感染性炎症粘膜上皮闭锁蛋白（occludin）表达及其DNA甲基化的研究
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摘要
第一部分感染性炎症粘膜上皮大鼠动物模型的建立
目的：建立不同类型(实验性糖尿病、非糖尿病)牙龈感染性炎症粘膜上皮大鼠模型，为进一步探讨粘膜上皮闭锁蛋白（occludin）的上皮屏障机制打下基础。
方法：选用雄性SPF级Wistar大鼠60只，四周龄，体重180~220克，按照体重随机分为N组（正常对照组）、D组（单纯糖尿病组）、P组（单纯牙周炎组）和DP组（糖尿病牙周炎组）四组。D组、DP组大鼠采用一次性尾静脉注射链脲佐菌素（Streptozotocin, STZ）的方法诱导生成大鼠糖尿病模型；P组、DP组大鼠采用丝线结扎法结合牙周接种牙龈卟啉单胞菌（Porphyromonasgingivalis, P.g）的方法，在双侧上颌第一磨牙制作炎症粘膜上皮模型，并且同时给予高糖饮食。造模四周后处死动物，取上颌第一磨牙及其牙龈粘膜上皮组织，4%多聚甲醛固定、石蜡包埋，进行病理组织学检查，以确立炎症粘膜上皮动物模型成立，并对各组大鼠牙龈粘膜上皮炎症情况进行分级比较。
结果：糖尿病大鼠出现多饮、多食、多尿、体重减轻、毛皮失去光泽、活动减少、易感染等明显的糖尿病临床症状。糖尿病组、糖尿病牙周炎组血糖值高于正常值，与正常对照组比较有显著性差异（P<0.05），糖尿病模型成立。病理HE染色显示正常大鼠牙周软组织、牙周膜附着正常，无炎细胞浸润；单纯糖尿病组大鼠牙周软组织有少量炎细胞浸润，未见明显牙周袋；单纯牙周炎模型组大鼠牙周软组织有大量炎细胞浸润；糖尿病牙周炎组大鼠软组织内可见出血、大量炎细胞浸润、组织纤维化。炎症粘膜上皮模型成立。糖尿病牙周炎大鼠牙龈粘膜上皮炎症分级病理评分与牙周炎组大鼠比较有显著性差异（P<0.05）。
结论：
1．成功建立感染性炎症粘膜上皮大鼠动物模型。
2．与正常对照组、单纯糖尿病组、单纯牙周炎组相比，合并糖尿病的牙周炎大鼠模型牙龈上皮炎症改变最重。
第二部分感染性炎症粘膜上皮闭锁蛋白表达的变化
目的：对大鼠牙龈感染性炎症粘膜上皮组织紧密连接蛋白闭锁蛋白（occludin）进行检测，了解牙龈粘膜上皮组织感染性炎症时闭锁蛋白（occludin）分布及表达水平的变化。
方法：取大鼠牙龈粘膜上皮组织，部分组织4%多聚甲醛固定、石蜡包埋，免疫组化检测各组大鼠牙龈粘膜上皮内闭锁蛋白（occludin）的分布表达的变化。另取部分新鲜组织置于液氮中保存，RT-PCR半定量检测闭锁蛋白（occludin）mRNA表达的变化。实验数据使用SPSS16.0统计软件进行统计学分析。
结果：闭锁蛋白（occludin）阳性着色主要位于粘膜表浅细胞的细胞膜及细胞质，为棕黄色。正常牙龈粘膜中，闭锁蛋白（occludin）排列致密连续，阳性表达着色均一；糖尿病组、牙周炎组及糖尿病牙周炎组闭锁蛋白（occludin）表达有中断、排序混乱，尤其糖尿病牙周炎组甚至有表达缺失。牙周炎组及糖尿病牙周炎组闭锁蛋白（occludin） mRNA的表达较正常对照组降低，差异具有统计学意义（P<0.05）；糖尿病牙周炎组比牙周炎组闭锁蛋白（occludin）mRNA的表达降低，差异具有统计学意义（P<0.05）。
结论：
1.糖尿病及牙周炎大鼠更易出现牙周粘膜屏障损伤，以糖尿病牙周炎大鼠最为明显。提示在牙龈粘膜慢性炎症过程中，闭锁蛋白（occludin）表达下降，糖尿病会加重以上变化，因而使病原微生物更容易侵入，引起牙龈粘膜屏障不同程度的破坏，这可能为预防和治疗牙周粘膜损伤提供依据。
2.粘膜上皮闭锁蛋白（occludin）水平在慢性感染性炎症时表达下调，且与粘膜屏障受损程度有关。
第三部分感染性炎症粘膜上皮闭锁蛋白DNA甲基化
目的：了解感染性炎症粘膜上皮闭锁蛋白（occludin）DNA甲基化状态，并探讨其对粘膜上皮屏障的影响。
方法：取大鼠模型牙龈粘膜上皮新鲜组织置于液氮中保存，采用甲基化特异性PCR技术（MSP）检测牙龈粘膜上皮闭锁蛋白（occludin）基因启动子甲基化情况。
结果：对闭锁蛋白（occludin）基因启动子区甲基化状态进行检测，所有经亚硫酸氢盐处理的标本都有闭锁蛋白（occludin）基因非甲基化产物，未发现闭锁蛋白（occludin）DNA甲基化产物。闭锁蛋白（occludin） DNA甲基化例数和甲基化率均为0。
结论：尽管闭锁蛋白（occludin）在不同体质大鼠牙龈粘膜组织中均有表达，但闭锁蛋白（occludin）DNA甲基化与牙龈粘膜感染性炎症的发生及严重程度可能不具有确切的相关关系。
PART1Establishment of infective inflammation mucousepithelium in rats models
Objective: To establish infective inflammation mucous epithelium ratsmodel with/without diabetes, and create foundations for the furtherstudy on immune mechanism of Occludin protein in infectiveinflammation mucous epithelium.
Method:60male Wistar rats were randomly divided into normalcontrol group (group N), diabetes mellitus group (group D),periodontitis group (group P) and diabetic periodontitis group (groupDP) of four groups. Single intravenous injection of streptozotocin(Streptozotocin, STZ) method was used to induce diabetic rat model in Group D and group DP; Group P and group DP were treated by tiringsilk thread to teeth combined with periodontal inoculation ofPorphyromonas gingivalis (Porphyromonas gingivalis, P.g), givinghigh sugar diet in the meantime. Maxillary first molar and gingivalepithelial tissue were taken after4weeks, fixed with4%paraformaldehyde, embedded in paraffin, Histopathologicalexamination were conducted to comfirm the success of animal model.situation of gingival epithelial and inflammatory classification of ratsin each group were compared.
Results: Clinical symptoms of diabetes,such as Polydipsia, polyphagia,polyuria, weight loss, easy to infection were observed in diabetic rats.Blood glucose of group D and DP was higher than that of group N（P<0.05）.In group N, HE staining results showed normal periodontalsoft tissue and periodontal attachment, no inflammatory cell infiltration.In group D, a small amount of inflammatory cell infiltration could beobserved in periodontal soft tissue, no significant periodontal pocketwere found. In group P, obvious inflammatory cell infiltration could befound in periodontal soft tissue. In group DP, the most seriousperiodontitis gingival epithelial inflammation could be found, significant difference could be found when compared with group P(P<0.05).
Conclusion: Infective inflammation mucous epithelium rats modelwith/without diabetes were successfully established. Diabeticperiodontitis rats suffer the most serious eriodontitis gingival epithelialinflammation.
PART2Expression of occludin in infective inflammatorymucosal epithelial
Objective: To detect the expression and distribution of occludin, a kindof tight junction protein of epithelial, in infective inflammation mucousepithelium rats model with/without diabetes.
Methods: Immunohistochemical methods were used to detect theexpression and distribution of occludin in infective inflammationmucous epithelium rats model with/without diabetes. RT-PCRsemi-quantitative detection were used to observe the mRNAexpression of occluding protein. SPSS16.0statistical analysis wasused to analyze the data.
Results: Occludin protein positive staining mainly located on themembrane and cytoplasm in superficial cells with brown color. Innormal gingival mucosa, the expression of occludin is compact andcontinuous with uniform coloring. In diabetes mellitus group,periodontitis group and diabetic periodontitis group, the expression ofoccludin were interrupted and disorder, especially in diabeticperiodontitis group, loss of occluding could be found. Expression ofoccludin mRNA in periodontitis group and diabetic periodontitis groupwere decreased compared to that of normal control group, thedifference was statistically significant (P<0.05); occludin mRNAexpression in diabetic periodontitis group is lower than that ofperiodontitis group, statistically significant difference could befound(P<0.05). Conclusion: Diabetes and periodontitis rats are more susceptible toperiodontal mucosal barrier injury, especially in diabetic rats.Decrease of occludin expression and diabetes may promote theprocess of infective inflammatory gingival mucosa injury, result in theinvasion of pathogenic microorganism, further cause the destructionof the gingival mucosa barrier. These results may provide basis for theprevention and treatment of periodontal mucosal injury.
PART3Study on occludin DNA methylation in infectiveinflammatory mucosal epithelial
Objective: Understanding of the infective inflammatory mucosalepithelial occludin (occludin) DNA methylation status, and its effect onthe mucosal epithelial barrier.
Methods: Methylation specific PCR (MSP) were used to dectectoccludin (occludin) DNA methylation. SPSS16.0statistical analysiswas used to analyze the data.
Results: The occludin gene promoter methylation status were detected,all specimens after bisulfite treatment had occludin DNAnon-methylatetd product, occludin DNA methylation products were notfound. The number of cases of occluding DNA methylation andmethylation rate were0.
Conclusion: Although occludin expressed in gingival mucosa tissue,but occludin DNA methylation may have not the exact relationshipwith the occurrence and the severity of gingival inflammation.
目的：建立不同类型(实验性糖尿病、非糖尿病)牙龈感染性炎症粘膜上皮大鼠模型，为进一步探讨粘膜上皮闭锁蛋白（occludin）的上皮屏障机制打下基础。
方法：选用雄性SPF级Wistar大鼠60只，四周龄，体重180~220克，按照体重随机分为N组（正常对照组）、D组（单纯糖尿病组）、P组（单纯牙周炎组）和DP组（糖尿病牙周炎组）四组。D组、DP组大鼠采用一次性尾静脉注射链脲佐菌素（Streptozotocin, STZ）的方法诱导生成大鼠糖尿病模型；P组、DP组大鼠采用丝线结扎法结合牙周接种牙龈卟啉单胞菌（Porphyromonasgingivalis, P.g）的方法，在双侧上颌第一磨牙制作炎症粘膜上皮模型，并且同时给予高糖饮食。造模四周后处死动物，取上颌第一磨牙及其牙龈粘膜上皮组织，4%多聚甲醛固定、石蜡包埋，进行病理组织学检查，以确立炎症粘膜上皮动物模型成立，并对各组大鼠牙龈粘膜上皮炎症情况进行分级比较。
结果：糖尿病大鼠出现多饮、多食、多尿、体重减轻、毛皮失去光泽、活动减少、易感染等明显的糖尿病临床症状。糖尿病组、糖尿病牙周炎组血糖值高于正常值，与正常对照组比较有显著性差异（P<0.05），糖尿病模型成立。病理HE染色显示正常大鼠牙周软组织、牙周膜附着正常，无炎细胞浸润；单纯糖尿病组大鼠牙周软组织有少量炎细胞浸润，未见明显牙周袋；单纯牙周炎模型组大鼠牙周软组织有大量炎细胞浸润；糖尿病牙周炎组大鼠软组织内可见出血、大量炎细胞浸润、组织纤维化。炎症粘膜上皮模型成立。糖尿病牙周炎大鼠牙龈粘膜上皮炎症分级病理评分与牙周炎组大鼠比较有显著性差异（P<0.05）。
结论：
1．成功建立感染性炎症粘膜上皮大鼠动物模型。
2．与正常对照组、单纯糖尿病组、单纯牙周炎组相比，合并糖尿病的牙周炎大鼠模型牙龈上皮炎症改变最重。
第二部分感染性炎症粘膜上皮闭锁蛋白表达的变化
目的：对大鼠牙龈感染性炎症粘膜上皮组织紧密连接蛋白闭锁蛋白（occludin）进行检测，了解牙龈粘膜上皮组织感染性炎症时闭锁蛋白（occludin）分布及表达水平的变化。
方法：取大鼠牙龈粘膜上皮组织，部分组织4%多聚甲醛固定、石蜡包埋，免疫组化检测各组大鼠牙龈粘膜上皮内闭锁蛋白（occludin）的分布表达的变化。另取部分新鲜组织置于液氮中保存，RT-PCR半定量检测闭锁蛋白（occludin）mRNA表达的变化。实验数据使用SPSS16.0统计软件进行统计学分析。
结果：闭锁蛋白（occludin）阳性着色主要位于粘膜表浅细胞的细胞膜及细胞质，为棕黄色。正常牙龈粘膜中，闭锁蛋白（occludin）排列致密连续，阳性表达着色均一；糖尿病组、牙周炎组及糖尿病牙周炎组闭锁蛋白（occludin）表达有中断、排序混乱，尤其糖尿病牙周炎组甚至有表达缺失。牙周炎组及糖尿病牙周炎组闭锁蛋白（occludin） mRNA的表达较正常对照组降低，差异具有统计学意义（P<0.05）；糖尿病牙周炎组比牙周炎组闭锁蛋白（occludin）mRNA的表达降低，差异具有统计学意义（P<0.05）。
结论：
1.糖尿病及牙周炎大鼠更易出现牙周粘膜屏障损伤，以糖尿病牙周炎大鼠最为明显。提示在牙龈粘膜慢性炎症过程中，闭锁蛋白（occludin）表达下降，糖尿病会加重以上变化，因而使病原微生物更容易侵入，引起牙龈粘膜屏障不同程度的破坏，这可能为预防和治疗牙周粘膜损伤提供依据。
2.粘膜上皮闭锁蛋白（occludin）水平在慢性感染性炎症时表达下调，且与粘膜屏障受损程度有关。
第三部分感染性炎症粘膜上皮闭锁蛋白DNA甲基化
目的：了解感染性炎症粘膜上皮闭锁蛋白（occludin）DNA甲基化状态，并探讨其对粘膜上皮屏障的影响。
方法：取大鼠模型牙龈粘膜上皮新鲜组织置于液氮中保存，采用甲基化特异性PCR技术（MSP）检测牙龈粘膜上皮闭锁蛋白（occludin）基因启动子甲基化情况。
结果：对闭锁蛋白（occludin）基因启动子区甲基化状态进行检测，所有经亚硫酸氢盐处理的标本都有闭锁蛋白（occludin）基因非甲基化产物，未发现闭锁蛋白（occludin）DNA甲基化产物。闭锁蛋白（occludin） DNA甲基化例数和甲基化率均为0。
结论：尽管闭锁蛋白（occludin）在不同体质大鼠牙龈粘膜组织中均有表达，但闭锁蛋白（occludin）DNA甲基化与牙龈粘膜感染性炎症的发生及严重程度可能不具有确切的相关关系。
PART1Establishment of infective inflammation mucousepithelium in rats models
Objective: To establish infective inflammation mucous epithelium ratsmodel with/without diabetes, and create foundations for the furtherstudy on immune mechanism of Occludin protein in infectiveinflammation mucous epithelium.
Method:60male Wistar rats were randomly divided into normalcontrol group (group N), diabetes mellitus group (group D),periodontitis group (group P) and diabetic periodontitis group (groupDP) of four groups. Single intravenous injection of streptozotocin(Streptozotocin, STZ) method was used to induce diabetic rat model in Group D and group DP; Group P and group DP were treated by tiringsilk thread to teeth combined with periodontal inoculation ofPorphyromonas gingivalis (Porphyromonas gingivalis, P.g), givinghigh sugar diet in the meantime. Maxillary first molar and gingivalepithelial tissue were taken after4weeks, fixed with4%paraformaldehyde, embedded in paraffin, Histopathologicalexamination were conducted to comfirm the success of animal model.situation of gingival epithelial and inflammatory classification of ratsin each group were compared.
Results: Clinical symptoms of diabetes,such as Polydipsia, polyphagia,polyuria, weight loss, easy to infection were observed in diabetic rats.Blood glucose of group D and DP was higher than that of group N（P<0.05）.In group N, HE staining results showed normal periodontalsoft tissue and periodontal attachment, no inflammatory cell infiltration.In group D, a small amount of inflammatory cell infiltration could beobserved in periodontal soft tissue, no significant periodontal pocketwere found. In group P, obvious inflammatory cell infiltration could befound in periodontal soft tissue. In group DP, the most seriousperiodontitis gingival epithelial inflammation could be found, significant difference could be found when compared with group P(P<0.05).
Conclusion: Infective inflammation mucous epithelium rats modelwith/without diabetes were successfully established. Diabeticperiodontitis rats suffer the most serious eriodontitis gingival epithelialinflammation.
PART2Expression of occludin in infective inflammatorymucosal epithelial
Objective: To detect the expression and distribution of occludin, a kindof tight junction protein of epithelial, in infective inflammation mucousepithelium rats model with/without diabetes.
Methods: Immunohistochemical methods were used to detect theexpression and distribution of occludin in infective inflammationmucous epithelium rats model with/without diabetes. RT-PCRsemi-quantitative detection were used to observe the mRNAexpression of occluding protein. SPSS16.0statistical analysis wasused to analyze the data.
Results: Occludin protein positive staining mainly located on themembrane and cytoplasm in superficial cells with brown color. Innormal gingival mucosa, the expression of occludin is compact andcontinuous with uniform coloring. In diabetes mellitus group,periodontitis group and diabetic periodontitis group, the expression ofoccludin were interrupted and disorder, especially in diabeticperiodontitis group, loss of occluding could be found. Expression ofoccludin mRNA in periodontitis group and diabetic periodontitis groupwere decreased compared to that of normal control group, thedifference was statistically significant (P<0.05); occludin mRNAexpression in diabetic periodontitis group is lower than that ofperiodontitis group, statistically significant difference could befound(P<0.05). Conclusion: Diabetes and periodontitis rats are more susceptible toperiodontal mucosal barrier injury, especially in diabetic rats.Decrease of occludin expression and diabetes may promote theprocess of infective inflammatory gingival mucosa injury, result in theinvasion of pathogenic microorganism, further cause the destructionof the gingival mucosa barrier. These results may provide basis for theprevention and treatment of periodontal mucosal injury.
PART3Study on occludin DNA methylation in infectiveinflammatory mucosal epithelial
Objective: Understanding of the infective inflammatory mucosalepithelial occludin (occludin) DNA methylation status, and its effect onthe mucosal epithelial barrier.
Methods: Methylation specific PCR (MSP) were used to dectectoccludin (occludin) DNA methylation. SPSS16.0statistical analysiswas used to analyze the data.
Results: The occludin gene promoter methylation status were detected,all specimens after bisulfite treatment had occludin DNAnon-methylatetd product, occludin DNA methylation products were notfound. The number of cases of occluding DNA methylation andmethylation rate were0.
Conclusion: Although occludin expressed in gingival mucosa tissue,but occludin DNA methylation may have not the exact relationshipwith the occurrence and the severity of gingival inflammation.
引文
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[16]姚丽艳,王文瑜,钟泉,等.大鼠实验性牙周病模型的建立及组织学评价[J].福建医药杂志,2009,31(3):63-65.
[17]刘颖凤,王小竞,吴礼安.大鼠尼古丁实验性牙周炎动物模型的建立[J].牙体牙髓牙周病学杂志,2008,18(3):148-152.
[18]任静,夏舟斌,欧炯光,等.云南白药对实验性牙周炎的影响[J].实用口腔医学杂志,2010,26(6):721-724.
[19] Nakajima K, Hamada N, Takahashi Y,et al. Restraint stress enhancesalveolar bone loss in an experimental rat model. JPeriodontal Res.2006;41(6):527-534.
[20] Struillou X, Boutigny H, Soueidan A, et al. Experimental animalmodels in periodontology: a review. Open Dent J.2010;4:37-47.
[21] Cai X, Li C, Du G,et al. Protective effects of baicalin onligature-induced periodontitis in rats. J Periodontal Res.2008;43(1):14-21.
[22] Oz HS, Ebersole JL. A novel murine model for chronic inflammatoryalveolar bone loss. J Periodontal Res.2010;45(1):94-99.
[23] Breivik T, Opstad PK, Gjermo P, et al. Effects ofhypothalamic-pituitary-adrenal axis reactivity on periodontal tissuedestruction in rats. Eur J Oral Sci.2000;108(2):115-122.
[24] Kesavalu L,Sathishkumar S,Bakthavatchalu V,et al. Rat model ofpolymicrobial infection, immunity, and alveolar bone resorption inperiodontal disease. Infect Immun.2007;75(4):1704-1712.
[25] Graves DT,Fine D,Teng YTA,et al.The use of rodent models toinvestigate host-bacteria interactions related to periodontaldiseases.J Clin Periodontol.2008;35(2):89-105.
[26] Lalla E, Lanster IB, Feit M, et al. Blockade of RAGE suppressesperiodontitis-associated bone loss in diabetic mice. J Clin Invest,2000,105(8):1117~1124.
[27] Mahamed DA, Marleau A, Alnaeeli M, et al. G (-) anaerobes-reactiveCD4+T-cell trigger RANKL-mediated enhanced alveolar bone loss indiabetic NOD mice. Diabetes,2005,54(5):1477~1486.
[28]Tsukita S, Furse M. Occludin and cladins in tight junction strands:leading or supporting players?[J]. Trends Cell Biol,1999,9(7):268-273.
[29] Wan H,Winton HL,Soeller C,et al. Der p1facilitatestransepithelial allergen delivery by disruption of tight junctions[J].J Clin Invest,1999,104(1):123-133.
[30] Wan H,Winton HL,Soeller C,et al. Quantitative structural andbiochemical analyses of tight junction dynamics following exposure ofepithelial cells to house dust mite allergen Der p1[J]. Clin ExpAllergy,2000,30(5):685-698.
[31]Wan H,Winton HL,Soeller C,et al.The transmembrane protein occludinof epithelial tight junctions is a functional target for serinepeptidases from faecal pellets of Dermatophagoides pteronyssinus[J].Clin Exp Allergy,2001,31(2):279-294.
[32] Ploss A,Evans MJ, Gaysinskaya VA,etal. Human occludin is ahepatitis C virusentry fact or required for infection of mouse cells.Nature,2009,457(7231):882-886.
[33] Coyne CB,Shen L, Turer JR, etal. Coxsackie virusentry acrossepithelial tight junctions requires occluding and the small GTP asesRab34and Rab5. Cell Host Microbe,2007,2(3):181-192.
[34] Bird A P. CpG islands as gene markers in the vertebrate nucleus.Trends Genet,1987,3:342～347
[35] Ooi SKT，Bestor TH. The colorful history of active DNAdemethylation［J］. Cell，2008，133（7）：45-48.
[36]Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, andfunction. Cell,2004,116(2):281-97
[37] Chekulaeva M, Filipowicz W. Mechanisms of miRNAmediatedpost-transcriptional regulation in animal cells.Curr Opin Cell Biol,2009,21(3):452-60
[38] O'Connell RM, Rao DS, Chaudhuri AA, et al. Physiological andpathological roles for microRNAs in the immune system. Nat Rev Immunol,2010,10(2):111-22
[39] Eisenberg LM, Eisenberg CA. Stem cell plasticity, cell fusion,and transdifferentiation. Birth Defects Res C Embryo Today,2003,69(3):209-18
[40]戴胜川,张玉梅,周同,等.细胞转分化的病理生理意义.生命科学,2006,18(1):58-61
[41] M. Furuse, T. Hirase, M. Itoh, A. Nagafuchi, S. Yonemura, S.Tsukita, S. Tsukita, Occludin a novel integral membrane proteinlocalizing at tight junctions, J. Cell Biol.123(1993)1777–1788.
[42] Ando-Akatsuka Y, Saitou M, Hirase T, Kishi M, Sakakibara A, ItohM, Yonemura S, Furuse M, Tsukita S (May1996)."Interspecies diversityof the occludin sequence: cDNA cloning of human, mouse, dog, andrat-kangaroo homologues". J Cell Biol133(1):43–47.
[43] Umeda K, Ikenouchi J, Katahira–Tayama S, et al. ZO-1andZO-2independently determine where claudins are polymerized in tight–junction strand formation[J]. Cell,2006,126(34):741-754.
[44] Umeda K, Ikenouchi J, Katahira–Tayama S, et al. ZO-1andZO-2independently determine where claudins are polymerized in tight–junction strand formation[J]. Cell,2006,126(34):741-754.
[45] Willis CL，Meske DS，Davis TP. Protein kinase C activation modulatesreversible increase in cortical blood-brain barrier permeability andtight junction protein expression during hypoxia and posthypoxicreoxygenation [J]. J Cereb Blood Flow Metab，2010，30（11）：1847-1859.
[46] Balda MS, Whitney JA,Flores C,et al. Functional dissociationof paracel–lular permeability and transepithelial electricalresistance and disruption of the apicalbasolateral intramenbranedi-Vusion barrier by expression of a mutant tight junction membraneprotein[J].J Cell Biol,1996,134(4):1031-1049.
[47] Hirase T, Kawashima S, Wong EY, et al. Regulation of tight junctionpermeability and occluding phosphorylation by Rhoap160ROCK dependentand independent mechanisms[J].J Biol Chem,2001,276(13):10423-10431.
[48] Van Itallie CM, Anderson JM. Occludin confers adhesiveness whenexpressed in fibroblasts[J]. J Cell Sci,1997,110(Pt9):1113-1121.
[49] Wang Z, Wang Z, Wade P, Mandell KJ, et al. Raf1repressesexpression of thw tight junction protein occluding via activation ofthe zinc-WngerTranscription factor slug[J]. Oncogene,2007,26(8):1222-1230.
[50] Kale G, Naren AP, Sheth P,et al. Tyrosinephosphorylation ofoccluding attenuatesits interaction swith ZO-1,ZO-2,and ZO-3.Biochem Biophys Res Commun,2003,302(2):324-329.
[51]Amasheh M, Grotjohann I,Amasheh S, et al.Regulation of mucosalstructure and barrier function in rat colone exposed to tumornecrosis fact or alpha and intergerongamma in vitro: anovel modelfor study ingthepatho mechanisms of inflammatory bowel diseasecytokines.Scand J Gastroenterol,2009,44(10):1226-1235.
[52]Sappington PL, Han X, Yang R, et al.Ethylpyruvateamelioratesintestinal epithelial barrierdys function inendotoxemicmice andimmunostimulatedCaco-2enterocyticmono layers.J Pharmacol Exp Ther,2003,304(1):464-476.
[53]Ma TY, Boivin MA, Ye D, et al. Mechanism of TNF-{alpha}modulationofCaco-2intestinal epithelial tight junction barrier: roleof myosin light-chain kinase protein expression. Am J PhysiolGastrointest Liver Physiol,2005,288(3):G422-430.
[54] Boivin MA, Ye D, Kennedy JC, et al. Mechanism of glucocorticoidregulation of the intestinal tight junction barrier. Am J PhysiolGastrointest Liver Physiol,2007,292(2):G590-G598.
[55] Li Q, Zhang Q, Wang M, et al. Interferon-gamma and tumor necrosisfactor-alpha disrupt epithelial barrier function by alter in glipidcomposition in membrane microdomains of tight junction. Clin Immunol,2008,126(1):67-80.
[56] Al-Sadi RM, Ma TY. IL-1betacausesan increase inintestinalepithelial tight junction permeability. J Immunol,2007,178(7):4641-4649.
[57] BruewerM, LuegeringA, KucharzikT, etal.Proin flammatorycytokines disrupt epithelial barrier function byapoptosis-independent mechanisms.JImmunol,2003,171(11):6164-6172.
[58] UtechM, IvanovAI, SamarinSN, etal.Mechanism ofIFN-gamma-induce dendocytosis of tight junctionproteins:myosinII-dependent vacuolarization of the apicalplasmamembrane. MolBiol Cell,2005,16(10):5040-5052.
[59] WalshSV, HopkinsAM, ChenJ, etal.Rhokinase regulates tightjunction function and is necessary for tight junction assembly inpolarizedintestinal epithelia.Gastroenterology,2001,121(3):566-579.
[60] LaukoetterMG, BruewerM, NusratA.Regulation of the intestinalepithelial barrierby theapicaljunctionalcomplex.CurrOpinGastroenterol,2006,22(2):85-89.
[61] Gassler N, Rohr C, Schneider A, et al. Inflammatory bowel diseaseis associated with changes of enterocytic junctions [J]. Am J PhysiolGastrointest Liver Physiol,2001,281（1）：G216-G228.
[62]24Bird A P. DNA methyl at ion and the fr equency of CpG in animalDNA. Nu cleic Acids Res,1980,8(7):1499-1504
[63]Bird A P. CpG ri ch isl ands an d the fun ct ion of DNA methylation.Nature,1986,321(6067):209-212
[64] Bird A P. The essent ials of DNA methylat ion. Cel l,1992,70(1):5-8
[65] Jones P A, Baylin S B. The fundamental role of epigenetic eventsin cancer. Nat Rev Genet,2002,3(6):415-428
[66] Newman PE. Can reduced folic acid and vitamin B12levels causedeficient DNA methylation producingmutations which initiateatherosclerosis［J］. Med Hypotheses，1999，53（5）：421-424.
[67] Hiltun en MO， Turunen MP， kinen TP， et al. DNA hy pomethylationand methylt ran sferase expr ess ion in atherosclerotic lesions［J］.Vasc Med，2002，7（1）：5-11.
[68]Leader JE， Wang C， PopovVM， et al. Epigenetics and the estrogenreceptor［J］. Ann N Y Acad Sci，2006，1089：73-87.
[69] Schisler DA, S lininger RJ, B ehle RW, et a.l Form u lation ofB acillusspp. for biological control of plant diseases. Phytopathology,2004,94:12671271.
[70] Cang S，Lu Q，Ma Y，et al． Clinical advances in hypomethylatingagents targeting epigenetic pathways ［J］． Curr Cancer Drug Targets，2010，10(5):539－45．
[71]王国庆.黄芪抑制内毒素诱导性动物牙周炎的组织学研究[D].陕西：陕西中医学院，2006.
［2］ Oshitani N，Watanabe K，Nakamura S，et al． Dislocation of tightjunction proteins without F-actin disruption in inactive Crohn'sdisease ［J］． Int J Mol Med，2005，15(3):407-410．
［3］ Suzuki T，Elias BC，Seth A， et al． PKC eta regulates occludinphosphorylation and epithelial tight junction integrity［J］．Proc NatlAcad Sci U S A，2009，106(1):61-66．
[4] Wan H,Winton HL,Soeller C,et al. Der p1facilitatestransepithelial allergen delivery by disruption of tight junctions[J].J Clin Invest,1999,104(1):123-133.
[5] Wan H,Winton HL,Soeller C,et al. Quantitative structural andbiochemical analyses of tight junction dynamics following exposure ofepithelial cells to house dust mite allergen Der p1[J]. Clin ExpAllergy,2000,30(5):685-698.
[6]Wan H,Winton HL,Soeller C,et al.The transmembrane protein occludinof epithelial tight junctions is a functional target for serinepeptidases from faecal pellets of Dermatophagoides pteronyssinus[J].Clin Exp Allergy,2001,31(2):279-294.
[7] Yang TQ,Takeuchi K,Hayashi S,et al.Effect of histamine on thepermeability of the nasal mucosa in vivo[J].ActaOtolaryngol,2003,123(3):401-405.
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[9]黄朝平.紧密连接蛋白Claudin-1、Occludin与变应性鼻炎发病机制的相关性研究[D].四川：川北医学院，2013.
[10] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, andfunction. Cell,2004,116(2):281-297.
[11] Chekulaeva M, Filipowicz W. Mechanisms of miRNAmediatedpost-transcriptional regulation in animal cells. Curr Opin Cell Biol,2009,21(3):452-60
[12] O'Connell RM, Rao DS, Chaudhuri AA, et al. Physiological andpathological roles for microRNAs in the immune system. Nat Rev Immunol,2010,10(2):111-22
[13] Presland RB, Jurevic RJ. Making sense of the epithelial barrier:what molecular biology and genetics t ell us about the funct ions oforal mucosal and epidermal tissues. J Dent Educ,2002,66:564-574.
[14] PreslandRB, Dale BA. Epithelial sturctural proteins of the skinand oral cavity: funct ion in health and disease. Crit Rev Oral BiolMed,2000,11:383-408.
[15]李华菁，付云.高级氧化蛋白产物在糖尿病相关性牙周炎中的作用［J］.国际口腔医学杂志，2011，6（38）：677-680.
[16]姚丽艳,王文瑜,钟泉,等.大鼠实验性牙周病模型的建立及组织学评价[J].福建医药杂志,2009,31(3):63-65.
[17]刘颖凤,王小竞,吴礼安.大鼠尼古丁实验性牙周炎动物模型的建立[J].牙体牙髓牙周病学杂志,2008,18(3):148-152.
[18]任静,夏舟斌,欧炯光,等.云南白药对实验性牙周炎的影响[J].实用口腔医学杂志,2010,26(6):721-724.
[19] Nakajima K, Hamada N, Takahashi Y,et al. Restraint stress enhancesalveolar bone loss in an experimental rat model. JPeriodontal Res.2006;41(6):527-534.
[20] Struillou X, Boutigny H, Soueidan A, et al. Experimental animalmodels in periodontology: a review. Open Dent J.2010;4:37-47.
[21] Cai X, Li C, Du G,et al. Protective effects of baicalin onligature-induced periodontitis in rats. J Periodontal Res.2008;43(1):14-21.
[22] Oz HS, Ebersole JL. A novel murine model for chronic inflammatoryalveolar bone loss. J Periodontal Res.2010;45(1):94-99.
[23] Breivik T, Opstad PK, Gjermo P, et al. Effects ofhypothalamic-pituitary-adrenal axis reactivity on periodontal tissuedestruction in rats. Eur J Oral Sci.2000;108(2):115-122.
[24] Kesavalu L,Sathishkumar S,Bakthavatchalu V,et al. Rat model ofpolymicrobial infection, immunity, and alveolar bone resorption inperiodontal disease. Infect Immun.2007;75(4):1704-1712.
[25] Graves DT,Fine D,Teng YTA,et al.The use of rodent models toinvestigate host-bacteria interactions related to periodontaldiseases.J Clin Periodontol.2008;35(2):89-105.
[26] Lalla E, Lanster IB, Feit M, et al. Blockade of RAGE suppressesperiodontitis-associated bone loss in diabetic mice. J Clin Invest,2000,105(8):1117~1124.
[27] Mahamed DA, Marleau A, Alnaeeli M, et al. G (-) anaerobes-reactiveCD4+T-cell trigger RANKL-mediated enhanced alveolar bone loss indiabetic NOD mice. Diabetes,2005,54(5):1477~1486.
[28]Tsukita S, Furse M. Occludin and cladins in tight junction strands:leading or supporting players?[J]. Trends Cell Biol,1999,9(7):268-273.
[29] Wan H,Winton HL,Soeller C,et al. Der p1facilitatestransepithelial allergen delivery by disruption of tight junctions[J].J Clin Invest,1999,104(1):123-133.
[30] Wan H,Winton HL,Soeller C,et al. Quantitative structural andbiochemical analyses of tight junction dynamics following exposure ofepithelial cells to house dust mite allergen Der p1[J]. Clin ExpAllergy,2000,30(5):685-698.
[31]Wan H,Winton HL,Soeller C,et al.The transmembrane protein occludinof epithelial tight junctions is a functional target for serinepeptidases from faecal pellets of Dermatophagoides pteronyssinus[J].Clin Exp Allergy,2001,31(2):279-294.
[32] Ploss A,Evans MJ, Gaysinskaya VA,etal. Human occludin is ahepatitis C virusentry fact or required for infection of mouse cells.Nature,2009,457(7231):882-886.
[33] Coyne CB,Shen L, Turer JR, etal. Coxsackie virusentry acrossepithelial tight junctions requires occluding and the small GTP asesRab34and Rab5. Cell Host Microbe,2007,2(3):181-192.
[34] Bird A P. CpG islands as gene markers in the vertebrate nucleus.Trends Genet,1987,3:342～347
[35] Ooi SKT，Bestor TH. The colorful history of active DNAdemethylation［J］. Cell，2008，133（7）：45-48.
[36]Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, andfunction. Cell,2004,116(2):281-97
[37] Chekulaeva M, Filipowicz W. Mechanisms of miRNAmediatedpost-transcriptional regulation in animal cells.Curr Opin Cell Biol,2009,21(3):452-60
[38] O'Connell RM, Rao DS, Chaudhuri AA, et al. Physiological andpathological roles for microRNAs in the immune system. Nat Rev Immunol,2010,10(2):111-22
[39] Eisenberg LM, Eisenberg CA. Stem cell plasticity, cell fusion,and transdifferentiation. Birth Defects Res C Embryo Today,2003,69(3):209-18
[40]戴胜川,张玉梅,周同,等.细胞转分化的病理生理意义.生命科学,2006,18(1):58-61
[41] M. Furuse, T. Hirase, M. Itoh, A. Nagafuchi, S. Yonemura, S.Tsukita, S. Tsukita, Occludin a novel integral membrane proteinlocalizing at tight junctions, J. Cell Biol.123(1993)1777–1788.
[42] Ando-Akatsuka Y, Saitou M, Hirase T, Kishi M, Sakakibara A, ItohM, Yonemura S, Furuse M, Tsukita S (May1996)."Interspecies diversityof the occludin sequence: cDNA cloning of human, mouse, dog, andrat-kangaroo homologues". J Cell Biol133(1):43–47.
[43] Umeda K, Ikenouchi J, Katahira–Tayama S, et al. ZO-1andZO-2independently determine where claudins are polymerized in tight–junction strand formation[J]. Cell,2006,126(34):741-754.
[44] Umeda K, Ikenouchi J, Katahira–Tayama S, et al. ZO-1andZO-2independently determine where claudins are polymerized in tight–junction strand formation[J]. Cell,2006,126(34):741-754.
[45] Willis CL，Meske DS，Davis TP. Protein kinase C activation modulatesreversible increase in cortical blood-brain barrier permeability andtight junction protein expression during hypoxia and posthypoxicreoxygenation [J]. J Cereb Blood Flow Metab，2010，30（11）：1847-1859.
[46] Balda MS, Whitney JA,Flores C,et al. Functional dissociationof paracel–lular permeability and transepithelial electricalresistance and disruption of the apicalbasolateral intramenbranedi-Vusion barrier by expression of a mutant tight junction membraneprotein[J].J Cell Biol,1996,134(4):1031-1049.
[47] Hirase T, Kawashima S, Wong EY, et al. Regulation of tight junctionpermeability and occluding phosphorylation by Rhoap160ROCK dependentand independent mechanisms[J].J Biol Chem,2001,276(13):10423-10431.
[48] Van Itallie CM, Anderson JM. Occludin confers adhesiveness whenexpressed in fibroblasts[J]. J Cell Sci,1997,110(Pt9):1113-1121.
[49] Wang Z, Wang Z, Wade P, Mandell KJ, et al. Raf1repressesexpression of thw tight junction protein occluding via activation ofthe zinc-WngerTranscription factor slug[J]. Oncogene,2007,26(8):1222-1230.
[50] Kale G, Naren AP, Sheth P,et al. Tyrosinephosphorylation ofoccluding attenuatesits interaction swith ZO-1,ZO-2,and ZO-3.Biochem Biophys Res Commun,2003,302(2):324-329.
[51]Amasheh M, Grotjohann I,Amasheh S, et al.Regulation of mucosalstructure and barrier function in rat colone exposed to tumornecrosis fact or alpha and intergerongamma in vitro: anovel modelfor study ingthepatho mechanisms of inflammatory bowel diseasecytokines.Scand J Gastroenterol,2009,44(10):1226-1235.
[52]Sappington PL, Han X, Yang R, et al.Ethylpyruvateamelioratesintestinal epithelial barrierdys function inendotoxemicmice andimmunostimulatedCaco-2enterocyticmono layers.J Pharmacol Exp Ther,2003,304(1):464-476.
[53]Ma TY, Boivin MA, Ye D, et al. Mechanism of TNF-{alpha}modulationofCaco-2intestinal epithelial tight junction barrier: roleof myosin light-chain kinase protein expression. Am J PhysiolGastrointest Liver Physiol,2005,288(3):G422-430.
[54] Boivin MA, Ye D, Kennedy JC, et al. Mechanism of glucocorticoidregulation of the intestinal tight junction barrier. Am J PhysiolGastrointest Liver Physiol,2007,292(2):G590-G598.
[55] Li Q, Zhang Q, Wang M, et al. Interferon-gamma and tumor necrosisfactor-alpha disrupt epithelial barrier function by alter in glipidcomposition in membrane microdomains of tight junction. Clin Immunol,2008,126(1):67-80.
[56] Al-Sadi RM, Ma TY. IL-1betacausesan increase inintestinalepithelial tight junction permeability. J Immunol,2007,178(7):4641-4649.
[57] BruewerM, LuegeringA, KucharzikT, etal.Proin flammatorycytokines disrupt epithelial barrier function byapoptosis-independent mechanisms.JImmunol,2003,171(11):6164-6172.
[58] UtechM, IvanovAI, SamarinSN, etal.Mechanism ofIFN-gamma-induce dendocytosis of tight junctionproteins:myosinII-dependent vacuolarization of the apicalplasmamembrane. MolBiol Cell,2005,16(10):5040-5052.
[59] WalshSV, HopkinsAM, ChenJ, etal.Rhokinase regulates tightjunction function and is necessary for tight junction assembly inpolarizedintestinal epithelia.Gastroenterology,2001,121(3):566-579.
[60] LaukoetterMG, BruewerM, NusratA.Regulation of the intestinalepithelial barrierby theapicaljunctionalcomplex.CurrOpinGastroenterol,2006,22(2):85-89.
[61] Gassler N, Rohr C, Schneider A, et al. Inflammatory bowel diseaseis associated with changes of enterocytic junctions [J]. Am J PhysiolGastrointest Liver Physiol,2001,281（1）：G216-G228.
[62]24Bird A P. DNA methyl at ion and the fr equency of CpG in animalDNA. Nu cleic Acids Res,1980,8(7):1499-1504
[63]Bird A P. CpG ri ch isl ands an d the fun ct ion of DNA methylation.Nature,1986,321(6067):209-212
[64] Bird A P. The essent ials of DNA methylat ion. Cel l,1992,70(1):5-8
[65] Jones P A, Baylin S B. The fundamental role of epigenetic eventsin cancer. Nat Rev Genet,2002,3(6):415-428
[66] Newman PE. Can reduced folic acid and vitamin B12levels causedeficient DNA methylation producingmutations which initiateatherosclerosis［J］. Med Hypotheses，1999，53（5）：421-424.
[67] Hiltun en MO， Turunen MP， kinen TP， et al. DNA hy pomethylationand methylt ran sferase expr ess ion in atherosclerotic lesions［J］.Vasc Med，2002，7（1）：5-11.
[68]Leader JE， Wang C， PopovVM， et al. Epigenetics and the estrogenreceptor［J］. Ann N Y Acad Sci，2006，1089：73-87.
[69] Schisler DA, S lininger RJ, B ehle RW, et a.l Form u lation ofB acillusspp. for biological control of plant diseases. Phytopathology,2004,94:12671271.
[70] Cang S，Lu Q，Ma Y，et al． Clinical advances in hypomethylatingagents targeting epigenetic pathways ［J］． Curr Cancer Drug Targets，2010，10(5):539－45．
[71]王国庆.黄芪抑制内毒素诱导性动物牙周炎的组织学研究[D].陕西：陕西中医学院，2006.