iNOS和VEGF在大鼠牙髓损伤修复中的表达和意义
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摘要
目的
诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS)是机体合成NO的最主要限速因子之一,可以使体内产生高出生理浓度的一氧化氮(nitric oxide,NO)。而NO是细胞信息传递的重要调节因子,犹如一把双刃剑广泛参与了机体的生理及病理过程。
牙齿在受各种刺激如损伤、根折、正畸力作用及可逆性牙髓炎时可引起牙髓内一些细胞分泌各种多肽类生长因子,从而增加血管通透性,促进血管生成。血管生成是牙本质成功修复的前提,血管内皮生长因子(vascular endothelial growth factor, VEGF)就是其中一种重要的血管生成因子。
牙髓受到各种刺激如创伤等可以使牙髓组织产生一系列的反应,具有一定的抵御刺激、进行自身修复反应的能力,但是牙髓修复的机制尚不明确。本研究旨在探讨iNOS和VEGF在大鼠牙髓机械损伤后修复过程中的表达和二者之间的关系,及不同盖髓剂直接盖髓后iNOS表达变化和分布规律,为iNOS在牙髓损伤修复中的作用机制提供实验依据。
材料和方法
制备大鼠牙髓机械损伤模型及氢氧化钙(calcium hydroxide, CH)和矿物三氧化物凝聚体(mineral trioxide aggregate, MTA)直接盖髓模型,分别于处理后即刻、1d、3d、7d、14d、21d、28d用4%多聚甲醛心脏灌流固定、取材、脱钙后常规石蜡包埋,制备切片,采用免疫组织化学染色(SABC法)观察牙髓机械损伤组牙髓的病理变化及iNOS和VEGF表达情况,和盖髓组iNOS的表达变化,用显微照相系统及图像分析软件测定各组标本阳性染色的平均光密度值(optical density,OD)。利用SPSS13.0统计软件对所得数据进行单因素方差分析(ANOVA)、Pearson相关分析及配对t-检验,P<0.05时有显著性差异。
结果
1、在正常牙髓组织中iNOS染色呈阴性,开髓即刻牙髓中iNOS出现低水平表达,3d组牙髓iNOS出现强阳性表达,损伤7d后iNOS表达开始减弱,损伤21d后接近正常水平。表达主要位于中性粒细胞,成纤维细胞,成牙本质细胞和少数血管内皮细胞。损伤后1d、3d、7d、14d iNOS表达强度与正常对照有差异。
2、正常牙髓VEGF在血管内皮细胞染色呈强阳性,成牙本质细胞染色弱阳性或无着色。牙髓损伤后,VEGF表达强度呈现先增加后降低的趋势,3d时表达最强,7d略有降低,后逐渐降低至28d时接近正常水平。表达主要位于血管内皮细胞,成纤维细胞,中性粒细胞,部分成牙本质细胞。损伤后Id、3d、7d、14d VEGF表达强度与正常对照有差异。
3、Pearson相关分析显示,iNOS与VEGF在牙髓损伤修复中的表达呈正相关关系(r=0.817,P<0.05)。
4、两盖髓组即刻牙髓中iNOS出现低水平表达并逐渐增强,3d时达到高峰,7d后表达开始减弱,21-28d接近正常水平。MTA直接盖髓同一时点牙髓炎症程度较CH组低,修复性牙本质形成较CH组多,CH盖髓后3diNOS表达显著强于MTA组。
结论
1、大鼠牙髓损伤后,iNOS和VEGF的表达均呈先增高后降低的趋势,二者的表达变化与牙髓早期炎症状态和后期修复程度有关,iNOS和VEGF的表达强度具有正相关性。
2、CH直接盖髓炎症及坏死程度较MTA组重,3d时iNOS显著高于MTA组,MTA可能通过某种途径降低牙髓iNOS活性,使牙髓炎症程度低于CH组,盖髓剂不同对牙髓修复程度和iNOS的表达强度有影响。
Objective
Inducible nitric oxide synthase (iNOS) is one of the most important limiting factor about the synthesis of nitric oxide (NO) in body, which can make the body to produce higher concentration of NO than physiological[1]. And NO is an important regulator in the process of cell information transmission, which extensively involved in the process of physiological and pathological like a double-edged sword[2].
After subject to various incentives, such as injury, root fracture, orthodontic force and reversible pulpitis, some of the pulp cells may secrete a variety of peptide growth factors, which can increase vascular permeability and promote angiogenesis. Angiogenesis is a prerequisite for successfully repair of dentin, and vascular endothelial growth factor (VEGF) is one of an important vascular endothelial growth factors.
When subject to various incentives, such as trauma, pulp can produce a series of reaction, including againsting stimulus and carrying out self-repair capacity. But the mechanism of pulp repair is not clear. This study aimed to explore the expression of iNOS and VEGF in rat dental pulp after injury, reveal the relationship between the two, and show the different distribution of iNOS expression after direct pulp capping with different pulp capping agent, in order to understand the role of iNOS in the process of dental pulp injury and repair, and find out the relationship between iNOS and VEGF.
Materials and Methdods
Preparation of rat dental pulp mechanical injury model and direct pulp capping model with CH (calcium hydroxide) and MTA (mineral trioxide aggregate). The rats will be executed at immediately, 1d,3d,7d,14d,21d and 28d, heart perfusion fixed with 4% paraformaldehyde, drawn, decalcification, embedded in paraffin, prepare sections, and to observe the pathological changes and the expression of iNOS and VEGF in the group of mechanical injure, and the expression of iNOS in capping groups after immunohistochemistry (SABC). Software for image analysis was used to test the optical density (OD) of each Specimen. The data were analyzed by one-way ANOVA, pearson correlation analysis and paired t-test with SPSS 13.0, P<0.05 was considered statistically significant.
Results
1、In normal pulp, the stain of iNOS was negative; immediately after pulp injury was low-level expression while 3days was strong positive; after 7 days the stain became less positive gradually till 21 days approach to normal. The expression mainly located in neutrophils, fibroblasts, odontoblasts, and a few endothelial cells. The expression intensity of iNOS was different from normal controls after injury 1d,3d,7d,14d.
2、The VEGF stained strongly positive, in vascular endothelial cells, weakly positive or no color in odontoblasts in normal pulp. After pulp injury, the staining of VEGF was markedly increased and then decreased, the strongest expression was at3 days,7days slightly decreased, and reduced to near normal levels at 28day. The expression mainly located in vascular endothelial cells, fibroblasts, neutrophils and some of the odontoblast. The expression intensity of VEGF was different from normal controls after injury 1d,3d,7d,14d.
3、Pearson correlation analysis showed that iNOS and VEGF were positive correlation in the dental pulp injury and repair (r=0.817, P<0.05).
4、In the two pulp capping groups, iNOS expression was weakly positive at immediately, while 3days was the most strongest positive; after 7 days the stain became less positive gradually till 21-28 days approach to normal. The degree of pulpitis in the group of MTA was lower than the CH, and reparative dentin was more than the CH group. The expression of iNOS in the group of CH was significantly higher than MTA, 3days after direct pulp capping.
Conclusions
1、In the process of rat dental pulp injury and repair, the expression of iNOS and VEGF expression was increased at first and then decreased, which has some relationship with the early inflammation and late repair degree and the expression of iNOS was correlated with VEGF.
2、After direct pulp capping with CH, the degree of pulp inflammation and necrosis was worse than MTA, the expression of iNOS was significantly higher than MTA at 3 days.Maybe MTA can reduce the activity of iNOS in pulp, so that the degree of inflammation lower than the group of CH. The kind of pulp capping agents was impact on the pulp repairation and the expression of iNOS.
诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS)是机体合成NO的最主要限速因子之一,可以使体内产生高出生理浓度的一氧化氮(nitric oxide,NO)。而NO是细胞信息传递的重要调节因子,犹如一把双刃剑广泛参与了机体的生理及病理过程。
牙齿在受各种刺激如损伤、根折、正畸力作用及可逆性牙髓炎时可引起牙髓内一些细胞分泌各种多肽类生长因子,从而增加血管通透性,促进血管生成。血管生成是牙本质成功修复的前提,血管内皮生长因子(vascular endothelial growth factor, VEGF)就是其中一种重要的血管生成因子。
牙髓受到各种刺激如创伤等可以使牙髓组织产生一系列的反应,具有一定的抵御刺激、进行自身修复反应的能力,但是牙髓修复的机制尚不明确。本研究旨在探讨iNOS和VEGF在大鼠牙髓机械损伤后修复过程中的表达和二者之间的关系,及不同盖髓剂直接盖髓后iNOS表达变化和分布规律,为iNOS在牙髓损伤修复中的作用机制提供实验依据。
材料和方法
制备大鼠牙髓机械损伤模型及氢氧化钙(calcium hydroxide, CH)和矿物三氧化物凝聚体(mineral trioxide aggregate, MTA)直接盖髓模型,分别于处理后即刻、1d、3d、7d、14d、21d、28d用4%多聚甲醛心脏灌流固定、取材、脱钙后常规石蜡包埋,制备切片,采用免疫组织化学染色(SABC法)观察牙髓机械损伤组牙髓的病理变化及iNOS和VEGF表达情况,和盖髓组iNOS的表达变化,用显微照相系统及图像分析软件测定各组标本阳性染色的平均光密度值(optical density,OD)。利用SPSS13.0统计软件对所得数据进行单因素方差分析(ANOVA)、Pearson相关分析及配对t-检验,P<0.05时有显著性差异。
结果
1、在正常牙髓组织中iNOS染色呈阴性,开髓即刻牙髓中iNOS出现低水平表达,3d组牙髓iNOS出现强阳性表达,损伤7d后iNOS表达开始减弱,损伤21d后接近正常水平。表达主要位于中性粒细胞,成纤维细胞,成牙本质细胞和少数血管内皮细胞。损伤后1d、3d、7d、14d iNOS表达强度与正常对照有差异。
2、正常牙髓VEGF在血管内皮细胞染色呈强阳性,成牙本质细胞染色弱阳性或无着色。牙髓损伤后,VEGF表达强度呈现先增加后降低的趋势,3d时表达最强,7d略有降低,后逐渐降低至28d时接近正常水平。表达主要位于血管内皮细胞,成纤维细胞,中性粒细胞,部分成牙本质细胞。损伤后Id、3d、7d、14d VEGF表达强度与正常对照有差异。
3、Pearson相关分析显示,iNOS与VEGF在牙髓损伤修复中的表达呈正相关关系(r=0.817,P<0.05)。
4、两盖髓组即刻牙髓中iNOS出现低水平表达并逐渐增强,3d时达到高峰,7d后表达开始减弱,21-28d接近正常水平。MTA直接盖髓同一时点牙髓炎症程度较CH组低,修复性牙本质形成较CH组多,CH盖髓后3diNOS表达显著强于MTA组。
结论
1、大鼠牙髓损伤后,iNOS和VEGF的表达均呈先增高后降低的趋势,二者的表达变化与牙髓早期炎症状态和后期修复程度有关,iNOS和VEGF的表达强度具有正相关性。
2、CH直接盖髓炎症及坏死程度较MTA组重,3d时iNOS显著高于MTA组,MTA可能通过某种途径降低牙髓iNOS活性,使牙髓炎症程度低于CH组,盖髓剂不同对牙髓修复程度和iNOS的表达强度有影响。
Objective
Inducible nitric oxide synthase (iNOS) is one of the most important limiting factor about the synthesis of nitric oxide (NO) in body, which can make the body to produce higher concentration of NO than physiological[1]. And NO is an important regulator in the process of cell information transmission, which extensively involved in the process of physiological and pathological like a double-edged sword[2].
After subject to various incentives, such as injury, root fracture, orthodontic force and reversible pulpitis, some of the pulp cells may secrete a variety of peptide growth factors, which can increase vascular permeability and promote angiogenesis. Angiogenesis is a prerequisite for successfully repair of dentin, and vascular endothelial growth factor (VEGF) is one of an important vascular endothelial growth factors.
When subject to various incentives, such as trauma, pulp can produce a series of reaction, including againsting stimulus and carrying out self-repair capacity. But the mechanism of pulp repair is not clear. This study aimed to explore the expression of iNOS and VEGF in rat dental pulp after injury, reveal the relationship between the two, and show the different distribution of iNOS expression after direct pulp capping with different pulp capping agent, in order to understand the role of iNOS in the process of dental pulp injury and repair, and find out the relationship between iNOS and VEGF.
Materials and Methdods
Preparation of rat dental pulp mechanical injury model and direct pulp capping model with CH (calcium hydroxide) and MTA (mineral trioxide aggregate). The rats will be executed at immediately, 1d,3d,7d,14d,21d and 28d, heart perfusion fixed with 4% paraformaldehyde, drawn, decalcification, embedded in paraffin, prepare sections, and to observe the pathological changes and the expression of iNOS and VEGF in the group of mechanical injure, and the expression of iNOS in capping groups after immunohistochemistry (SABC). Software for image analysis was used to test the optical density (OD) of each Specimen. The data were analyzed by one-way ANOVA, pearson correlation analysis and paired t-test with SPSS 13.0, P<0.05 was considered statistically significant.
Results
1、In normal pulp, the stain of iNOS was negative; immediately after pulp injury was low-level expression while 3days was strong positive; after 7 days the stain became less positive gradually till 21 days approach to normal. The expression mainly located in neutrophils, fibroblasts, odontoblasts, and a few endothelial cells. The expression intensity of iNOS was different from normal controls after injury 1d,3d,7d,14d.
2、The VEGF stained strongly positive, in vascular endothelial cells, weakly positive or no color in odontoblasts in normal pulp. After pulp injury, the staining of VEGF was markedly increased and then decreased, the strongest expression was at3 days,7days slightly decreased, and reduced to near normal levels at 28day. The expression mainly located in vascular endothelial cells, fibroblasts, neutrophils and some of the odontoblast. The expression intensity of VEGF was different from normal controls after injury 1d,3d,7d,14d.
3、Pearson correlation analysis showed that iNOS and VEGF were positive correlation in the dental pulp injury and repair (r=0.817, P<0.05).
4、In the two pulp capping groups, iNOS expression was weakly positive at immediately, while 3days was the most strongest positive; after 7 days the stain became less positive gradually till 21-28 days approach to normal. The degree of pulpitis in the group of MTA was lower than the CH, and reparative dentin was more than the CH group. The expression of iNOS in the group of CH was significantly higher than MTA, 3days after direct pulp capping.
Conclusions
1、In the process of rat dental pulp injury and repair, the expression of iNOS and VEGF expression was increased at first and then decreased, which has some relationship with the early inflammation and late repair degree and the expression of iNOS was correlated with VEGF.
2、After direct pulp capping with CH, the degree of pulp inflammation and necrosis was worse than MTA, the expression of iNOS was significantly higher than MTA at 3 days.Maybe MTA can reduce the activity of iNOS in pulp, so that the degree of inflammation lower than the group of CH. The kind of pulp capping agents was impact on the pulp repairation and the expression of iNOS.
引文
1 刘建国,王宪,陈明哲.一氧化氮和细胞因子之间的相互调节作用.生理科学进展,2000,31(1)：61-64.
2 Colasanti M, Suzuki H. The dual personality of NO. Trends Pharmacol Sci,2000,21(7): 249-252.
3 于世凤.口腔组织病理学.6版.北京：人民卫生出版社,2007,64.
4 王忠东.牙髓自身修复潜能的研究进展.国外医学口腔医学分册,1996,23(2)：65-69.
5 Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature,1980,88(5789):373-376.
6 Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature,1987,327(6122):524-526.
7 Ka Bian, Yan Ke, Yoshinori Kamisaki, et al. Proteomic Modification by Nitric Oxide. J Pharmacol Sci,2006,101(4):271-279.
8 Nathan C, Xie QW. Regulation of biosynthesis of nitric oxide. J Biol Chem,1994,269(19): 13725-13728.
9 苏涛.一氧化氮的生物信使作用与口腔相关疾病.中国组织工程研究与临床康复,2008,12(11)：2148-2152.
10张琼,张士胜,王玲.一氧化氮与血管生成的研究进展.国际眼科杂志,2007,7(3)：788-790.
11 Collins PD, Connolyl DT, Willima TJ. Charaeterization of the increase in vascular permeability induced by vascular permeability factor in vivo. Br J Pharmacol,1993,109(1): 195-199.
12 Matsushita K, Motnai R, Sakuta T, et al. The role of vaseular endohtelial growth factor in human dental pulp cells:inudetion of chemoxatis, prolierfation, and dieffrentiation and activation of the AP-1-dependent signaling pathway. J Dent Res,2000,79(8):1596-1603.
13 Weinberg JB. Nitric oxide as an inflammatory mediator in autoimmune MRL-lpr/lpr mice. Environ Health Perspect.1998,106(Suppl5):1131-1137.
14 Kawashima N, Nakano-Kawanishi H, Suzuki N, et al. Effect of NOS inhibitor on cytokine and COX2 expression in rat pulpitis. J Dent Res,2005,84(8):762-767.
15 Yasuhara R, Suzawa T, Miyamoto Y, et al. Nitric oxide in pulp cell growth, differentiation, and mineralization. J Dent Res,2007,86(2):163-168.
16钟锡鹏,韦文波,周琳,等.人滞留乳牙牙髓组织中一氧化氮合酶的分布特征及图像分析.实用医学杂志,2007,23(10)：1484-1486.
17梅予锋.修复性牙本质形成过程中一氧化氮的功能-Ⅰ：一氧化氮合成酶的表达.临床口腔医学杂志,2007,23(1)：29-32.
18 Tiseher E, GosPedoarwiez D, Mitehell R, et al. Vaseular endohetlial gorwth afctor:a new member of the Platelet-derived growth factor gene finily. Bioehem Biophys Res Commum, 1989,165(3):1198-1206.
19 Veillette CJ, von Schroeder HP. Endothelin-1 down regulates the expression of vascular endothelial growth factor-A associated with osteoprogenitor proliferation and differentiation. Bone,2004,34(2):288-296.
20 Breier G, Albrecht U, Sterrer S, et al. Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation. Development,1992,114(2): 521-532.
21 Elias PM, Arbiser J, Brown BE, et al. Epidermal vascular endothelial growth factor production is required for permeability barrier homeostasis, dermal angiogenesis, and the development of epidermal hyperplasia:implications for the pathogenesis of psoriasis. Am J Pathol,2008,173(3):689-699.
22 Yang LC, Tsai CH, Huang FM, et al. Induction of vascular endothelial growth factor expression in human pulp fibroblasts stimulated with black-pigmented Bacteroides. Int Endod J,2004,37(9):588-592.
23 Artese L, Rubini C, Ferrero G, et al. Vascular endothelial growth fator (VEGF) expression in healthy and inflamed human dental pulps. J Endod,2002,28(1):20-23.
24 Matsushita K, Motani R, Sakuta T, et al. Lipopolysaccharide enhances the production of vascular endothelial growth factor by human pulp cells in culture. Infect Immun,1999,67(4): 1633-1639.
25周建清,王永忠,蒋红艳,等.大鼠创伤组织中CGRP,VEGF表达的变化及意义.江苏大学学报：医学版,2008,18(5)：386-388.
26韩雪峰,李建宁,扬大平,等.跨区供血皮瓣成活过程中血管构筑及内源性VEGF变化的实验研究.中国美容医学杂志,2008,17(2)：232-234.
27赵辉,鲁继增,张松林,等.任意皮瓣局部注射血管内皮生长因子对其微循环及血管再生的影响.中国临床康复,2005,9(6)：50-52.
28 Puzserova A, Kopincova J, Bernatova I. The role of endothelium and nitric oxide in the regulation of vascular tone. Cesk Fysiol,2008,57(2-3):53-60.
29 Kimura H, Weisz A, Kurashima Y, et al. Hypoxia response element of the human vascular endothelial growth factor gene mediates transcriptional regulation by nitric oxide:control of hypoxia-inducible factor-1 activity by nitric oxide. Blood,2000,95(1):189-197.
30 Muorhara T, Horowitz J R, Silver M, et al. Vascular endothelial growth factor/vascular permedility factor enhance vascules permeadility via nitric oxide and porstacyclin. Circulation, 1998,97(1):99.
31张志纯,胡东旭.兔下颌骨牵张成骨中VEGF与NOS的相关性研究.中国口腔种植学杂志,2008,13(1)：6-9.
32王嘉德,高学军.牙体牙髓病学.北京：北京大学医学出版社,2006,357-358,
33 Briso AL, Rahal V, Mestrener SR, et al. Biological response of pulps submitted to different capping materials. Braz Oral Res,2006,20(3):219-225.
34 Camilleri J, Pitt Ford TR. Mineral trioxide aggregate:a review of the constituents and biological properties of the material. Int Endod J,2006,39(10):747-754.
35 Byers M, Taylor P, Khayat B, et al. Effects of injury and inflammation on pulp and periapical nerves. J Endodon,1990,16(2):78-84.
36 Green SJ, Seheller LF, Marletta MA, et al. Nitrie Oxide:eytokine Regulation of nitrie oxide in host resistnaee to intraeellular pathogens. Immunol Lett,1994,43(1-2):87-94.
37 Gobert AP, Dauloueds S, LePoivre M, et al. L-arginine availability modulates local nitric oxide production and parasite killing in experimental trypanosomiasis. Infect Immun,2000, 68(8):4653-4657.
38 Xie QW, Leung M, Fuortes M, et al. Complementation analysis of mutants of nitric oxide synthase reveals that the active site requires hemes. Proc Natl Acad Sci USA,1996,93(10): 4891-4896.
39 Perrella MA, Hsich CM, Lee WS, et al. Arrest of endotoxin indueed hypertention by TGF-β. Proc Natl Aead Sci USA,1996,93(5):2054-2059.
40 Di Nardo Di Maio F, Lohinai Z, D'Arcangelo C, et al. Nitric oxide synthase in healthy and inflamed human dental pulp. J Dent Res,2004,83(4):312-316.
41 严焱,刘正.大鼠实验性牙髓炎组织中诱导型一氧化氮合成酶的mRNA表达.牙体牙髓牙周病学杂志,1998,8(4)：234-235.
42 D'Arcangelo C, Di Nardo-Di Maio F, Patrono C, et al. NOS evaluations in human dental pulp capping with MTA and calcium hydroxide. Int J Immunopathol Pharmacol,2007,20(1 Suppl 1):27-32.
43张玲,张侬,南小思,等.Vitapex糊剂一次法乳牙根管治疗的临床疗效观察.中国实用口腔科杂志,2009,2(3)：155-166.
44 Albina JE, Henry WL Jr, Mastrofrancesco B, et al. Macrophage activation by culture in an anoxic environment. J Immunol,1995,155(9):4391-4396.
1 Culotta E, Koshland DE Jr. NO news is good news. Science,1992,258(5090):1862-1865.
2 Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine.Nature,1980,288(5789):373-376.
3 Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature,1987,327(6122):524-526.
4 Ricclardolo FL. Multiple roles of nitric oxide in the airways. Thorax,2003,88(2):175-182.
5 Ugar-Cankal D, Ozmeric N. A multifaceted molecule, nitric oxide in oral and periodontal diseases. Clin Chim Acta,2006,366(1-2):90-100.
6 Nathan C, Xie QW. Regulation of biosynthesis of nitric oxide. J Biol Chem.1994,269(19): 13725-13728.
7 Aktan F. iNOS-mediated nitric oxide production and its regulation. Life Sci.2004,75(6): 639-653.
8 武宏敏,张建新.一氧化氮与心肌缺血再灌注损伤.河北医药,2008,30(6)：836-838.
9 阚文宏,赵克森.线粒体一氧化氮合酶研究现状.国际检验医学杂志,2006,27(8)：698-700.
10 Song MY, Zwemer CF, Whitesall SE, et al. Acute and conditioned hypoxic tolerance augmented by endothelial nitric oxide synthase inhibition in mice. J Appl Physiol,2007,102(2): 610-615.
11 Lacza Z, Snipes JA, Zhang J, et al. Mitochondrial nitric oxide synthase is not eNOS, nNOS or iNOS. Free Radic Biol Med,2003,35(10):1217-1228.
12 Ghafourifar P, Sen CK. Mitochondrial nitric oxide synthase. Front Biosci,2007,12: 1072-1078.
13 Bringold U, Ghafourifar P, Richter C. Peroxynitrite formed by mitochondrial NO synthase promotes mitochondrial Ca2+release. Free Radic Biol Med,2000,29(3-4):343-348.
14 Dedkova EN, Blatter LA. Modulation of mitochondrial Ca2+by nitric oxide in cultured bovine vascular endothelial cells. Am J Physiol Cell Physiol,2005,289(4):836-845.
15 Dzurik R, Krivosikova Z, Stefikova K, et al. Mitochondria and mitochondrial nitric oxide synthase alterations participate in energetical dysbalance, aging and age-related diseases. Bratisl Lek Listy,2006,107(11-12):405-411.
16刘文武,孙学军,徐伟刚.线粒体一氧化氮合酶及其生物学作用.第二军医大学学报,2006,27(6)：656-659.
17温继梨,承喜凤,陈凤英.一氧化氮与心血管疾病.内蒙古医学院学报2008,30(3)：223-228.
18 Ka Bian, Yan Ke, Yoshinori Kamisaki, et al. Proteomic modification by nitric oxide. J Pharmacol Sci,2006,101(4):271-279.
19陈东林.一氧化氮在生物体内的化学过程.生物学教学,2000,25(3)：1-3.
20任艳丽,高冬梅.一氧化氮与健康.中国地方病防治杂志,2006,21(6)：344-347.
21 Assreuy J, Cunha FQ, Liew FY, et al. Feedback inhibition of nitric oxide synthase activity by nitric oxide. Br J Pharmacol,1993,108(3):833-837.
22 Weinberger B, Weiss K, Heck DE, et al. Pharmacologic therapy of persistent pulmonary hypertension of the newborn. Pharmacol Ther,2001,89(1):67-79.
23刘晓宇,高和.一氧化氮对呼吸系统炎症与病原微生物影响研究进展.国际呼吸杂志,2007,27(5)：338-341.
24 Colasanti M, Suzuki H. The dual personality of NO. Trends Pharmacol Sci,2000,21(7): 249-252.
25唐卫忠,彭玉田,林珠,等.正常人、龋病和牙髓炎患者牙髓组织中一氧化氮含量的检测分析.牙体牙髓牙周病学杂志,1997,7(3)：162-164.
26严焱,刘正.大鼠实验性牙髓炎组织中诱导型一氧化氮合成酶的mRNA表达.牙体牙髓牙周病学杂志,1998,8(4)：234-235.
27 Di Nardo Di Maio F, Lohinai Z, D'Arcangelo C, et al. Nitric oxide synthase in healthy and inflamed human dental pulp. J Dent Res,2004,83(4):312-316.
28 Anggard E. Nitric oxide:mediator, murderer, and medicine. Lancet,1994,343(8907): 1199-1206.
29 Baylis C, Mitruka B, Deng A. Chronic blockade of nitric oxide synthesis in the rat produces systemic hypertension and glomerular damage. J Clin Invest,1992,90(1):278-281.
30 Albina JE, Abate JA, Henry WL Jr. Role of IFN-gamma in the induction of the nitric oxide-synthesizing pathway. J immunol,1991,147(1):144-148.
31 Clancy RM, Abramson SB. Nitric oxide:a novel mediator of inflammation. Proc Soc Exp Biol Med,1995,210(2):93-101.
32 Kispelyi B, Lohinai Z, Ivanyi I, et al. The effect of local nitric oxide synthase inhibition on the diameter of pulpal arteriole in dental bond materialinduced vasodilation in rat. Life Sci,2005, 77(12):1367-1374.
33 Kimura H, Esumi H. Reciprocal regulation between nitric oxide and vascular endothelial growth factor in angiogenesis. Acta Biochim Pol,2003,50(1):49-59.
34 Fukumura D, Gohongi T, Kadambi A, et al. Predominant role of endothelial nitric oxide synthase in vascular endotllelial growth factorinduced angiogensesis and vascular permeability. Proc Natl Acad Sci USA,2001,98(5):2604-2609.
35 Shen BQ, Lee DY, Zioncheck TF. Vascular endothelial growth factor governs endothelial nitricoxide synthase expression via a KDR/Fik-1 receptor and a protein kinase C signaling pathway. J Biol Chem,1999,274(46):33057-33063.
36于世凤.口腔组织病理学.6版.北京：人民卫生出版社.2007：64-69.
37 Burssens P, Forsyth R, Steyaert A, et al. Influence of burst TENS stimulation on the healing of Achilles tendon suture in man. Acta Orthop Belg,2003,69(6):528-532.
38 Wu H, Guan C, Qin X, et al. Upregulation of substance P receptor expression by calcitonin gene-related peptide, a possible cooperative action of two neuropeptides involved in airway inflammation. Pulm Pharmacol Ther,2007,20(5):513-524.
39 Chen S, Wu J, Song Z, et al. An investigation of immunocompetence substances in normal gingival and periodontitis tissue. Chin Med J(Engl),2000,113(9):844-847.
40 Moore RM, Sedrish SA, Holmes EP, et al. Role of endothelium and nitric oxide in modulating in vitro responses of colonic arterial and venous rings to vasodilatory neuropeptides in horses. Can J Vet Res,2005,69(2):116-122.
41 Furness JB, Pompolo S, Shuttleworth CW, et al. Light and electron microscopic immunochemical analysis of nerve fibre types innervating the taenia of the guinea-pig caecum. Cell Tissue Res,1992,270(1):125-137.
42 Law AS, Baumgardner KR, Meller ST, et al. Localization and changes in NADPH-diaphorase reactivity and nitric oxide synthase immunore-activity in rat pulp following tooth preparation. J Dent Res,1999,78(10):1585-1595.
43谢晖,毛靖,齐娟.大鼠正畸牙移动中牙髓iNOS表达的免疫组化研究.临床口腔医学杂志,2005,21(5)：283-285.
44王忠东.牙髓自身修复潜能的研究进展.国外医学口腔医学分册,1996,23(2)：65-69.
45王忠东,文玲英,金岩,等.人牙髓组织中NOS的分布特征和图像分析研究.牙体牙髓牙周病学杂志,2001,11(1)：1-4.
46钟锡鹏,韦文波,周琳,等.人滞留乳牙牙髓组织中一氧化氮合酶的分布特征及图像分析.实用医学杂志,2007,23(10)：1484-1486.
47 Damoulis PD, Hauschka PV. Cytokines induce nitric oxide production in mouse osteoblasts. Biochem Biophys Res Commun,1994,201(2):924-931.
48梅予锋.修复性牙本质形成过程中一氧化氮的功能-Ⅰ：一氧化氮合成酶的表达.临床口腔医学杂志,2007,23(1)：29-32.
49 Byers MR, Taylor PE, Khayat BG, et al. Effects of injury and inflammation on pulp and periapical nerves. J Endod,1990,16(2):78-83.
2 Colasanti M, Suzuki H. The dual personality of NO. Trends Pharmacol Sci,2000,21(7): 249-252.
3 于世凤.口腔组织病理学.6版.北京：人民卫生出版社,2007,64.
4 王忠东.牙髓自身修复潜能的研究进展.国外医学口腔医学分册,1996,23(2)：65-69.
5 Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature,1980,88(5789):373-376.
6 Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature,1987,327(6122):524-526.
7 Ka Bian, Yan Ke, Yoshinori Kamisaki, et al. Proteomic Modification by Nitric Oxide. J Pharmacol Sci,2006,101(4):271-279.
8 Nathan C, Xie QW. Regulation of biosynthesis of nitric oxide. J Biol Chem,1994,269(19): 13725-13728.
9 苏涛.一氧化氮的生物信使作用与口腔相关疾病.中国组织工程研究与临床康复,2008,12(11)：2148-2152.
10张琼,张士胜,王玲.一氧化氮与血管生成的研究进展.国际眼科杂志,2007,7(3)：788-790.
11 Collins PD, Connolyl DT, Willima TJ. Charaeterization of the increase in vascular permeability induced by vascular permeability factor in vivo. Br J Pharmacol,1993,109(1): 195-199.
12 Matsushita K, Motnai R, Sakuta T, et al. The role of vaseular endohtelial growth factor in human dental pulp cells:inudetion of chemoxatis, prolierfation, and dieffrentiation and activation of the AP-1-dependent signaling pathway. J Dent Res,2000,79(8):1596-1603.
13 Weinberg JB. Nitric oxide as an inflammatory mediator in autoimmune MRL-lpr/lpr mice. Environ Health Perspect.1998,106(Suppl5):1131-1137.
14 Kawashima N, Nakano-Kawanishi H, Suzuki N, et al. Effect of NOS inhibitor on cytokine and COX2 expression in rat pulpitis. J Dent Res,2005,84(8):762-767.
15 Yasuhara R, Suzawa T, Miyamoto Y, et al. Nitric oxide in pulp cell growth, differentiation, and mineralization. J Dent Res,2007,86(2):163-168.
16钟锡鹏,韦文波,周琳,等.人滞留乳牙牙髓组织中一氧化氮合酶的分布特征及图像分析.实用医学杂志,2007,23(10)：1484-1486.
17梅予锋.修复性牙本质形成过程中一氧化氮的功能-Ⅰ：一氧化氮合成酶的表达.临床口腔医学杂志,2007,23(1)：29-32.
18 Tiseher E, GosPedoarwiez D, Mitehell R, et al. Vaseular endohetlial gorwth afctor:a new member of the Platelet-derived growth factor gene finily. Bioehem Biophys Res Commum, 1989,165(3):1198-1206.
19 Veillette CJ, von Schroeder HP. Endothelin-1 down regulates the expression of vascular endothelial growth factor-A associated with osteoprogenitor proliferation and differentiation. Bone,2004,34(2):288-296.
20 Breier G, Albrecht U, Sterrer S, et al. Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation. Development,1992,114(2): 521-532.
21 Elias PM, Arbiser J, Brown BE, et al. Epidermal vascular endothelial growth factor production is required for permeability barrier homeostasis, dermal angiogenesis, and the development of epidermal hyperplasia:implications for the pathogenesis of psoriasis. Am J Pathol,2008,173(3):689-699.
22 Yang LC, Tsai CH, Huang FM, et al. Induction of vascular endothelial growth factor expression in human pulp fibroblasts stimulated with black-pigmented Bacteroides. Int Endod J,2004,37(9):588-592.
23 Artese L, Rubini C, Ferrero G, et al. Vascular endothelial growth fator (VEGF) expression in healthy and inflamed human dental pulps. J Endod,2002,28(1):20-23.
24 Matsushita K, Motani R, Sakuta T, et al. Lipopolysaccharide enhances the production of vascular endothelial growth factor by human pulp cells in culture. Infect Immun,1999,67(4): 1633-1639.
25周建清,王永忠,蒋红艳,等.大鼠创伤组织中CGRP,VEGF表达的变化及意义.江苏大学学报：医学版,2008,18(5)：386-388.
26韩雪峰,李建宁,扬大平,等.跨区供血皮瓣成活过程中血管构筑及内源性VEGF变化的实验研究.中国美容医学杂志,2008,17(2)：232-234.
27赵辉,鲁继增,张松林,等.任意皮瓣局部注射血管内皮生长因子对其微循环及血管再生的影响.中国临床康复,2005,9(6)：50-52.
28 Puzserova A, Kopincova J, Bernatova I. The role of endothelium and nitric oxide in the regulation of vascular tone. Cesk Fysiol,2008,57(2-3):53-60.
29 Kimura H, Weisz A, Kurashima Y, et al. Hypoxia response element of the human vascular endothelial growth factor gene mediates transcriptional regulation by nitric oxide:control of hypoxia-inducible factor-1 activity by nitric oxide. Blood,2000,95(1):189-197.
30 Muorhara T, Horowitz J R, Silver M, et al. Vascular endothelial growth factor/vascular permedility factor enhance vascules permeadility via nitric oxide and porstacyclin. Circulation, 1998,97(1):99.
31张志纯,胡东旭.兔下颌骨牵张成骨中VEGF与NOS的相关性研究.中国口腔种植学杂志,2008,13(1)：6-9.
32王嘉德,高学军.牙体牙髓病学.北京：北京大学医学出版社,2006,357-358,
33 Briso AL, Rahal V, Mestrener SR, et al. Biological response of pulps submitted to different capping materials. Braz Oral Res,2006,20(3):219-225.
34 Camilleri J, Pitt Ford TR. Mineral trioxide aggregate:a review of the constituents and biological properties of the material. Int Endod J,2006,39(10):747-754.
35 Byers M, Taylor P, Khayat B, et al. Effects of injury and inflammation on pulp and periapical nerves. J Endodon,1990,16(2):78-84.
36 Green SJ, Seheller LF, Marletta MA, et al. Nitrie Oxide:eytokine Regulation of nitrie oxide in host resistnaee to intraeellular pathogens. Immunol Lett,1994,43(1-2):87-94.
37 Gobert AP, Dauloueds S, LePoivre M, et al. L-arginine availability modulates local nitric oxide production and parasite killing in experimental trypanosomiasis. Infect Immun,2000, 68(8):4653-4657.
38 Xie QW, Leung M, Fuortes M, et al. Complementation analysis of mutants of nitric oxide synthase reveals that the active site requires hemes. Proc Natl Acad Sci USA,1996,93(10): 4891-4896.
39 Perrella MA, Hsich CM, Lee WS, et al. Arrest of endotoxin indueed hypertention by TGF-β. Proc Natl Aead Sci USA,1996,93(5):2054-2059.
40 Di Nardo Di Maio F, Lohinai Z, D'Arcangelo C, et al. Nitric oxide synthase in healthy and inflamed human dental pulp. J Dent Res,2004,83(4):312-316.
41 严焱,刘正.大鼠实验性牙髓炎组织中诱导型一氧化氮合成酶的mRNA表达.牙体牙髓牙周病学杂志,1998,8(4)：234-235.
42 D'Arcangelo C, Di Nardo-Di Maio F, Patrono C, et al. NOS evaluations in human dental pulp capping with MTA and calcium hydroxide. Int J Immunopathol Pharmacol,2007,20(1 Suppl 1):27-32.
43张玲,张侬,南小思,等.Vitapex糊剂一次法乳牙根管治疗的临床疗效观察.中国实用口腔科杂志,2009,2(3)：155-166.
44 Albina JE, Henry WL Jr, Mastrofrancesco B, et al. Macrophage activation by culture in an anoxic environment. J Immunol,1995,155(9):4391-4396.
1 Culotta E, Koshland DE Jr. NO news is good news. Science,1992,258(5090):1862-1865.
2 Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine.Nature,1980,288(5789):373-376.
3 Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature,1987,327(6122):524-526.
4 Ricclardolo FL. Multiple roles of nitric oxide in the airways. Thorax,2003,88(2):175-182.
5 Ugar-Cankal D, Ozmeric N. A multifaceted molecule, nitric oxide in oral and periodontal diseases. Clin Chim Acta,2006,366(1-2):90-100.
6 Nathan C, Xie QW. Regulation of biosynthesis of nitric oxide. J Biol Chem.1994,269(19): 13725-13728.
7 Aktan F. iNOS-mediated nitric oxide production and its regulation. Life Sci.2004,75(6): 639-653.
8 武宏敏,张建新.一氧化氮与心肌缺血再灌注损伤.河北医药,2008,30(6)：836-838.
9 阚文宏,赵克森.线粒体一氧化氮合酶研究现状.国际检验医学杂志,2006,27(8)：698-700.
10 Song MY, Zwemer CF, Whitesall SE, et al. Acute and conditioned hypoxic tolerance augmented by endothelial nitric oxide synthase inhibition in mice. J Appl Physiol,2007,102(2): 610-615.
11 Lacza Z, Snipes JA, Zhang J, et al. Mitochondrial nitric oxide synthase is not eNOS, nNOS or iNOS. Free Radic Biol Med,2003,35(10):1217-1228.
12 Ghafourifar P, Sen CK. Mitochondrial nitric oxide synthase. Front Biosci,2007,12: 1072-1078.
13 Bringold U, Ghafourifar P, Richter C. Peroxynitrite formed by mitochondrial NO synthase promotes mitochondrial Ca2+release. Free Radic Biol Med,2000,29(3-4):343-348.
14 Dedkova EN, Blatter LA. Modulation of mitochondrial Ca2+by nitric oxide in cultured bovine vascular endothelial cells. Am J Physiol Cell Physiol,2005,289(4):836-845.
15 Dzurik R, Krivosikova Z, Stefikova K, et al. Mitochondria and mitochondrial nitric oxide synthase alterations participate in energetical dysbalance, aging and age-related diseases. Bratisl Lek Listy,2006,107(11-12):405-411.
16刘文武,孙学军,徐伟刚.线粒体一氧化氮合酶及其生物学作用.第二军医大学学报,2006,27(6)：656-659.
17温继梨,承喜凤,陈凤英.一氧化氮与心血管疾病.内蒙古医学院学报2008,30(3)：223-228.
18 Ka Bian, Yan Ke, Yoshinori Kamisaki, et al. Proteomic modification by nitric oxide. J Pharmacol Sci,2006,101(4):271-279.
19陈东林.一氧化氮在生物体内的化学过程.生物学教学,2000,25(3)：1-3.
20任艳丽,高冬梅.一氧化氮与健康.中国地方病防治杂志,2006,21(6)：344-347.
21 Assreuy J, Cunha FQ, Liew FY, et al. Feedback inhibition of nitric oxide synthase activity by nitric oxide. Br J Pharmacol,1993,108(3):833-837.
22 Weinberger B, Weiss K, Heck DE, et al. Pharmacologic therapy of persistent pulmonary hypertension of the newborn. Pharmacol Ther,2001,89(1):67-79.
23刘晓宇,高和.一氧化氮对呼吸系统炎症与病原微生物影响研究进展.国际呼吸杂志,2007,27(5)：338-341.
24 Colasanti M, Suzuki H. The dual personality of NO. Trends Pharmacol Sci,2000,21(7): 249-252.
25唐卫忠,彭玉田,林珠,等.正常人、龋病和牙髓炎患者牙髓组织中一氧化氮含量的检测分析.牙体牙髓牙周病学杂志,1997,7(3)：162-164.
26严焱,刘正.大鼠实验性牙髓炎组织中诱导型一氧化氮合成酶的mRNA表达.牙体牙髓牙周病学杂志,1998,8(4)：234-235.
27 Di Nardo Di Maio F, Lohinai Z, D'Arcangelo C, et al. Nitric oxide synthase in healthy and inflamed human dental pulp. J Dent Res,2004,83(4):312-316.
28 Anggard E. Nitric oxide:mediator, murderer, and medicine. Lancet,1994,343(8907): 1199-1206.
29 Baylis C, Mitruka B, Deng A. Chronic blockade of nitric oxide synthesis in the rat produces systemic hypertension and glomerular damage. J Clin Invest,1992,90(1):278-281.
30 Albina JE, Abate JA, Henry WL Jr. Role of IFN-gamma in the induction of the nitric oxide-synthesizing pathway. J immunol,1991,147(1):144-148.
31 Clancy RM, Abramson SB. Nitric oxide:a novel mediator of inflammation. Proc Soc Exp Biol Med,1995,210(2):93-101.
32 Kispelyi B, Lohinai Z, Ivanyi I, et al. The effect of local nitric oxide synthase inhibition on the diameter of pulpal arteriole in dental bond materialinduced vasodilation in rat. Life Sci,2005, 77(12):1367-1374.
33 Kimura H, Esumi H. Reciprocal regulation between nitric oxide and vascular endothelial growth factor in angiogenesis. Acta Biochim Pol,2003,50(1):49-59.
34 Fukumura D, Gohongi T, Kadambi A, et al. Predominant role of endothelial nitric oxide synthase in vascular endotllelial growth factorinduced angiogensesis and vascular permeability. Proc Natl Acad Sci USA,2001,98(5):2604-2609.
35 Shen BQ, Lee DY, Zioncheck TF. Vascular endothelial growth factor governs endothelial nitricoxide synthase expression via a KDR/Fik-1 receptor and a protein kinase C signaling pathway. J Biol Chem,1999,274(46):33057-33063.
36于世凤.口腔组织病理学.6版.北京：人民卫生出版社.2007：64-69.
37 Burssens P, Forsyth R, Steyaert A, et al. Influence of burst TENS stimulation on the healing of Achilles tendon suture in man. Acta Orthop Belg,2003,69(6):528-532.
38 Wu H, Guan C, Qin X, et al. Upregulation of substance P receptor expression by calcitonin gene-related peptide, a possible cooperative action of two neuropeptides involved in airway inflammation. Pulm Pharmacol Ther,2007,20(5):513-524.
39 Chen S, Wu J, Song Z, et al. An investigation of immunocompetence substances in normal gingival and periodontitis tissue. Chin Med J(Engl),2000,113(9):844-847.
40 Moore RM, Sedrish SA, Holmes EP, et al. Role of endothelium and nitric oxide in modulating in vitro responses of colonic arterial and venous rings to vasodilatory neuropeptides in horses. Can J Vet Res,2005,69(2):116-122.
41 Furness JB, Pompolo S, Shuttleworth CW, et al. Light and electron microscopic immunochemical analysis of nerve fibre types innervating the taenia of the guinea-pig caecum. Cell Tissue Res,1992,270(1):125-137.
42 Law AS, Baumgardner KR, Meller ST, et al. Localization and changes in NADPH-diaphorase reactivity and nitric oxide synthase immunore-activity in rat pulp following tooth preparation. J Dent Res,1999,78(10):1585-1595.
43谢晖,毛靖,齐娟.大鼠正畸牙移动中牙髓iNOS表达的免疫组化研究.临床口腔医学杂志,2005,21(5)：283-285.
44王忠东.牙髓自身修复潜能的研究进展.国外医学口腔医学分册,1996,23(2)：65-69.
45王忠东,文玲英,金岩,等.人牙髓组织中NOS的分布特征和图像分析研究.牙体牙髓牙周病学杂志,2001,11(1)：1-4.
46钟锡鹏,韦文波,周琳,等.人滞留乳牙牙髓组织中一氧化氮合酶的分布特征及图像分析.实用医学杂志,2007,23(10)：1484-1486.
47 Damoulis PD, Hauschka PV. Cytokines induce nitric oxide production in mouse osteoblasts. Biochem Biophys Res Commun,1994,201(2):924-931.
48梅予锋.修复性牙本质形成过程中一氧化氮的功能-Ⅰ：一氧化氮合成酶的表达.临床口腔医学杂志,2007,23(1)：29-32.
49 Byers MR, Taylor PE, Khayat BG, et al. Effects of injury and inflammation on pulp and periapical nerves. J Endod,1990,16(2):78-83.