不同牙周状态下牙龈组织内质网应激水平的差异性探究

发布时间：2018-01-09 04:15

本文关键词：不同牙周状态下牙龈组织内质网应激水平的差异性探究　出处：《第四军医大学》2016年硕士论文　论文类型：学位论文

【摘要】：大量研究表明细胞凋亡参与了牙周疾病的病理生理过程,而与细胞凋亡相关的通路主要分为线粒体途径、死亡受体途径、颗粒酶B途径以及近年来新发现的内质网应激(endoplasmic reticulum stress,ERS)途径。ERS是组织及细胞在不利的生存环境下产生的一种应激性反应,其目的在于维持细胞稳态,主要途径为保护性的非折叠蛋白反应(unfolded protein response,UPR)。UPR可以通过蛋白质生成减慢、折叠功能加强、未折叠蛋白降解等途径适应内质网应激,对细胞起保护作用,但是当刺激持续存在或强度过大,超过细胞自我调节能力时则会诱导细胞发生凋亡,细胞整体由促生存向促凋亡方向发生转变。牙龈作为唯一暴露于口腔及各类牙周疾病起始部位的牙周组织,探究牙龈组织从健康到慢性牙龈炎再到慢性牙周炎及侵袭性牙周炎的发展过程中其内质网应激水平发生的改变具有重要的临床意义。当牙周疾病发生时,牙周致病菌的致病成分及其产生的毒素等可以使牙龈上皮的多种粘结蛋白如上皮钙黏素和封闭蛋白-1等的表达水平出现异常,上皮细胞间的相互连接受到破坏发生,牙龈结合上皮通透性显著增加,导致菌斑微生物更易于穿过牙龈上皮的物理屏障,到达上皮深层组织,从而促进牙周炎的发展;此外,牙龈上皮分泌的多种免疫因子如抗菌肽LL-37、人β防御素等在抵御菌斑微生物的入侵,预防牙周病的发展过程中均起到了重要作用。目前已有文献报道ers参与了慢性牙周炎的发病过程,有研究报道指出在慢性牙周炎情况下内质网应激上游标志物grp78及下游凋亡相关因子chop升高,但是健康与慢性牙龈炎,侵袭性牙周炎情况下ers水平的差异表达情况尚无相关报道。目的初步探讨健康、慢性牙龈炎、慢性牙周炎及侵袭性牙周炎牙龈组织中内质网应激水平是否存在差异。方法探究不同状态下牙龈组织中内质网应激标志物grp78、perk、atf4、chop的表达变化:临床收集健康、慢性牙龈炎、慢性牙周炎及侵袭性牙周炎患者的牙龈,通过免疫组化染色对牙龈组织中内质网应激标志物的表达水平进行定性分析;实时定量pcr检测牙龈组织中grp78、perk、atf4、chop的mrna表达量;蛋白质印迹法检测牙龈组织中grp78、perk、atf4、chop的蛋白表达量。结果1.免疫组化结果显示:grp78及chop阳性表达细胞率均为侵袭性牙周炎组慢性牙周炎组慢性牙龈炎组牙周健康组,差异具有统计学意义(p0.05);perk及atf4牙龈组织中阳性细胞表达率的统计结果为慢性牙周炎慢性牙龈炎健康及侵袭性牙周炎组,且差异有统计学意义(p0.05),健康组及侵袭性牙周炎组中perk及atf4两种因子表达量的差异没有统计学意义(p0.05)。2.real-timepcr结果显示:grp78、chopmrna表达量呈现一致性,均表现为健康组慢性牙龈炎组慢性牙周炎组侵袭性牙周炎组,且差异均有统计学意义(p0.05)。perkmrna表达量为健康组与侵袭性牙周炎组间无显著性差异(p0.05),慢性牙龈炎组及慢性牙周炎组表达均有升高,且慢性牙周炎组高于慢性牙龈炎组,差异均有统计学意义(p0.05)。atf-4mrna的表达结果为健康组及侵袭性牙周炎组间表达量未见显著性差异(p0.05);慢性牙龈炎组与慢性牙周炎组高于健康及侵袭性牙周炎组,差异有统计学意义(P0.05);但慢性牙龈炎组及慢性牙周炎组间表达未见显著性差异(P0.05)。3.蛋白印迹法结果显示:GRP78、CHOP蛋白在健康组、慢性牙龈炎组、慢性牙周炎组及侵袭性牙周炎组的表达量为:健康组慢性牙龈炎组慢性牙周炎组侵袭性牙周炎组,且差异有统计学意义(P0.05);PERK、ATF-4蛋白的表达情况为:健康组与侵袭性牙周炎组慢性牙龈炎组慢性牙周炎组,组间差异有统计学意义(P0.05),而健康组及侵袭性牙周炎两组间PERK、ATF-4蛋白的表达量的差异没有统计学意义(P0.05)。结论1、不同状态下牙龈组织中的内质网应激水平存在差异,且表现为健康组慢性牙龈炎组慢性牙周炎组侵袭性牙周炎。2、内质网应激PERK通路的水平为健康组、侵袭性牙周炎组慢性牙龈炎组慢性牙周炎组。3、侵袭性牙周炎患者牙龈的内质网应激水平高于慢性牙周炎,但两者之间引起内质网应激反应的通路可能有所不同。
[Abstract]:A large number of studies show that apoptosis is involved in the pathogenesis of periodontal disease, and apoptosis related pathways are mainly divided into the mitochondrial pathway, death receptor pathway, Granzyme B pathway and recently discovered endoplasmic reticulum stress (endoplasmic reticulum stress, ERS).ERS is a stress reaction of tissue and cell in the adverse environment, its purpose is to maintain the cell homeostasis, the main way for the protection of the unfolded protein response (unfolded protein response, UPR.UPR) can be generated by slow protein folding function strengthen, unfolded protein degradation pathway to endoplasmic reticulum stress, protective effect on cells, but when the stimulus persist or intensity is too large, more than cell self-regulation would induce cell apoptosis, cell apoptosis by promoting the survival to the overall direction of occurrence of gum change. As the only exposure to the oral cavity and all kinds of periodontal disease initiation site of periodontal tissue, explore the gingival tissue from healthy to chronic gingivitis and chronic periodontitis development and aggressive periodontitis in the endoplasmic reticulum stress level changes has important clinical significance. When periodontal disease occurs, the pathogenic composition of periodontal pathogens and the toxin can make a variety of adhesive protein such as gingival epithelial expression of E-cadherin and -1 protein closed abnormal epithelial cells, the inter connection damage occurs, the junctional epithelial permeability increased significantly, resulting in plaque microorganisms more easily through the physical barrier of gingival epithelium, epithelial to deep tissue thus, to promote the development of periodontitis; in addition, a variety of immune factors such as gingival epithelial secretion of antimicrobial peptide LL-37 of human beta defensin in resisting microbial plaque The invasion, development process of prevention of periodontal disease has played an important role. At present, it has been reported that ers is involved in the pathogenesis of chronic periodontitis, studies have reported that in the case of chronic periodontitis upstream of endoplasmic reticulum stress related factors of GRP78 signs and downstream apoptosis of chop increased, but the health and chronic gingivitis, there is no related situation reported the differential expression of ers level of aggressive periodontitis cases. Objective to explore the health, chronic gingivitis, the existence of ER stress levels in chronic periodontitis and aggressive periodontitis gingival tissue. Methods on gingival tissue differences under different states of endoplasmic reticulum stress markers GRP78, perk, ATF4, chop expression changes: collection of clinical health, chronic gingivitis and chronic periodontitis and aggressive periodontitis gingival, by immunohistochemical staining of the endoplasmic reticulum stress markers in gingival tissue The qualitative analysis of the expression level; GRP78, real-time quantitative PCR detection of perk ATF4 in gingival tissue, the expression of chop, mRNA; GRP78, Western blotting detection of gingival tissue in perk, ATF4, chop protein expression was 1.. The results of immunohistochemistry showed that GRP78 and chop positive cells were invasive periodontitis group of chronic periodontitis group of chronic gingivitis group periodontal health group, the difference was statistically significant (P0.05); perk and ATF4 positive cells in gingival tissue expression rate of statistical results for chronic gingivitis and chronic periodontitis and healthy aggressive periodontitis group, and the difference was statistically significant (P0.05), perk and ATF4 in health group and invasive in periodontitis group two factor expression difference was not statistically significant (P0.05.2.real-timepcr) the results showed that GRP78, chopmrna expression showed consistency, showed a healthy group of chronic gingivitis group of chronic periodontal The inflammation group of aggressive periodontitis group, and the differences were statistically significant (P0.05) the expression level of.Perkmrna was no significant health group and aggressive periodontitis group differences (P0.05), chronic gingivitis and chronic periodontitis group group expression were increased, and the chronic periodontitis group was higher than that of chronic gingivitis group, there were statistically significant differences (P0.05) results.Atf-4mrna the expression is no significant difference between healthy group and aggressive periodontitis group (P0.05 group); chronic gingivitis and chronic periodontitis group was higher than that of healthy and aggressive periodontitis group, the difference was statistically significant (P0.05); but the chronic gingivitis group and chronic periodontitis group was no significant difference between the expression of.3. protein (P0.05) Western blot results showed that GRP78, CHOP protein in healthy group, chronic gingivitis group, chronic periodontitis group and the expression of aggressive periodontitis group were: group of chronic gingivitis group of chronic periodontal health Periodontitis group of aggressive periodontitis group, and the difference was statistically significant (PERK, P0.05); the expression of ATF-4 protein: healthy group and aggressive periodontitis group of chronic gingivitis group of chronic periodontitis group, there was significant difference between the groups (P0.05), and the healthy group and aggressive periodontitis PERK between the two groups, the difference expression the expression of ATF-4 was not statistically significant (P0.05). Conclusion: 1, endoplasmic reticulum stress level in gingival tissue under different conditions in the existence of differences, and performance for the healthy group of chronic gingivitis group of chronic periodontitis group of aggressive periodontitis.2, PERK pathway of endoplasmic reticulum stress levels as healthy group, aggressive periodontitis group of chronic gingivitis and chronic periodontitis group.3, endoplasmic reticulum stress levels in patients with aggressive periodontitis gingival than chronic periodontitis, but between the two pathways caused by endoplasmic reticulum stress response may be different.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R781.4

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