so was CD 18 of neutrophil. The expression of these adhesion molecules was effectively blocked by treatment with anti-TNF-a monoclonal antibody.
In the third part of this study, standard assays were performed for plasma levels of malondialdehyde (MDA), creatine kinase (CK) and nitric oxide (NO), skeletal muscle and lung myeloperoxidase (MPO) activity was scored as well. The oedema degree was quantified by calculating the wet/dry weight ratio of skeletal muscle. Skeletal muscle and lung were also observed histologically and ultrastructurally. Terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labeling (TUNEL) method was used to identify apoptosis in skeletal muscle. Results showed that skeletal muscle ischemia-reperfusion resulted in a significant increase in the level of MDA, NO, CK, MPO and wet/dry weight ratio. Treatment with anti-TNF-a monoclonal antibody significantly lowered the levels of MDA, NO, CK and MPO, but couldn't reduce the oedema degree of skeletal muscle. Intravenous administration of anti-TNF-a monoclonal antibody at the time of reperfusion provided significant protection against histological and ultrastructural injury in both skeletal muscle and lung. A great number of TUNEL-positive cells in skeletal muscle were detected at 4-hour reperfusion. Monoclonal antibody targeting TNF-a distinctly reduced the rate of apoptotic cells. Skeletal muscle ischemia-reperfusion initiates a systemic TNF-a response. Both local
(skeletal muscle) and remote organ (lung) injury after ischemia-reperfusion requires participation of TNF-a, and the injury can be effectively attenuated by treatment with anti-TNF-a monoclonal antibody. This will be informative for new prevention and cure of SMIRI.
引文
1． 孙建民，蒋米尔．五十年来我国血管外科的进展。中华外科杂志，1999，37(9) ：531-532
2． Kyriakides C, Austen WG Jr, Wamg Y, et al. Neutrophil mediated remote organ injury after lower torso ischemia and reperfusion is selectin and complement dependent. J Trauma, 2000, 48(1) : 32-38
3． Ferrante A Nandoskar M, Walz A, et al. Effects of tumor necrosis factor alpha and interleukin-1 alpha and beta on human neutrophil migration, respiratory burst and degranulation. Int Arch Allergy Appl Immunol, 1988; 86(1) : 82-91
4． Horvath CJ, Ferro TJ, Jesmok G, et al. Recombinant tumor necrosis factor increase pulmonary vascular permeability independent of neutrophils. Proc Natl Acard Sci (USA), 1988; 85: 9219-9223
5． Frolkis I, Gurevitch J, Yuhas Y, et al. Interaction between paracrine tumor necrosis factor-alpha and paracrine angiotensin Ⅱ during myocardial ischemia. J Am Coll Cardiol, 2001; 37(4) : 316-322
6． Peralta C, Fernandez L, Panes J, et al. Preconditioning protects against systemic disorders associated with hepatic ischemia-reperfusion through blockade of tumor necrosis factor-induced P-selectin up-regulation in the rat. Hepatology, 2001; 33(1) : 100-113
7． Donnahoo KK, Meldrum DR, Shenkar R, et al. Early renal ischemia, with or without reperfusion, activates NFkappaB and increases TNF-alpha bioactivity in the kidney. J Urol, 2000; 163: 1328-1332
8． Welbourn R, Goldman G, O'Riordain M, et al. Role for tumor necrosis factor as mediator of lung injury following lower torso ischemia. J
Appl Physiol, 1991; 70: 2645-2649
9． Ascer EA, Gennaro M, Cupo S, et al. Do cytokines play a role in skeletal muscle ischemia and reperfusion?. Cardiovasc Surg, 1992; 33: 588-592
10． Seekamp A, Warren JS Remick DG, et al. Requirements for tumor necrosis factor-alpha and interleukin-1 in limb ischemia/reperfusion injury and associated lung injury. Am J Pathol, 1993; 143(2) : 453-463
11． Herskowitz A, Choi S, Ansari AA, et al. Cytokine mRNA expression in postischemic/reperfused myocardium. Am J Pathol, 1995; 146(2) : 419-428
12． Crinnion JN, Homer-Vanniasinkam S, Parkin SM, et al. Role of neutrophil-endothelial adhesion in skeletal muscle reperfusion injury. Br J Surg, 1996; 83(2) : 251-254
13． 吴其夏，余应年，卢建。新编病理生理学。北京：中国协和医科大学出版社，1999：242-259
14． Goris RJA. MODS/SIRS: Result of an overwhelming inflammatory response? World J Surg, 1996, 20:418
15． Bone RC. Immunologic dissonance: A continuing evolution in our understanding of the systemic inflammatory response syndrome (SIRS) and the multiple organ dysfunction syndrome (MODS). Ann Int Med, 1996, 125(8) : 681
16． Bone RC. Toward a theory regarding the pathogenesis of the systemic inflammatory response syndrome. Crit Care Med, 1996, 24(2) : 163
17． Gute DC, Ishida T, Yarimizu K, et al. Inflammatory response to ischemia and reperfusion in skeletal muscle. Mol Cell Biochem, 1998; 179(1-2) : 169-187
18． Colletti LM, Remick DG, Burtch GD, et al. Role of tumor necrosis
factor-α in the pathophysiologic alterations after hepatic ischemia/reperfusion injury in the rat. J Clin Invest, 1990; 85: 1936-1943
19． Gaines GC, Welborn MBⅢ, Moldawer LL, et al. Attenuation of skeletal muscle ischemia/reperfusion injury by inhibition of tumor necrosis factor. J Vase Surg, 1999; 29(2) : 370-376
20． 孙卫民，王惠琴。细胞因子研究方法学。北京：人民卫生出版社，1999：587-597
21． Testa M, De Ruvo E, Russo A, et al. Induction of interleukin-1 beta and interleukin-6 gene expression in hypoperfused skeletal muscle of patients with peripheral arterial disease. Ital Heart J, 2000; 1(1) : 64-67
22． Wang WZ, Guo SZ, Tsai TM, et al. Platelet-activating factor contributes postischemic vasospasm. J Surg Res, 2000; 89(2) : 139-146
23． Kyriakides C, Woodcock SA, Wang Y, et al. Soluble P-selectin moderates complement-dependent reperfusion injury of ischemic skeletal muscle. Am J Physiol Cell Physiol, 2000; 279(2) : c520-c528
2． Kyriakides C, Austen WG Jr, Wamg Y, et al. Neutrophil mediated remote organ injury after lower torso ischemia and reperfusion is selectin and complement dependent. J Trauma, 2000, 48(1) : 32-38
3． Ferrante A Nandoskar M, Walz A, et al. Effects of tumor necrosis factor alpha and interleukin-1 alpha and beta on human neutrophil migration, respiratory burst and degranulation. Int Arch Allergy Appl Immunol, 1988; 86(1) : 82-91
4． Horvath CJ, Ferro TJ, Jesmok G, et al. Recombinant tumor necrosis factor increase pulmonary vascular permeability independent of neutrophils. Proc Natl Acard Sci (USA), 1988; 85: 9219-9223
5． Frolkis I, Gurevitch J, Yuhas Y, et al. Interaction between paracrine tumor necrosis factor-alpha and paracrine angiotensin Ⅱ during myocardial ischemia. J Am Coll Cardiol, 2001; 37(4) : 316-322
6． Peralta C, Fernandez L, Panes J, et al. Preconditioning protects against systemic disorders associated with hepatic ischemia-reperfusion through blockade of tumor necrosis factor-induced P-selectin up-regulation in the rat. Hepatology, 2001; 33(1) : 100-113
7． Donnahoo KK, Meldrum DR, Shenkar R, et al. Early renal ischemia, with or without reperfusion, activates NFkappaB and increases TNF-alpha bioactivity in the kidney. J Urol, 2000; 163: 1328-1332
8． Welbourn R, Goldman G, O'Riordain M, et al. Role for tumor necrosis factor as mediator of lung injury following lower torso ischemia. J
Appl Physiol, 1991; 70: 2645-2649
9． Ascer EA, Gennaro M, Cupo S, et al. Do cytokines play a role in skeletal muscle ischemia and reperfusion?. Cardiovasc Surg, 1992; 33: 588-592
10． Seekamp A, Warren JS Remick DG, et al. Requirements for tumor necrosis factor-alpha and interleukin-1 in limb ischemia/reperfusion injury and associated lung injury. Am J Pathol, 1993; 143(2) : 453-463
11． Herskowitz A, Choi S, Ansari AA, et al. Cytokine mRNA expression in postischemic/reperfused myocardium. Am J Pathol, 1995; 146(2) : 419-428
12． Crinnion JN, Homer-Vanniasinkam S, Parkin SM, et al. Role of neutrophil-endothelial adhesion in skeletal muscle reperfusion injury. Br J Surg, 1996; 83(2) : 251-254
13． 吴其夏，余应年，卢建。新编病理生理学。北京：中国协和医科大学出版社，1999：242-259
14． Goris RJA. MODS/SIRS: Result of an overwhelming inflammatory response? World J Surg, 1996, 20:418
15． Bone RC. Immunologic dissonance: A continuing evolution in our understanding of the systemic inflammatory response syndrome (SIRS) and the multiple organ dysfunction syndrome (MODS). Ann Int Med, 1996, 125(8) : 681
16． Bone RC. Toward a theory regarding the pathogenesis of the systemic inflammatory response syndrome. Crit Care Med, 1996, 24(2) : 163
17． Gute DC, Ishida T, Yarimizu K, et al. Inflammatory response to ischemia and reperfusion in skeletal muscle. Mol Cell Biochem, 1998; 179(1-2) : 169-187
18． Colletti LM, Remick DG, Burtch GD, et al. Role of tumor necrosis
factor-α in the pathophysiologic alterations after hepatic ischemia/reperfusion injury in the rat. J Clin Invest, 1990; 85: 1936-1943
19． Gaines GC, Welborn MBⅢ, Moldawer LL, et al. Attenuation of skeletal muscle ischemia/reperfusion injury by inhibition of tumor necrosis factor. J Vase Surg, 1999; 29(2) : 370-376
20． 孙卫民，王惠琴。细胞因子研究方法学。北京：人民卫生出版社，1999：587-597
21． Testa M, De Ruvo E, Russo A, et al. Induction of interleukin-1 beta and interleukin-6 gene expression in hypoperfused skeletal muscle of patients with peripheral arterial disease. Ital Heart J, 2000; 1(1) : 64-67
22． Wang WZ, Guo SZ, Tsai TM, et al. Platelet-activating factor contributes postischemic vasospasm. J Surg Res, 2000; 89(2) : 139-146
23． Kyriakides C, Woodcock SA, Wang Y, et al. Soluble P-selectin moderates complement-dependent reperfusion injury of ischemic skeletal muscle. Am J Physiol Cell Physiol, 2000; 279(2) : c520-c528
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