摘要
目的:通过探讨口腔扁平苔藓(oral lichen planus, OLP)中CD4+、CD8+T细胞及CD4/CD8比例的变化,了解细胞免疫在OLP发病中的作用;通过对细胞凋亡的研究,进一步了解细胞凋亡与OLP的发病关系;通过研究OLP中TGF-β1及Smad4、Smad7蛋白的表达和分布,分析OLP中细胞免疫与细胞凋亡的关系,进一步了解OLP发病机制。
方法:采用免疫组化SP法检测27例OLP患者病变组织、10例正常口腔粘膜(normal oral mucosa, NOM)组织中CD4+T细胞、CD8+T细胞、TGF-β1及Smad4、Smad7蛋白的表达水平,并用脱氧核糖核甘酸末端转移酶介导的原位缺口末端标记法(TUNEL法)检测OLP中细胞凋亡指数(apoptotic index, AI),并对实验结果进行统计分析。
结果:①OLP固有层中CD8+T细胞数为35.57±4.25,比正常对照组7.57±3.14显著增高(P<0.05); CD4+T细胞数为17.81±5.13,比正常对照组10.68±4.81显著增高(P<0.05);CD4/CD8为0.51±0.17与正常对照组1.54±0.78相比差异有统计学意义(P<0.05)。②OLP中上皮细胞的AI为14.26±2.32,比正常对照组6.69±0.64显著增加(P<0.05)。固有层淋巴细胞的AI为8.52±2.34,比正常对照组20.14±7.27显著减少(P<0.05)。③OLP中上皮细胞TGF-β1、Smad7及Smad4的平均染色强度分别为0.4839±0.2545、0.5852±0.2504、0.3681±0.1827,其中TGF-β1、Smad7与正常对照组相比表达增高,差异有统计学意义(P<0.05)。但糜烂型与非糜烂型OLP中TGF-β1、Smad7及Smad4的表达差异均无统计学意义(P>0.05)。④OLP固有层CD8+T细胞数与病程呈正相关(r=0.553,P<0.05);与淋巴细胞凋亡呈负相关关系(r=-0.674,P<0.05);上皮内TGF-β1的表达与上皮细胞凋亡呈正相关关系(r=0.691,P<0.05)
结论:1、OLP固有层中CD4+、CD8+T细胞浸润的增加,CD4/CD8比值的变化,说明细胞免疫紊乱在OLP的发生、发展中起一定的作用。
2、OLP中上皮细胞及固有层淋巴细胞凋亡存在异常,上皮细胞凋亡增加而固有层淋巴细胞凋亡减少,说明细胞凋亡异常与OLP的发生、发展有一定的关系。
3、OLP上皮内Smad7、TGF-β1高表达提示TGF-β1/Smad信号转导通路调节紊乱
4、OLP中上皮细胞凋亡增加可能机制为:①TGF-β1/Smad信号通路调节紊乱,TGF-β1高表达可能导致上皮细胞的凋亡增加;②CD8+T细胞增加,凋亡减少,可能使上皮细胞凋亡增加。
5、细胞免疫功能紊乱和细胞凋亡异常在OLP的发病中起重要作用。
Objective: To evaluate the expression of Smad7, Smad4, TGF-β1, CD4+and CD8+T lymphocytes and the changes of the ratio of CD4/CD8 in oral lichen planus(OLP), and to investigate the role and the relationship of immunological reaction and cell apoptosis in the pathogenesis of oral lichen planus.
Methods: Immunohistochemical technique was employed to study the expression of CD4+,CD8+Tcells in specimen from 27 OLP cases and 10 normal oral mucosa (NOM) cases, and the expression of Smad7 ,Smad4, TGF-β1 in 17 OLP cases and 5 NOM cases. TUNEL was used for detecting the cell apoptotic index (AI) in 17 OLP cases. All OLP cases were divided into erosion-atrophy group (13 cases) and non-erosion group (14 cases) according to their clinical features.
Results: The amount of CD8+ (35.57±4.25) and CD4+ (17.81±5.13) T cells are obviously elevated in lamina propria of OLP group compared with CD8+ (7.57±3.14) and CD4+ (10.68±4.81) in the NOM(P<0.05). There is a strong significance (P<0.05) when compared the ratio of CD4/CD8 in both group. AI was remarkably increased in epithelia cells (14.26±2.32) and significantly decreased in lymphocytes in lamina propria (8.52±2.34) in OLP cases compared with its expression in the NOM (6.69±0.64) and (20.14±7.27) respectively. The mean stain of TGF-β1 (0.4839±0.2545), Smad7 (0.5852±0.2504) and Smad4(0.3681±0.1827) were analyzed in OLP epithelia cells, in which TGF-β1 and Smad7 demonstrated a strong significance(P<0.05) compared the NOM, but their expression in erosion-type and non-erosion-type of OLP showed no significant difference (P>0.05). The amount of CD8+ T cells in OLP was positively correlated with pathological development of OLP(r=0.553,P<0.05),while it has a negative relationship with cell apoptosis of lymphocytes(r=-0.674,P<0.05).There is a positive correlation between TGF-β1 expression and cell apoptosis in epithelial cells (r=0.691,P<0.05).
Conclusion: The increased amount of CD4+, CD8+T cells in lamina propria of OLP and the change of ration of CD4/CD8 suggest that immune response is involved in the pathogenesis of OLP. There is a significant difference of cell apoptotic index between epithelial cells and lymphocytes in lamina propria in OLP, which indicated the abnormal cell apoptosis plays an important role in the pathogenesis of OLP. The high expression of Smad7 and TGF-β1 in OLP showed that TGF-β1/Smad normal pathway was disturbed. Elevated production with low apoptosis in CD8 + T cells in lymphocytes and high expression of TGF-β1 in epithelial cells together with the imbalance of TGF-β1/Smad pathway may all contribute in the mechanisms of cell apoptosis of epithelial cells in OLP. Dysfunctional immune response and abnormal cell apoptosis may play important roles in the pathogenesis of OLP.
引文
1.Femiano F,Cozzolino F,Gaeta GM,et al.Recent advances on the pathogenesis of oral lichen planus.The adhesion molecules.Minerva Stomatol,1999,48(4):151-159
2.Takeuchi Y, Tohnai I, Kaneda T,etal.Immunohistochemical analysis of cells in mucosal lesions of oral lichen planus.J Oral Pathol,1988;17:367～373
3.Van Loon LAJ,Krieg SR,Davidson CL,et al.Quantification and distribution of lymphocyte subsets and Langerhans cells in normal human oral mucosa and skin.J Oral Pathol Med,1989;18:197～201
4.吴保萱,陈莹。口腔扁平苔藓患者 T 淋巴细胞及其亚群和 NK 细胞活性测定。口腔医学纵横,1992,8(2):69
5.Walsh LJ,Savage W,Ishii T,et al.Immunopahogenesis of oral lichen planus.J Oral Pathol Med,1990,19:389
6. Columbano A.Cell death:Current clifficuties in discriminating apoptosis from necrosis in the context of pathologicl processes in vivo[J].cell Biochem,1995,92:9363-9368
7.Dekker NP,Lozadar Nur F,Lagenaur LA,et al .Apoptosis associated markers in oral lichen planus.J Oral Pathol Med,1997;26:170-5.
8. Hirota M,Itot, O kudela K,et al . Cell proliferation activity and the expression of cell cycleregulatory proteins in oral lichen planus.J Oral Pathol Med,2002,31(4):204-12
9.周威,金岩,吴织芬,等。口腔扁平苔藓上皮细胞凋亡状况的分析和意义。牙体牙髓牙周病学杂志,2001,11(5):321-3
10.沈丽佳,阮萍,谢方方,等 Fas/Fasl 和颗粒酶 B 在口腔扁平苔藓中的表达及 意义。第一军医大学学报,2004;24(12)1362-1366
11. Kilpi AM,Activation maker analysis of mononuclear cell infiltrates of oral lichen planus in situ.Scand J Dent Res,1987,(95):174-80
12. Jungell P,Konttinen YT,Nortamo P er al.Immunoelectron microscopicstudy of distribution of T cell subsets in oral lichen planus.Scand J Dent Res,1989,(97)361-7
13. Khan ,Farah JCS,Savage NW et al. Th1 cytokines in oral lichen planus.J Oral Pathol Med,2003,32(2):77-83
14.Riberio A,Bronk SF,Roberts PJ,et al.The transformin growth factor beta(1)-inducible trandcription factor TIEC1,mediates apoptosis through oxidative stress.Hepatology,1990,30(6):1490-7
15.Min BM,Woo KM,Lee G et al.Terminal differentiation of normal human oralkeratinocytes is associated with enhanced cellular TGF-β and phospholipase c-γ1 levels and apoptotic cell death.Exp Cell Res,1999,249:377-385
16.Schuster N,Krieglstein K,Mechanisms of TGF-β mediated apoptosis.J Cell Tissue Res,2002,307(1):1-14
17.Karatsaidis A,Schreurs O,Axell T,et al Inhibition of transforming growth factor-beta/Smad signaling pathway in the epthelium of oral lichen.[J]invest Dermatol.2003Dec;121(6):1283-90.
18.陈剑声,柳志文等。OLP组织中Smad7蛋白的表达研究。[J]临床口腔医学杂志,2005,12(21)747-9
19. Pindborg JJ,Reichart PA,Smith CJ,et al.Histological typing of cancer and precancer of the oral mucosa.Springer,1996,
20.Bresalier RS,Ho SB,Schoeppner HL,et al,Ehanced sialyation of mucin-associated carbohydrate structures in human colon cancer metastasis.Gastroenterology,1996,110:1354～1367
21. Mariana VD,Maria C,Rafael D,et al.Oral lichen planus:immunohistology of mucosal lesions.[J]Oral Pathol Med 2002,31:410-4
22.Matthews JB,Scully CM,Potts AJ.Oral lichen planus:an immunoperoxidase study using monoclonal antibodies to lymphocyte subsets.[J]Br J Dermatol,1984,Nov;111(5):587-95
23. Kelekar A,Thompson CB.Bcl-2-family proteins:the role of the BH3 domainin apoptosis.Trends Cell Biol.1998,8:324
24. Adams JM,Cory-S.The Bcl-2 protein family:arbiters of cell survival.Science.1998,81:1322
25.刘青,金岩,吴织芬,肿瘤坏死因子α与口腔扁平苔藓。牙体牙髓牙周病学杂志,2000(10):242-244
26.Neppelberg E,Johannessen Ac,Jonsson R.Apoptosis in oral lichen planus.J Oral Sci,2001,109:361-364.
27.Tanda N,Mori S,Sarr K,et al. Expression of apoptotic signaling proteins in leukoplakia and oral lichen planus:quantitative and topographical studies.J Oral Pathol Med,2000,29,385-393
28. Majorana A,Facchetti F,Pelligrini W,et al.Apoptosis-associated markers in oral lichen planus.J Oral Pathol Med,1999,28,47-48
29. Cristina BI,Miguel Angel GM.Importance of Apoptotic Mechanisms in Inflammatory Infiltrate of Oral Lichen Planus Lesions.J Anticancer Research,2006,26,357-362
30.雷蕾,钟真珍,张海伟等,OLP上皮中TNF-α和TGF-β1的表达及其与细胞凋亡的关系。临床口腔医学杂志,2004,20(11),645-47
31. Sugerma PB,Savage NW,Zhou X,et al.Oral lichen planus.Clin Dermatol,2000(18)533-9
32. Shimizu M,Higaki Y.et al.The roal of granzyme-B-expressing CD8-positive T cells in apoptosis of keratinocytes in lichen planus[J].Arcb Dematol Res,1997,289(9),527-32
1.Gandara Rey J,Garcia Garcia A,Blanco Carrion A,et al.Cellular immune alterations in fifty-two patients with oral lichen planus.[J]Med Oral.2001(6):246-252
2.周刚 樊明文 蒋李懿等,口腔扁平苔藓Th 细胞亚群功能状态的初探 ,[J]中华医学杂志2003(83)1830-1832
3.W Iijima, H Ohtani, T Nakayama,et al.Infiltrating CD8+ T Cells in Oral Lichen Planus Predominantly Express CCR5 and CXCR3 and Carry Respective Chemokine Ligands RANTES/CCL5 and IP-10/CXCL10 in Their Cytolytic Granules.[J]AmPathol,2003 (163):261-8
4.Chou MJ,Daniels TE.Langerhans cells expressing HLA-DQ,HLA-DR and T6 antigens in normal oral mucosa and lichen planus.[J]Oral Pathol Med.1989 Dec;18(10):573-6
5.Mariana Villarroel D,Maria C,Rafael D et al .Oral lichen planus:immunohistology of mucosal lesions.[J]Oral Pathol Med.2002(31)410-4.
6.ZZ Zhao,PB Sugerman,XJ Zhou, LJ Walsh,et al. Mast cell degranulation and the roleof T cell RANTES in oral lichen planus.[J]Oral Disease,2001(7),246-251.
7.Zhang Y,Wang Z,Yan L,et al. A study on morphology and distribution of mast cells in oral lichen planus.[J]Hua Xi Kou Qiang Yi Xue Za Zhi.2002 Oct;20(5):346-352
8.SI Tobon-Arroyave,FA Villegas-Acosta,SM Ruiz-Restrepo et al .Expression of caspase-3and structural changes associated with apoptotic cell death of keratinocytes in ora lichen plauns.[J]Oral Disease,2004,173-8.
9.Bascones-Ilundain C,Gonzalez-Moles MA,Esparza-Gomez et al . Importance of apoptotic mechanisms in inflammatory infiltrate of oral lichn planus lesions. [J]Anticancer Res.2006Jan-Febl26(1A):357-62
10.李玲,细胞凋亡的分子机制及其调控因子。国外医学免疫学分册,2002,25(6):307-320
11.Khan A,Farah CS,Savage NW et al . Th1 cytokines in oral lichen planus. [ J]Oral Pathol Med.2,003 Feb,32(2):77-83
12.Pezelj-Ribaric S,Prso IB,Abram M,et al Salivary levels of tumor necrosis factor-alpha in oral lichen planus.[J]Mediators Inflamm.2004,13(2):131-3
13.Fan Y,Zhan Z,Peng T et al . The expression of apoptosis-associated proteins Bcl-2,Bax in oral leukoplakia and lichen planus.[J]Shanghai Kou qiang Yi Xue.2004,(13)497—501
14.A.Sklavounou-Andrikopoulou,E.Chrysomail,M.Iakovou,et al.Elveate serum evels of the apoptosis related molecules TNF-α,Fas/Apo-1 and Bcl-2 in oral lichen planus.J Oral Pathol Med2004(33);386-90
15.Neppelberg E, Johannessen AC,Jonsson R,Apoptosis in oral lichen planus.[J] Eur J Oral Sci.2001,109(5):361-4
16.Shen LJ,Ruan P,Xie FF.et al.Expression of Fas/Fasl and granzyme B in oral lichen planus and their significance.[J]Di Yi Jun Yi Da Xue Xue Bao.2004,(24)1362-6
17.Riberio A,Bronk SF,Roberts PJ,et al.The transformin growth factor beta(1)-inducible trandcription factor TIEC1,mediates apoptosis through oxidative stress.Hepatology,1990,30(6):1490-7
18.Schuster N,Krieglstein K,Mechanisms of TGF-β mediated apoptosis.J Cell Tissue Res,2002,307(1):1-14
19.Karatsaidis A,Schreurs O,Axell T,et al Inhibition of transforming growthfactor-beta/Smad signaling pathway in the epthelium of oral lichen.[J]invest Dermatol.2003Dec;121(6):1283-90.
20.陈剑声,柳志文等。OLP组织中Smad7蛋白的表达研究。[J]临床口腔医学杂志,2005,12(21)747-9