谷氨酰胺对新生鼠坏死性小肠结肠炎的影响
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摘要
目的探讨谷氨酰胺(glutamine，Gln)对新生鼠坏死性小肠结肠(neonatal necrotizing enterocolitis，NEC)肠粘膜修复的保护作用。
方法新生1日龄Wister大鼠30只随机分为三组，A组为正常对照组；B组为NEC模型组；C组为NEC模型后灌胃给Gln组(2.0g／kg.d)。建立NEC模型，给予新生鼠100％二氧化碳5min，然后再给予100％氧气5min，放回母鼠身边喂养，第4天断头处死新生鼠，取肠道组织待检。分别取近回盲段2～3cm肠道组织固定、包埋、切片。HE染色光镜下作病理学检查，应用免疫组化技术检测肠粘膜增殖细胞核抗原(PCNA)表达情况，TUNEL法检测肠粘膜细胞凋亡的变化。
结果B组HE染色切片见肠壁有不同程度的损伤，病理评分的中位积分为3分；C组有的肠粘膜正常，有的轻度肠上皮细胞脱落，有的绒毛轻度坏死，病理评分的中位积分为1分。B组PCNA数量均低于A组(P＜0.01)；C组的PCNA数量高于B组(P＜0.01)。B组的肠粘膜细胞凋亡的数量高于A组(P＜0.01)；C组的肠粘膜细胞凋亡的数量低于B组(P＜0.01)。
结论NEC时，新生鼠肠粘膜受损，增殖减慢，细胞凋亡的数量增加；补充Gln可促进NEC新生鼠肠粘膜隐窝细胞的增殖，减少肠粘膜细胞凋亡的数量，使肠粘膜修复加快。
Objective To investigate the nutrient effects of glutamine (Gln) on small intestinal repair in neonatal rats after necrotizing enterocolitis (NEC).
Methods 1st day thirty neonatal rats were devided into three groups, (A) control group. (B) NEC and non-intervention. (C) NEC and glutamine (Gln)-group(2.0g/kg.d). NEC model was established as following method: neonatal rats were put into 100% carbon dioxide for 5 min and then 100% oxygen for 5 min before returned to their mothers. This was done once daily for 3 days. On the 4th day, all the subjects were sacrificed. Intestinal tissue located at the boundary of ileum and cecum was obtained to do pathological study. Morphological changes were measured under a light microscope, expression of proliferating cell nuclear antigen (PCNA) as an index of cell proliferation was observed using immunohistochemical staining and image analysis, expression of apoptosis was studied using TUNEL staining and image analysis.
Results In group B, the histological lesions was different, and was graded 3 point. In group C, some intestinal mucous was normal, and some stratum submucosum and lamina propria was separate, or stratum submucosum and basement layer was dropsy, and was graded 1 point. The number of PCNA in group B lower than group A(P<0.01); The number of PCNA in group C higher than group B(P<0.05).The number of apoptosis in group B higher than group A(P<0.01); The number of apoptosis in group C lower than group B(P<0.05). Conclusions NEC can made neonate rat intestinal mucosa damage, step down proliferation of intestinal mucous, and to increase number of apoptosis in intestinal mucous. Oral glutamine could improve the proliferation of crypt cell, reduce apoptosis of intestinal mucosa, and promote repair of intestinal mucosa after NEC.
方法新生1日龄Wister大鼠30只随机分为三组，A组为正常对照组；B组为NEC模型组；C组为NEC模型后灌胃给Gln组(2.0g／kg.d)。建立NEC模型，给予新生鼠100％二氧化碳5min，然后再给予100％氧气5min，放回母鼠身边喂养，第4天断头处死新生鼠，取肠道组织待检。分别取近回盲段2～3cm肠道组织固定、包埋、切片。HE染色光镜下作病理学检查，应用免疫组化技术检测肠粘膜增殖细胞核抗原(PCNA)表达情况，TUNEL法检测肠粘膜细胞凋亡的变化。
结果B组HE染色切片见肠壁有不同程度的损伤，病理评分的中位积分为3分；C组有的肠粘膜正常，有的轻度肠上皮细胞脱落，有的绒毛轻度坏死，病理评分的中位积分为1分。B组PCNA数量均低于A组(P＜0.01)；C组的PCNA数量高于B组(P＜0.01)。B组的肠粘膜细胞凋亡的数量高于A组(P＜0.01)；C组的肠粘膜细胞凋亡的数量低于B组(P＜0.01)。
结论NEC时，新生鼠肠粘膜受损，增殖减慢，细胞凋亡的数量增加；补充Gln可促进NEC新生鼠肠粘膜隐窝细胞的增殖，减少肠粘膜细胞凋亡的数量，使肠粘膜修复加快。
Objective To investigate the nutrient effects of glutamine (Gln) on small intestinal repair in neonatal rats after necrotizing enterocolitis (NEC).
Methods 1st day thirty neonatal rats were devided into three groups, (A) control group. (B) NEC and non-intervention. (C) NEC and glutamine (Gln)-group(2.0g/kg.d). NEC model was established as following method: neonatal rats were put into 100% carbon dioxide for 5 min and then 100% oxygen for 5 min before returned to their mothers. This was done once daily for 3 days. On the 4th day, all the subjects were sacrificed. Intestinal tissue located at the boundary of ileum and cecum was obtained to do pathological study. Morphological changes were measured under a light microscope, expression of proliferating cell nuclear antigen (PCNA) as an index of cell proliferation was observed using immunohistochemical staining and image analysis, expression of apoptosis was studied using TUNEL staining and image analysis.
Results In group B, the histological lesions was different, and was graded 3 point. In group C, some intestinal mucous was normal, and some stratum submucosum and lamina propria was separate, or stratum submucosum and basement layer was dropsy, and was graded 1 point. The number of PCNA in group B lower than group A(P<0.01); The number of PCNA in group C higher than group B(P<0.05).The number of apoptosis in group B higher than group A(P<0.01); The number of apoptosis in group C lower than group B(P<0.05). Conclusions NEC can made neonate rat intestinal mucosa damage, step down proliferation of intestinal mucous, and to increase number of apoptosis in intestinal mucous. Oral glutamine could improve the proliferation of crypt cell, reduce apoptosis of intestinal mucosa, and promote repair of intestinal mucosa after NEC.
引文
1. Caplan MS, Jilling T. The role of polyunsaturated fatty acid sup-plementation in intestinal and neonatal necrotizinge terocolitis[J].Lipids, 2001,36(9):1053-1057.
2. Barnard J.Epidermal growth factor receptor blockade: anemerging The rapeutic modality in gastroenterology[J].Gastroenterology, 2001,120(7): 1872-1874.
3. Hackel PO,-Zwick E,Prenzel N,etal. Epidermal growth factor receptors:critical ediators of multiple receptor path ways[J]. Curr Opin Cell Biol, 999,11(2):184-189.
4. Luttrell LM , Daaka Y, Lefkowitz RJ. Regulation of tyrosine kinase Cascades by G-protein coupied receptors[J].Curr Opin Cell Biol,1999,ll(2):177-183.
5. Shin CE,Falcone RA Jr,Stuart L,etal. Diminished epidermal Growth factor levels in nfants with necrotizing enterocolitis [J].J Pediatr Surg,2000,35(2):173-176.
6. Miyazaki Y. Epidermal growth factor receptor mediates stress-in-duced expression of its ligands in rat gastri cepithelial cells[J].Gastroenterology, 2001, 120(1):108-116.
7. Miettinen PJ, Berger JE,Meneses J ,etal. Epithelial immatunity and multiorgan failure in mice lacking epidermal growth factor receptor[J] .Nature, 1995,376(6538):337-341.
8. Duh G, Mouri N,Warburton D,etal.EgF regulates early embryonic mouse gut development in chemically defined organ culture[J].Pediatr Res, 2000, 48(6):794-802.
9. Akisu M, Kullahcioglu Girgin F, Baka M, etal. The role of recombinant human erythropoietin in lipid peroxidation and plateletactivating factor generation in a rat model of necrotizing enterocolitis[J]. EurJ Pediatr Surg, 2001, 11(3): 167.
10. Muguruma K. The centralrole of PAF innecrotizing enterocolitis development[J]. Adv Exp Med Biol, 1997, 407: 379.
11. Romagnoli C Frezza S, Cingonali A, etal. Plasma level of interleukin-6 and interleukin-10 inpreterm neonates evaluated for sepsis[J]. EurJ Pediatr, 2001, 160(6): 345-350.
12. Nadler EP, Stanford A, Zhang XR, etal. Intestinal cytokine gene expression in infants with acut enecrotizing enterocolitis: interleukin-11 mRNA expression inversely correlates with extent of disease[J]. J Pediatr Surg, 2001, 36(8): 11-2.
13. Romagnoli C, Frezza S, Cingolani A, et al. Plasma levels of interleukin-6 and interleukin-10 in preterm neonates evaluated for sepsis. Eur J Pediatr. [J]. Eur J Pediatr, 2001, 160 (6): 345-50.
14. Becher RM, Wu G, Calanko JA, et al. Reduced serum amino acid concentrations in infants with necrotizing enterocolitis [J]. J Pediatr, 2000, 137(6): 785-93.
15. EliaM, LunnPG. Theuse of glutamine in the treatment of gastrointest inldisorder sinman[J]. Nuer, 1997, 13: 743-747.
16．蒋朱明，于康．肠粘膜屏障损害与肠外／肠内营养的近代概念[J]．医学研究通讯，2000，29(2)：25．
17. Nala. Buius. Physiologic importance of glutamine metaboliam, 1989, 38: 1-5
18. Buchman AL. Glutamine: commercially essential or conditionally Essential Acritical appraisail of the human data[J]. AmJ Clin Nutr, 2001, 74(1): 25-32.
19．蒋朱明，于康．肠粘膜屏障损害与肠外／肠内营养的近代概念[J]．医学研究通讯，2000，29(2)：225．
20. Jiang ZM, Cao JD, Zh uXG etal. The impact of alany-glutamine On clinical safaty, nitrogen balance, intestinal permea bility, and Clinical out come in post operative patients: a randomized, double- blind, controlled study of 120 patients[J]. JPEN, 1999, 23(5): S62.
21. Papaconstantinou HT, Hwang KO, Rajaraman S etal. Glutamine Deprivation induces apoptosis in intestinal epithelial cell [J]. Surgery, 1998, 124(2): 152-160.
22. Wischmeyer PE, Lynch J, Liedel Jetal. Glutamine administration reduces Gramnegative bactere miain severely burned patients: aprospective, randomized, double-blind trial versus isonitrogenous control[J]. Crit Care Med, 2001, 29(11): 2075-2080.
23. Daniele B, Perrone F, Gallo C etal. Oral glutamine in the Prevention of fluorouracil induced intestinal toxicity: a double blind, Placebo controlled, randomized trail[J]. Gut, 2001, 48(1): 282
24. Calder PC. Glutamine and the immunesystem [J]. Amino Acids, 1999, 17(3): 227-241.
25. Furukawa S, Saito H, Fukatsu K etal. Glutamine-enhanced bacterial killing by neutrophis from post operative patients[J]. Nut, 1997, 13(10): 863-869.
26．施鑫，刘放南，陈永明．谷氨酰胺对内毒素激活大鼠枯否细胞的免疫调理作用[J]．中华实验外科杂志，2001，18(2)：152-153．
27. Wu Y, Kudsk KA, Gocinski B etal. Route and type of nutrition Influence IgA-mediating intestinal cytokines [J]. Ann Surg, 1999, 229(5): 662-668.
28. Fukatsu K, Lundberg AH, Hanna MK etal. Route of nutrition Influences intercellular adhesion moleculel expression and neutrophil accumulation in intestine[J]. ArchSurg, 1999, 134(10): 1055-1064.
29．张启良．新编病理生理学教程[M]．上海：上海科学技术出版社，2000．237．
30. Mcclave SA, Ritchie CS. Artificial nutrition in pancreatic disease: What lessons have we learn from the literature [J]. Clin Nutr, 2000, 19(1): 126.
31. Koike K, Moore EE, Moore FA etal. Gut phospholipase A2mediates neutrophile priming and lung injury after mesenteror ischemia-reperfusion[J]. Am J Physiol, 1995, 268(3Pt1): G397-G403.
32．吴肇汉．现代临床营养治疗学[M]．上海：上海科学技术出版社，2001．211-231．
33. Kenneth A. Currenta spects of mucosal immunology and its in fluence by nutrition[J]. AmJ Surg, 2002, 183(4): 390-398.
34. Xu Y, Nguyen Q, Lo DC etal. C-myc-Dependent hepatoma cell Apoposis results from oxidative stress and not adeficiency of Growth factors[J]. JCell Physiol, 1997, 170(2): 192-199.
35. Shigeo IK, Ben L, Cheryl D etal. Total Parenteral nutrition supple mentation with glutamine improvess urvival after gut Ischemia/reperfusion[J]. JPEN, 2002, 26(3): 169-172.
36. Eric A, Philip C. The proposed role of glutamine in cells of the Immune system and speculative consequences for the whole animal[J]. Nutrition, 1997, 13(7): 728-737.
37. Okur H, Kucukaydin M, Kose K, et al. Hypoxia Induced necrotizing Enterocolitis in the immature Rat: The Role of Lipid peroxidation and Management by vitamin E[J]. J Pediatr Surg, 1995, 30: 1416-9.
38. Dvorak B, Halpern MD, Holubec H, et al. Epidermal growth factor reduces the development of necrotizing enterocolitis in a neonatal rat model[J]. Am J Physiol Gastrointest Liver Physiol. 2002, 282(1): G156-64.
39. Horvath K, Jami M, Hill ID, et al. Isocaloric glutamine-free diet and the morphology and function of rat small intestine[J]. JPEN J Parenter Enteral Nutr, 1996, 20 (2): 128-34.
40. Neu J, Demuro V, Li N. Glutamine: clinical applications and mechanisms of action[J]. Curr Opin Clin Nutr Metab Care. 20025(1): 69-75.
41．石慧琳，程义勇，李树田等．补充谷氨酰胺对束缚大鼠肠屏障功能的影响[J]．营养学报．2004，25(1)：31-35．
42．何桂珍，杨乃发，刘跃武等．不同途径补充谷氨酰胺对大鼠肠粘膜和通透性的影响[J]．中国临床营养杂志．2004，12(1)：13-17．
43．赵云．王凤君，王裴等．大鼠烧伤后补充胰高血糖素样多肽-2 对其相关基因表达的影响[J]．第三军医大学学报．2003，25(19)：1741-1744．
44. Orazi A, Du X, Yang Z, et al. Interleukin-11 prevents apoptosis and accelerates recovery of small intestine mucosa in mice treated with combined chemotherapy and radiation[J]. Lab Invest, 1996, 75(1): 32-42.
45. Clark JA, Lane RH, Maclennan NK. Epidermal growth factor reduces intestinal apoptosis in an experimental model of necrotizing enterocolitis [J]. Am J Physiol Gastrointest Liver Physiol. 2005,288 (4):G755-62.
46. Jilling T, Lu J, Jackson M. Intestinal Epithelial Apoptosis Initiates Gross Bowel Necrosis in an Experimental Rat Model of Neonatal Necrotizing Enterocolitis. Pediatr Res, 2004, 55 (4):622-9.
47. Ko YG, Kim EY, Kim T, et al. Glutamine-dependent anti apoptotic interaction of human glutaminyl-tRNA synthetase with apoptosis signal-regulating kinase 1[J], J Biol Chem, 2001,276 (8):6030-6.
48. Exner R,Weingartmann G, Eliasem MM, et al. Glutamine deficiency renders human monocytic cells more susceptible to specific apoptosis triggers [J]. Surgery, 2002,131(1):75-80.
2. Barnard J.Epidermal growth factor receptor blockade: anemerging The rapeutic modality in gastroenterology[J].Gastroenterology, 2001,120(7): 1872-1874.
3. Hackel PO,-Zwick E,Prenzel N,etal. Epidermal growth factor receptors:critical ediators of multiple receptor path ways[J]. Curr Opin Cell Biol, 999,11(2):184-189.
4. Luttrell LM , Daaka Y, Lefkowitz RJ. Regulation of tyrosine kinase Cascades by G-protein coupied receptors[J].Curr Opin Cell Biol,1999,ll(2):177-183.
5. Shin CE,Falcone RA Jr,Stuart L,etal. Diminished epidermal Growth factor levels in nfants with necrotizing enterocolitis [J].J Pediatr Surg,2000,35(2):173-176.
6. Miyazaki Y. Epidermal growth factor receptor mediates stress-in-duced expression of its ligands in rat gastri cepithelial cells[J].Gastroenterology, 2001, 120(1):108-116.
7. Miettinen PJ, Berger JE,Meneses J ,etal. Epithelial immatunity and multiorgan failure in mice lacking epidermal growth factor receptor[J] .Nature, 1995,376(6538):337-341.
8. Duh G, Mouri N,Warburton D,etal.EgF regulates early embryonic mouse gut development in chemically defined organ culture[J].Pediatr Res, 2000, 48(6):794-802.
9. Akisu M, Kullahcioglu Girgin F, Baka M, etal. The role of recombinant human erythropoietin in lipid peroxidation and plateletactivating factor generation in a rat model of necrotizing enterocolitis[J]. EurJ Pediatr Surg, 2001, 11(3): 167.
10. Muguruma K. The centralrole of PAF innecrotizing enterocolitis development[J]. Adv Exp Med Biol, 1997, 407: 379.
11. Romagnoli C Frezza S, Cingonali A, etal. Plasma level of interleukin-6 and interleukin-10 inpreterm neonates evaluated for sepsis[J]. EurJ Pediatr, 2001, 160(6): 345-350.
12. Nadler EP, Stanford A, Zhang XR, etal. Intestinal cytokine gene expression in infants with acut enecrotizing enterocolitis: interleukin-11 mRNA expression inversely correlates with extent of disease[J]. J Pediatr Surg, 2001, 36(8): 11-2.
13. Romagnoli C, Frezza S, Cingolani A, et al. Plasma levels of interleukin-6 and interleukin-10 in preterm neonates evaluated for sepsis. Eur J Pediatr. [J]. Eur J Pediatr, 2001, 160 (6): 345-50.
14. Becher RM, Wu G, Calanko JA, et al. Reduced serum amino acid concentrations in infants with necrotizing enterocolitis [J]. J Pediatr, 2000, 137(6): 785-93.
15. EliaM, LunnPG. Theuse of glutamine in the treatment of gastrointest inldisorder sinman[J]. Nuer, 1997, 13: 743-747.
16．蒋朱明，于康．肠粘膜屏障损害与肠外／肠内营养的近代概念[J]．医学研究通讯，2000，29(2)：25．
17. Nala. Buius. Physiologic importance of glutamine metaboliam, 1989, 38: 1-5
18. Buchman AL. Glutamine: commercially essential or conditionally Essential Acritical appraisail of the human data[J]. AmJ Clin Nutr, 2001, 74(1): 25-32.
19．蒋朱明，于康．肠粘膜屏障损害与肠外／肠内营养的近代概念[J]．医学研究通讯，2000，29(2)：225．
20. Jiang ZM, Cao JD, Zh uXG etal. The impact of alany-glutamine On clinical safaty, nitrogen balance, intestinal permea bility, and Clinical out come in post operative patients: a randomized, double- blind, controlled study of 120 patients[J]. JPEN, 1999, 23(5): S62.
21. Papaconstantinou HT, Hwang KO, Rajaraman S etal. Glutamine Deprivation induces apoptosis in intestinal epithelial cell [J]. Surgery, 1998, 124(2): 152-160.
22. Wischmeyer PE, Lynch J, Liedel Jetal. Glutamine administration reduces Gramnegative bactere miain severely burned patients: aprospective, randomized, double-blind trial versus isonitrogenous control[J]. Crit Care Med, 2001, 29(11): 2075-2080.
23. Daniele B, Perrone F, Gallo C etal. Oral glutamine in the Prevention of fluorouracil induced intestinal toxicity: a double blind, Placebo controlled, randomized trail[J]. Gut, 2001, 48(1): 282
24. Calder PC. Glutamine and the immunesystem [J]. Amino Acids, 1999, 17(3): 227-241.
25. Furukawa S, Saito H, Fukatsu K etal. Glutamine-enhanced bacterial killing by neutrophis from post operative patients[J]. Nut, 1997, 13(10): 863-869.
26．施鑫，刘放南，陈永明．谷氨酰胺对内毒素激活大鼠枯否细胞的免疫调理作用[J]．中华实验外科杂志，2001，18(2)：152-153．
27. Wu Y, Kudsk KA, Gocinski B etal. Route and type of nutrition Influence IgA-mediating intestinal cytokines [J]. Ann Surg, 1999, 229(5): 662-668.
28. Fukatsu K, Lundberg AH, Hanna MK etal. Route of nutrition Influences intercellular adhesion moleculel expression and neutrophil accumulation in intestine[J]. ArchSurg, 1999, 134(10): 1055-1064.
29．张启良．新编病理生理学教程[M]．上海：上海科学技术出版社，2000．237．
30. Mcclave SA, Ritchie CS. Artificial nutrition in pancreatic disease: What lessons have we learn from the literature [J]. Clin Nutr, 2000, 19(1): 126.
31. Koike K, Moore EE, Moore FA etal. Gut phospholipase A2mediates neutrophile priming and lung injury after mesenteror ischemia-reperfusion[J]. Am J Physiol, 1995, 268(3Pt1): G397-G403.
32．吴肇汉．现代临床营养治疗学[M]．上海：上海科学技术出版社，2001．211-231．
33. Kenneth A. Currenta spects of mucosal immunology and its in fluence by nutrition[J]. AmJ Surg, 2002, 183(4): 390-398.
34. Xu Y, Nguyen Q, Lo DC etal. C-myc-Dependent hepatoma cell Apoposis results from oxidative stress and not adeficiency of Growth factors[J]. JCell Physiol, 1997, 170(2): 192-199.
35. Shigeo IK, Ben L, Cheryl D etal. Total Parenteral nutrition supple mentation with glutamine improvess urvival after gut Ischemia/reperfusion[J]. JPEN, 2002, 26(3): 169-172.
36. Eric A, Philip C. The proposed role of glutamine in cells of the Immune system and speculative consequences for the whole animal[J]. Nutrition, 1997, 13(7): 728-737.
37. Okur H, Kucukaydin M, Kose K, et al. Hypoxia Induced necrotizing Enterocolitis in the immature Rat: The Role of Lipid peroxidation and Management by vitamin E[J]. J Pediatr Surg, 1995, 30: 1416-9.
38. Dvorak B, Halpern MD, Holubec H, et al. Epidermal growth factor reduces the development of necrotizing enterocolitis in a neonatal rat model[J]. Am J Physiol Gastrointest Liver Physiol. 2002, 282(1): G156-64.
39. Horvath K, Jami M, Hill ID, et al. Isocaloric glutamine-free diet and the morphology and function of rat small intestine[J]. JPEN J Parenter Enteral Nutr, 1996, 20 (2): 128-34.
40. Neu J, Demuro V, Li N. Glutamine: clinical applications and mechanisms of action[J]. Curr Opin Clin Nutr Metab Care. 20025(1): 69-75.
41．石慧琳，程义勇，李树田等．补充谷氨酰胺对束缚大鼠肠屏障功能的影响[J]．营养学报．2004，25(1)：31-35．
42．何桂珍，杨乃发，刘跃武等．不同途径补充谷氨酰胺对大鼠肠粘膜和通透性的影响[J]．中国临床营养杂志．2004，12(1)：13-17．
43．赵云．王凤君，王裴等．大鼠烧伤后补充胰高血糖素样多肽-2 对其相关基因表达的影响[J]．第三军医大学学报．2003，25(19)：1741-1744．
44. Orazi A, Du X, Yang Z, et al. Interleukin-11 prevents apoptosis and accelerates recovery of small intestine mucosa in mice treated with combined chemotherapy and radiation[J]. Lab Invest, 1996, 75(1): 32-42.
45. Clark JA, Lane RH, Maclennan NK. Epidermal growth factor reduces intestinal apoptosis in an experimental model of necrotizing enterocolitis [J]. Am J Physiol Gastrointest Liver Physiol. 2005,288 (4):G755-62.
46. Jilling T, Lu J, Jackson M. Intestinal Epithelial Apoptosis Initiates Gross Bowel Necrosis in an Experimental Rat Model of Neonatal Necrotizing Enterocolitis. Pediatr Res, 2004, 55 (4):622-9.
47. Ko YG, Kim EY, Kim T, et al. Glutamine-dependent anti apoptotic interaction of human glutaminyl-tRNA synthetase with apoptosis signal-regulating kinase 1[J], J Biol Chem, 2001,276 (8):6030-6.
48. Exner R,Weingartmann G, Eliasem MM, et al. Glutamine deficiency renders human monocytic cells more susceptible to specific apoptosis triggers [J]. Surgery, 2002,131(1):75-80.