吡那地尔对严重烧伤大鼠肠上皮细胞线粒体功能的保护作用
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摘要
目的 肠道是对缺血、缺氧损伤敏感的器官之一，其缺氧性损伤普遍存在于严重烧创伤的病理生理过程中，在严重烧创伤中发生最早、恢复最迟。严重烧伤后，肠粘膜屏障损害、免疫功能受抑制、肠道菌群紊乱，肠道中的微生物和内毒素可通过受损的肠粘膜屏障进入体循环，形成肠源性感染，导致全身性炎症反应综合征(SIRS)乃至多器官功能不全(MODS)的发生。
线粒体作为能量转换的细胞器对缺血、缺氧等因素十分敏感，缺血、缺氧损伤可致线粒体结构和功能障碍，进一步导致肠粘膜损伤的发生。近来研究发现ATP依赖性钾通道(K_(ATP))开放剂对心肌缺血、缺氧性损伤具有保护作用，其作用靶点可能在线粒体膜ATP依赖性钾通道(mitoK_(ATP))，但却未见该类药物对肠粘膜缺血、缺氧性损伤具有保护作用的报道。
因此本研究旨在通过实验观察mitoK_(ATP)开放剂吡那地尔(Pin)对肠粘膜上皮细胞是否具有保护作用，寻找严重烧创伤致肠道缺血、缺氧性损伤的防治药物。
方法 ①建立活性氧损伤人肠上皮细胞的体外模型，运用MTT法、免疫组化法、激光共聚焦法，观察活性氧损伤肠上皮细胞线粒体的整体功能、细胞色素C的释放、线粒体膜电位的变化及吡那地尔对肠上皮细胞线粒体的保护作用；②复制大鼠30％TBSA Ⅲ度烫伤模型，观察大鼠血浆和肠粘膜组织ROS、MDA、SOD的含量，肠粘膜组织细胞色素C的释放、线粒体的呼吸功能、病理形态改变以及吡那地尔的保护作用。
结果 ①成功建立活性氧损伤肠上皮细胞的体外模型；②体外实验表明，采用高浓度(4mmol／L)及低浓度(400lμmol／L)H_2O_2损伤肠上皮细胞后，细胞线粒体整体功能下降、细胞色素释放增加、线粒体膜电位降低，吡那地尔可显著改善线粒体功能；③动物实验结果表明，烫伤大鼠血浆和肠粘膜组织ROS、MDA呈现不同程度增高、SOD降低，线粒体RCR下降、线粒体肿胀明显，上述变化在伤后6h尤为明显，吡那地尔可显著改善上述指标、减轻肠粘膜组织的病理学改变。
第三军医大学硕士学位论文
结论①毗那地尔对活性氧损伤的肠上皮细胞具有显著保护作用;
②毗那地尔对严重烧伤早期的肠粘膜组织具有显著保护作用。
Object: The intestine is one of the most sensitive organs on ischemia and anoxia. Since intestinal mucous membrane barrier was damaged after burn, microorganism and endotoxin entered into blood from intestinal tract, which easily resulted in systemic inflammatory response syndrome (SIRS) and even more, multiple organ dysfunction syndrome (MODS).
Mitochondrion, an organelle for translating energy, is very sensitive to ischemia and anoxia, which can cause disturbance of mitochondrial structure and function.
Recent studies found that ATP sensitive K+ channel (KATP) opener could protect myocardia from ischemia and hypoxia. Its pharmacological role targeted myocardial mitochondrion membrane KATP channel (mitoKArp). However, there wasn't any report about this kind of protection on intestinal mucous membrane damaged by ischemia and anoxia.
Therefore, this study aimed at searching for pharmacological protection of pinacidil (Pin), a mitoKATp opener, on intestinal mucous membrane damaged by severe burn.
Methods:
(1) After establishing a model using intestinal epithelial cells (HIC) damaged by reactive oxygen species, intestinal epithelial cells mitochondrial allomeric function, cytochrome C release, mitochondrial membrane potential and pinacidil protection on intesting epithelial cells mitochondria were observed.
(2) After establishing 30% TBSA III0 burn model, ROS, MDA and SOD in plasma and intestinal mucous as well as cytochorme C, mitochondrial respiratory function and pathological morphology changes in rats treated or un-treated with pinacidil were observed.
Results:
(1) In vitro, It was found that HIC cells' mitochondrial functions descented; cytochrome C release increased, while mitochondrial membrane potential reduced. However, pinacidil could improve mitochondrial function significantly.
(2) In burnt rats, ROS and MDA elevated in plasma and intestinal mucosa. SOD and mitochondrial RCR decreased. Mitochondrion swelling occurred in burned rats, too. The remarkable changes appeared at 6 hour-point after burn. Pinacidil could improve
mitochondrial functions and decrease pathomorphology damage of intestinal mucosa.
Conclusion:
(1) Pinacidil could protect intestinal epithelial cells from reactive oxygen species' injury in vitro
(2) Pinacidil could significantly protect intestinal mucous membrane at early stage in burnt rats in vivo.
线粒体作为能量转换的细胞器对缺血、缺氧等因素十分敏感，缺血、缺氧损伤可致线粒体结构和功能障碍，进一步导致肠粘膜损伤的发生。近来研究发现ATP依赖性钾通道(K_(ATP))开放剂对心肌缺血、缺氧性损伤具有保护作用，其作用靶点可能在线粒体膜ATP依赖性钾通道(mitoK_(ATP))，但却未见该类药物对肠粘膜缺血、缺氧性损伤具有保护作用的报道。
因此本研究旨在通过实验观察mitoK_(ATP)开放剂吡那地尔(Pin)对肠粘膜上皮细胞是否具有保护作用，寻找严重烧创伤致肠道缺血、缺氧性损伤的防治药物。
方法 ①建立活性氧损伤人肠上皮细胞的体外模型，运用MTT法、免疫组化法、激光共聚焦法，观察活性氧损伤肠上皮细胞线粒体的整体功能、细胞色素C的释放、线粒体膜电位的变化及吡那地尔对肠上皮细胞线粒体的保护作用；②复制大鼠30％TBSA Ⅲ度烫伤模型，观察大鼠血浆和肠粘膜组织ROS、MDA、SOD的含量，肠粘膜组织细胞色素C的释放、线粒体的呼吸功能、病理形态改变以及吡那地尔的保护作用。
结果 ①成功建立活性氧损伤肠上皮细胞的体外模型；②体外实验表明，采用高浓度(4mmol／L)及低浓度(400lμmol／L)H_2O_2损伤肠上皮细胞后，细胞线粒体整体功能下降、细胞色素释放增加、线粒体膜电位降低，吡那地尔可显著改善线粒体功能；③动物实验结果表明，烫伤大鼠血浆和肠粘膜组织ROS、MDA呈现不同程度增高、SOD降低，线粒体RCR下降、线粒体肿胀明显，上述变化在伤后6h尤为明显，吡那地尔可显著改善上述指标、减轻肠粘膜组织的病理学改变。
第三军医大学硕士学位论文
结论①毗那地尔对活性氧损伤的肠上皮细胞具有显著保护作用;
②毗那地尔对严重烧伤早期的肠粘膜组织具有显著保护作用。
Object: The intestine is one of the most sensitive organs on ischemia and anoxia. Since intestinal mucous membrane barrier was damaged after burn, microorganism and endotoxin entered into blood from intestinal tract, which easily resulted in systemic inflammatory response syndrome (SIRS) and even more, multiple organ dysfunction syndrome (MODS).
Mitochondrion, an organelle for translating energy, is very sensitive to ischemia and anoxia, which can cause disturbance of mitochondrial structure and function.
Recent studies found that ATP sensitive K+ channel (KATP) opener could protect myocardia from ischemia and hypoxia. Its pharmacological role targeted myocardial mitochondrion membrane KATP channel (mitoKArp). However, there wasn't any report about this kind of protection on intestinal mucous membrane damaged by ischemia and anoxia.
Therefore, this study aimed at searching for pharmacological protection of pinacidil (Pin), a mitoKATp opener, on intestinal mucous membrane damaged by severe burn.
Methods:
(1) After establishing a model using intestinal epithelial cells (HIC) damaged by reactive oxygen species, intestinal epithelial cells mitochondrial allomeric function, cytochrome C release, mitochondrial membrane potential and pinacidil protection on intesting epithelial cells mitochondria were observed.
(2) After establishing 30% TBSA III0 burn model, ROS, MDA and SOD in plasma and intestinal mucous as well as cytochorme C, mitochondrial respiratory function and pathological morphology changes in rats treated or un-treated with pinacidil were observed.
Results:
(1) In vitro, It was found that HIC cells' mitochondrial functions descented; cytochrome C release increased, while mitochondrial membrane potential reduced. However, pinacidil could improve mitochondrial function significantly.
(2) In burnt rats, ROS and MDA elevated in plasma and intestinal mucosa. SOD and mitochondrial RCR decreased. Mitochondrion swelling occurred in burned rats, too. The remarkable changes appeared at 6 hour-point after burn. Pinacidil could improve
mitochondrial functions and decrease pathomorphology damage of intestinal mucosa.
Conclusion:
(1) Pinacidil could protect intestinal epithelial cells from reactive oxygen species' injury in vitro
(2) Pinacidil could significantly protect intestinal mucous membrane at early stage in burnt rats in vivo.
引文
1．李建明，蔡黔，周红，等．过氧化氢对肠上皮细胞线粒体的损伤作用．第三军医大学学报，2002；24(2)：142-145．
2. Yakes FM, Van Houten B. Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proc Natl Acad Sci U.S.A, 1997;94(2):514-519.
3. Fujii H, Cody SH, Seydel U, et al. Recording of mitochondrial transmembrane potential
4. and volume in cultured rat osteoclasts by confocal laser scanning microscopy. Histochem, 1997;29:571-581.
5. Ramzy PI, Wolf SE, Irtun O, et al. Gut epithelial apoptosis after severe burn: effects of gut hypoperfusion. J Am Coll Surg, 2000; 190(3):281-287
6. Wolf SE, Ikeda H, Matin S, et al. Cutaneous burn increases apoptosis in the gut epithelium of mice. J Am Coll Surg, 1999; 188(1):10-16
7. Lightfoot E, Horton JW, Maass DL, et al. Major burn trauma in rats promotes cardiac and gastrointestinal apoptosis. Shock,1999 ; 11(1): 29-34
8．袁建成，李艳萍，秦孝健等．细胞凋亡在严重烧伤大鼠肠粘膜屏障损伤中的作用．中国危重病急救医学 1999；11(12)：715-717
9. Varedi M, George HG, David NH,et al. A thermal injury induced circulating factor compromises intestinal cell morphology, proliferation and migration. Am J Physiol, 1999; 277(40):G 175-G 182
10. Janssen YM, Van Houten B, Borm PJ, et al. Lab Invest. 1993;69:261-274
11. Noda T, Iwakiri R, Fujimono K, et al. Programmed cell death induced by ischemia-reperfusion in rat intestinal mucosa. Am J Physiol,1998,274:g270-g276.
12. Ikeda H, Suzuki M, kioke M, et al. Apoptosis is a major mode of cell death caused ischemia and ischemia/reperfusion injury to the rat intestinal epithelium. Gut, 1998,42:530-537.
13. Herz J,et al. Proc Natl Acad Sci USA,1993;90:2812-2819
14. Buttle M T, Sandstrom P A. Oxidative stress as a mediator of apoptosis. Immunol Today, 1994,15(1):7-10.
15. Chen,Q, & Ames B(1994) Proc Natl Acad Sci. USA. 91:4130-4134
16. Clopton DA, & Saltman P. (1995) Biochem Biophys Res Commun.210:189-196
17. Corcaran GB, Fix L, Iones DP, Moslen MT, Nicotcra P, Oberhammer FA, & Buttyan R. (1994) Toxicol Appl Pharmacol. 128:169-181
18. de Bono DP & Yang WD. (1995) Atherosclerosis (Ireland) 114:
19. Whittemore ER, Loo DT, & Cotman PM. (1994) Neuro-Report. 5:1485-1488
20. Marano CW, Lewis SA, Garulacan LA, et al. Tumor necrosis factor-alpha increases sodium and chloride conductance across the tight junction of CACO-2 BBE, a human intestinal epithelial cell line. J Membr Biol, 1998 ; 161(3): 263-274
21. Ruemmele FM, Dionne S, Levy E, et al. TNFalpha-induced IEC-6 cell apoptosis requires activation of ICE caspases whereas complete inhibition of the caspase cascade leads to necrotic cell death. Biochem Biophys Res Commun,, 1999 ; 260(1): 159-166
22. Le'Negrate G, Selva E, Auberger P, et al. Sustained polymorphonuclear leukocyte transmigration induces apoptosis in T84 intestinal epithelial cells. J Cell Biol, 2000 ; 150(6): 1479-1488
23. Araki Y, Fujiyama Y, Andoh A, et al. Hydrophilic and hydrophobic bile acids exhibit different cytotoxicities through cytolysis, interleukin-8 synthesis and apoptosis in the intestinal epithelial cell lines: IEC-6 and Caco-2 cells. Stand J Gastroenterol, 2001; 36(5): 533-539
24. Funda DP, Tuckova L, Farre MA, et al. CD14 is expressed and released as soluble CD14 by human intestinal epithelial cells in vitro: lipopolysaccharide activation of epithelial cells revisited. Infect Immun, 2001; 69(6): 3772-3781
25．杨瑞华，海春旭，李瑞珍等。过氧化氢与细胞生长状态的剂量反应关系研究。癌变畸变突变，1997；9(2)：92-99
26．曹纯章，杨绍娟，朴丽莎等．过氧化氢诱导心肌细胞凋亡性和非凋亡性死亡的研究．中国病理生理杂志，2000：16(5)：457-461
27. Noma A. ATP-regulated K~+ channels in cardiac muscle. Nature, 1983;305:147-8.
28．刘蔚，汪海．抗心肌缺血缺氧药物的新靶点：线粒体ATP敏感性钾通道．中国药理学通报，2001；17(6)：610—613。
29. Liu Y, Sato T, O Rourke B et al. Mitochondrial ATP-dependent potassium channel: Novel effectors of cardioprotection. Circulation,1998;97:2463-9.
30. Sato T, Sassaki N, Rourke B et al. Nicorandil, a potent cardioprotective agent, acts by
opening mitochondrial ATP-dependent potassium channels. J Am Coll Cardiol, 2000;35: 514-518.
31. Simon HU, Haj Yehia A, Levi Schaffer F.Role of reactive oxygen species (ROS) in apoptosis induction. Apoptosis. 2000 ; 5(5): 415-8
32. Kluck RM, Bossy Wetzel E, Green DR, et al. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis . Science, 1997;275 (5303) :1132-1134
33. Susin SA, Zamzami N, Castedo M, et al. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J Exp Med, 1996; 184(4): 1331-1335.
34．蔡黔，徐淑芬，周红等．过氧化氢对肠上皮细胞线粒体功能及细胞凋亡的影响．中国现代医学杂志，2002；12(21)：4-10．
35. Javadov SA; Lim KH; Kerr PM; et al. Protection of hearts from reperfusion injury by propofol is associated with inhibition of the mitochondrial permeability transition. Cardiovasc Res. 2000 ; 45(2): 360-9J
36. Levraut J; Iwase H; Shao ZH; et al. Cell death during ischemia: relationship to mitochondrial depolarization and ROS generation. Am J Physiol Heart Circ Physiol. 2003 ; 284(2): H549-58.
37. Crestanello JA; Doliba NM; Babsky AM; et al. Ischemic preconditioning improves mitochonddal tolerance to experimental calcium overload. J Surg Res. 2002;103(2): 243-51
38．梁晚益，黄跃生，杨宗诚，等．二氮嗪对严重烧伤大鼠心肌损害的保护作用及其机制[J]．第三军医大学学报，2003，25(19)：1731-1733．
39. Wan-Yi Liang, Li-Xin Tang, Zong-Cheng Yang, et al. Calcium induced the damage of myocardial mitochondrial respiratory function in the early stage aher severe burns.Burns,2002;28:143-146.
40. Park DA. Ischemic injury in the cat small intestine: Role of superoxide radicals.Gastroenterology 1982; 82(1): 9-15.
41. Toda N, et al. Cardiowase pharmacol[J]. 1985;7:1118.
42. Smallwood KJ,et al. Cardiowase pharmacol [J]. 1988; 12:102
43. Petit PX, Susin SA, Zamzami N ,et al. Mitochondria and programmed cell death: back to the future. FEBS Letters, 1996; 396(1):7-13
44. Reed JC. Cytochrome c : can't live with it, can't live without it. Cell, 1997; 91(5): 559-562
45. Vander Heiden MG, Chandel NS, Williamson EK, et al. BcI-XL regulates the membrane potential and volume homeostasis of mitochondria. Cell, 1997; 91(5):627-637.
46．蔡循，陈国强，陈竺等。线粒体跨膜电位与细胞凋亡。生物化学与生物物理进展，2001；28(1)：3—6．
47. Border JR. Multiple system organ failure[J]. Ann Surg, 1992; 216(2): 111-116.
48. Saito H, Trocki O, Alexander JW, et al. The effect route of nutrient administration on the nutritional state, catabolic hormone secretion, and gutmucosal integrity after burn injury. JPEN, 1987; 11:1.
2. Yakes FM, Van Houten B. Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proc Natl Acad Sci U.S.A, 1997;94(2):514-519.
3. Fujii H, Cody SH, Seydel U, et al. Recording of mitochondrial transmembrane potential
4. and volume in cultured rat osteoclasts by confocal laser scanning microscopy. Histochem, 1997;29:571-581.
5. Ramzy PI, Wolf SE, Irtun O, et al. Gut epithelial apoptosis after severe burn: effects of gut hypoperfusion. J Am Coll Surg, 2000; 190(3):281-287
6. Wolf SE, Ikeda H, Matin S, et al. Cutaneous burn increases apoptosis in the gut epithelium of mice. J Am Coll Surg, 1999; 188(1):10-16
7. Lightfoot E, Horton JW, Maass DL, et al. Major burn trauma in rats promotes cardiac and gastrointestinal apoptosis. Shock,1999 ; 11(1): 29-34
8．袁建成，李艳萍，秦孝健等．细胞凋亡在严重烧伤大鼠肠粘膜屏障损伤中的作用．中国危重病急救医学 1999；11(12)：715-717
9. Varedi M, George HG, David NH,et al. A thermal injury induced circulating factor compromises intestinal cell morphology, proliferation and migration. Am J Physiol, 1999; 277(40):G 175-G 182
10. Janssen YM, Van Houten B, Borm PJ, et al. Lab Invest. 1993;69:261-274
11. Noda T, Iwakiri R, Fujimono K, et al. Programmed cell death induced by ischemia-reperfusion in rat intestinal mucosa. Am J Physiol,1998,274:g270-g276.
12. Ikeda H, Suzuki M, kioke M, et al. Apoptosis is a major mode of cell death caused ischemia and ischemia/reperfusion injury to the rat intestinal epithelium. Gut, 1998,42:530-537.
13. Herz J,et al. Proc Natl Acad Sci USA,1993;90:2812-2819
14. Buttle M T, Sandstrom P A. Oxidative stress as a mediator of apoptosis. Immunol Today, 1994,15(1):7-10.
15. Chen,Q, & Ames B(1994) Proc Natl Acad Sci. USA. 91:4130-4134
16. Clopton DA, & Saltman P. (1995) Biochem Biophys Res Commun.210:189-196
17. Corcaran GB, Fix L, Iones DP, Moslen MT, Nicotcra P, Oberhammer FA, & Buttyan R. (1994) Toxicol Appl Pharmacol. 128:169-181
18. de Bono DP & Yang WD. (1995) Atherosclerosis (Ireland) 114:
19. Whittemore ER, Loo DT, & Cotman PM. (1994) Neuro-Report. 5:1485-1488
20. Marano CW, Lewis SA, Garulacan LA, et al. Tumor necrosis factor-alpha increases sodium and chloride conductance across the tight junction of CACO-2 BBE, a human intestinal epithelial cell line. J Membr Biol, 1998 ; 161(3): 263-274
21. Ruemmele FM, Dionne S, Levy E, et al. TNFalpha-induced IEC-6 cell apoptosis requires activation of ICE caspases whereas complete inhibition of the caspase cascade leads to necrotic cell death. Biochem Biophys Res Commun,, 1999 ; 260(1): 159-166
22. Le'Negrate G, Selva E, Auberger P, et al. Sustained polymorphonuclear leukocyte transmigration induces apoptosis in T84 intestinal epithelial cells. J Cell Biol, 2000 ; 150(6): 1479-1488
23. Araki Y, Fujiyama Y, Andoh A, et al. Hydrophilic and hydrophobic bile acids exhibit different cytotoxicities through cytolysis, interleukin-8 synthesis and apoptosis in the intestinal epithelial cell lines: IEC-6 and Caco-2 cells. Stand J Gastroenterol, 2001; 36(5): 533-539
24. Funda DP, Tuckova L, Farre MA, et al. CD14 is expressed and released as soluble CD14 by human intestinal epithelial cells in vitro: lipopolysaccharide activation of epithelial cells revisited. Infect Immun, 2001; 69(6): 3772-3781
25．杨瑞华，海春旭，李瑞珍等。过氧化氢与细胞生长状态的剂量反应关系研究。癌变畸变突变，1997；9(2)：92-99
26．曹纯章，杨绍娟，朴丽莎等．过氧化氢诱导心肌细胞凋亡性和非凋亡性死亡的研究．中国病理生理杂志，2000：16(5)：457-461
27. Noma A. ATP-regulated K~+ channels in cardiac muscle. Nature, 1983;305:147-8.
28．刘蔚，汪海．抗心肌缺血缺氧药物的新靶点：线粒体ATP敏感性钾通道．中国药理学通报，2001；17(6)：610—613。
29. Liu Y, Sato T, O Rourke B et al. Mitochondrial ATP-dependent potassium channel: Novel effectors of cardioprotection. Circulation,1998;97:2463-9.
30. Sato T, Sassaki N, Rourke B et al. Nicorandil, a potent cardioprotective agent, acts by
opening mitochondrial ATP-dependent potassium channels. J Am Coll Cardiol, 2000;35: 514-518.
31. Simon HU, Haj Yehia A, Levi Schaffer F.Role of reactive oxygen species (ROS) in apoptosis induction. Apoptosis. 2000 ; 5(5): 415-8
32. Kluck RM, Bossy Wetzel E, Green DR, et al. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis . Science, 1997;275 (5303) :1132-1134
33. Susin SA, Zamzami N, Castedo M, et al. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J Exp Med, 1996; 184(4): 1331-1335.
34．蔡黔，徐淑芬，周红等．过氧化氢对肠上皮细胞线粒体功能及细胞凋亡的影响．中国现代医学杂志，2002；12(21)：4-10．
35. Javadov SA; Lim KH; Kerr PM; et al. Protection of hearts from reperfusion injury by propofol is associated with inhibition of the mitochondrial permeability transition. Cardiovasc Res. 2000 ; 45(2): 360-9J
36. Levraut J; Iwase H; Shao ZH; et al. Cell death during ischemia: relationship to mitochondrial depolarization and ROS generation. Am J Physiol Heart Circ Physiol. 2003 ; 284(2): H549-58.
37. Crestanello JA; Doliba NM; Babsky AM; et al. Ischemic preconditioning improves mitochonddal tolerance to experimental calcium overload. J Surg Res. 2002;103(2): 243-51
38．梁晚益，黄跃生，杨宗诚，等．二氮嗪对严重烧伤大鼠心肌损害的保护作用及其机制[J]．第三军医大学学报，2003，25(19)：1731-1733．
39. Wan-Yi Liang, Li-Xin Tang, Zong-Cheng Yang, et al. Calcium induced the damage of myocardial mitochondrial respiratory function in the early stage aher severe burns.Burns,2002;28:143-146.
40. Park DA. Ischemic injury in the cat small intestine: Role of superoxide radicals.Gastroenterology 1982; 82(1): 9-15.
41. Toda N, et al. Cardiowase pharmacol[J]. 1985;7:1118.
42. Smallwood KJ,et al. Cardiowase pharmacol [J]. 1988; 12:102
43. Petit PX, Susin SA, Zamzami N ,et al. Mitochondria and programmed cell death: back to the future. FEBS Letters, 1996; 396(1):7-13
44. Reed JC. Cytochrome c : can't live with it, can't live without it. Cell, 1997; 91(5): 559-562
45. Vander Heiden MG, Chandel NS, Williamson EK, et al. BcI-XL regulates the membrane potential and volume homeostasis of mitochondria. Cell, 1997; 91(5):627-637.
46．蔡循，陈国强，陈竺等。线粒体跨膜电位与细胞凋亡。生物化学与生物物理进展，2001；28(1)：3—6．
47. Border JR. Multiple system organ failure[J]. Ann Surg, 1992; 216(2): 111-116.
48. Saito H, Trocki O, Alexander JW, et al. The effect route of nutrient administration on the nutritional state, catabolic hormone secretion, and gutmucosal integrity after burn injury. JPEN, 1987; 11:1.