Expression of MHC Class II, CD70, CD80, CD86 and pro-inflammatory cytokines is differentially regulated in oral epithelial cells following bacterial challenge

Oral epithelium may play a regulatory role in local immune responses when interacting with bacteria. The present study was undertaken to investigate the effects of selected bacterial pathogens found in periodontal and endodontic infections on oral epithelial cells. Expression of cell surface molecules (major histocompatibility complex (MHC) Class II, CD54, CD70, CD80 and CD86) and secretion of inflammatory cytokines (interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha) in response to selected bacterial challenge were examined on an immortalized oral epithelial cell line, HOK-18A and a skin epithelial cell line, HaCaT. Actinomyces viscosus, Actinomyces israelii, Fusobacterium nucleatum lipopolysaccharide (LPS) or primary human periradicular exudate from a granuloma were co-cultured with epithelial cells for 4 or 24 h. Subsequently, cell surface expression of MHC Class II, CD54, CD70, CD80 and CD86, along with pro-inflammatory cytokine levels were determined using flow cytometry, ELISA and RT-PCR. Results indicated that the selected oral bacteria have greater effects on oral versus skin epithelial cells. F. nucleatum increased MHC Class II and CD54 (ICAM-1) cell surface expression on HOK-18A and HaCaT cells. A. israelii also had enhancing effects on the expression of CD54 and MHC Class II. A. israelii and LPS induced a 2.8-fold (P < 0.001) and 4.4-fold (P < 0.005) TNF-alpha secretion, respectively, while F. nucleatum and LPS induced a 10-fold (P < 0.0004) and 6-fold (P < 0.01) IL-1beta secretion, respectively by HOK-18A. Interestingly, CD70, CD80, and CD86 were generally decreased upon bacteria and LPS challenge on HOK-18A. The effects of increased MHC Class II and decreased CD70 were also evident with challenge of human periradicular exudate on HOK-18A. The implications of the study are unique in that oral epithelial cells may play both activating and inhibitory roles in the host immune response towards infection by oral bacteria. We introduce a concept of 'dormancy' where the differential expression of key cell surface antigens on oral epithelial cells may keep the recruited immune effector cells in a state of unresponsiveness, thus contributing to the long term quiescent period observed in many periodontal and endodontic lesions.     

角质细胞生长因子对口腔黏膜上皮细胞凋亡的作用研究

目的 研究不同浓度角质细胞生长因子（KGF）对口腔黏膜上皮细胞凋亡的作用,为探讨KGF在口腔黏膜病发生发展中的作用提供依据。方法 将不同浓度的KGF（对照组0 ng·mL-1,实验1组5 ng·mL-1,实验2组25 ng·mL-1,实验3组50 ng·mL-1）分别加入体外培养的口腔黏膜上皮细胞,培养12、24、48 h后,倒置显微镜下观察其对细胞形态的影响,并用流式细胞仪检测细胞凋亡情况,荧光实时定量检测细胞凋亡相关基因Bcl-2、Bax mRNA的表达水平。结果 1）实验组较对照组细胞贴壁明显,且48 h时实验3组细胞核仁明显。2）培养48 h时,4组之间的细胞凋亡率、Bcl-2 mRNA、Bax mRNA表达均有统计学差异,随着KGF浓度的增加,细胞凋亡率和Bax mRNA表达逐渐降低,Bcl-2 mRNA表达逐渐升高（P<0.05）。结论 KGF可通过上调Bcl-2 mRNA和下调Bax mRNA的表达抑制上皮细胞的凋亡。     

口腔粘膜白斑和鳞癌组织凋亡相关蛋白Bcl-2和Bax的表达研究

目的研究凋亡相关蛋白Bcl-2、Bax在口腔正常粘膜,上皮异常增生和鳞癌组织中的表达及意义。方法采用免疫组织化学方法检测10例正常口腔粘膜,10例单纯性增生上皮,30例异常增生上皮,33例鳞癌石蜡包埋样本中Bcl-2、Bax的表达。结果Bcl-2在正常粘膜、单纯增生和异常增生白斑表达情况基本一致,显弱阳性表达且局限于基底层细胞.鳞癌组织中Bcl-2表达明显高于正常组和白斑组(P＜0.05),且随组织分化程度的降低,Bcl-2的表达逐步加强。Bax在正常粘膜、白斑和鳞癌组中总的阳性率无明显差异,但表达强度不同。正常组Bax弱表达局限于角化层,异常增生白斑组中随异常增生程度的增高,Bax表达加强且遍布上皮全层。鳞癌组织中高分化鳞癌的Bax表达明显高于低分化鳞癌。结论Bax蛋白表达水平代偿性增高是口腔癌前病损发生发展中的早期事件,Bcl-2可能并不参与癌前病变的发生,Bcl-2表达加强和Bax表达下降在促进肿瘤细胞异常增殖的同时,进一步抑制细胞的凋亡导致口腔癌最终的发生和发展。     

凋亡蛋白Bcl-2、Bax在白斑、口腔扁平苔藓中的表达

目的:观察凋亡蛋白Bcl-2、Bax在白斑、口腔扁平苔藓上皮细胞中的表达,探讨其在口腔白斑、口腔扁平苔藓癌变过程中的作用机制。方法:采用免疫组化法检测10例正常口腔黏膜上皮、18例口腔扁平苔藓、23例白斑、22例口腔鳞癌上皮组织中凋亡相关蛋白Bcl-2、Bax的表达水平。结果:Bcl-2在白斑、口腔扁平苔藓上皮细胞层无异常表达,但在口腔扁平苔藓淋巴细胞浸润带过度表达。Bcl-2在鳞癌组织中呈高表达,与正常黏膜相比有显著性差异(P<0.05)。Bax在上皮单纯增生、轻度、中度不典型增生和低分化鳞癌及糜烂型口腔扁平苔藓组织中呈过度表达,与正常黏膜相比有显著性差异(P<0.05)。结论:Bax参与了口腔白斑癌变的早期事件,而Bcl-2在不典型增生转化为鳞癌的阶段并未发生作用。口腔扁平苔藓的发病机制可能与Bcl-2抑制淋巴细胞凋亡,使细胞免疫亢进,从而刺激上皮细胞Bax过度表达,诱导角朊细胞凋亡有关。     

Immunohistochemical analysis of lymphocyte subpopulations in cyclosporin A-induced gingival overgrowth

BACKGROUND: Cyclosporin A (CsA) is an immunosuppressive agent that is known to induce gingival overgrowth (GO). Pharmacological, genetic, immunologic, and inflammatory factors seem to be involved in the complex pathogenesis of drug-induced GO. Lymphocyte subpopulations in human gingival connective tissue have been implicated in the pathogenesis of inflammatory periodontal diseases. One purpose of this study was to quantify CD4, CD8-, CD57-, and epithelial membrane antigen (EMA)-positive cells in the gingiva of renal transplant recipients treated with CsA, and compare them to findings in healthy controls. A second aim was to correlate cell numbers with clinical findings. METHODS: The study included 19 kidney recipients who were taking CsA and had significant GO (CsAGO+), 13 recipients who were taking CsA but showed no GO (CsAGO-), and 14 systemically healthy individuals with gingivitis (C). Sections from gingival biopsies were incubated with monoclonal antibodies for CD4, CD8, EMA, and CD57, and then analyzed using the avidin-biotin complex method. In each specimen, the mononuclear cell types were quantified and their distribution was evaluated in 3 separate tissue zones: S = subepithelial connective tissue beneath the sulcular epithelium; O = subepithelial connective tissue beneath the oral epithelium; and M = middle connective tissue. RESULTS: There were no significant differences among the groups with respect to the numbers of CD4+ and CD8+ cells in each of the 3 zones (P >0.05). In zone S, the CsAGO+ group had significantly more EMA-positive cells than either the C or CsAGO- groups (P <0.05). There were significant differences among the groups regarding numbers of CD57+ (natural killer) cells in zone M, with the lowest cell numbers in the CsAGO+ patients (P<0.05). CONCLUSIONS: The results showed that low numbers of natural killer cells are important in the expression of plaque-induced inflammatory changes in CsA-associated GO. It appears that these cells may influence the drug's ability to induce proliferative activity.     

Apoptosis in cyclosporin A-induced gingival overgrowth: a histological study

BACKGROUND: Cyclosporin A (CsA) is known to induce gingival overgrowth. Apoptosis plays a critical role in the regulation of inflammation and the host immune response. The aim of this study was to investigate apoptosis in CsA-induced gingival enlargement using electron microscopy examination of keratinocytes. METHODS: Gingiva specimens were collected from 12 CsA-treated renal transplant patients with gingival overgrowth and eight healthy controls with gingivitis. Clinical findings (probing depth, gingival index, and plaque index) were compared in the two groups. Histological and ultrastructural features of the specimens were also compared, and extent of keratinocyte apoptosis was scored on a three-tier scale: 0 = no apoptotic cells; 1 = one or two apoptotic cells; 2 = more than two cells. RESULTS: There were no significant differences between groups with respect to gingiva-related clinical findings or extent of keratinocyte apoptosis. CONCLUSIONS: The results indicate that the extent of keratinocyte apoptosis in the gingiva of kidney recipients with CsA-induced gingival overgrowth is similar to that observed in inflamed gingiva of healthy individuals. Further studies on apoptosis of different cell types in the presence of CsA should clarify this agent's role in the pathogenesis of drug-induced gingival enlargement.     

环孢素A引发牙龈过度生长过程中IL-6的表达

目的:对体外培养的牙龈上皮细胞和成纤维细胞施加环孢素A(cyclosporin,CsA)刺激,应用免疫组化方法探讨CsA引发药物性牙龈过度生长(gingival overgrowth,GO)的病理机制。方法:对体外培养的牙龈上皮细胞和成纤维细胞分别施加浓度为600、800和1000ng/mL,作用时间为48、72h的CsA刺激。在观察细胞生长曲线及其变化的基础上,通过对细胞铺片的免疫酶染色(ABC法)定量分析和对细胞培养液的酶联免疫吸附检测(ELISA法),分别对牙龈组织细胞IL-6的表达和分泌进行测定,应用SAS 6.0软件包对数据进行统计学分析。结果:牙龈上皮细胞接受CsA刺激后,细胞数量明显增加,与对照组相比具有显著差异(P<0.05)。在接受相同条件CsA刺激下,牙龈上皮细胞和成纤维细胞胞内IL-6表达无显著差异,细胞胞内IL-6表达量与CsA作用时间、浓度间相关。接受CsA刺激的最初24h内,牙龈成纤维细胞分泌IL-6的总量在各CsA浓度实验组及对照组间均无显著差异(P>0.05);牙龈成纤维细胞接受刺激超过24h后,CsA浓度为1000ng/mL的实验组与对照组间在细胞分泌IL-6总量上有显著增加...     

口腔扁平苔藓细胞凋亡及凋亡蛋白Bcl-2、Bax表达的变化

目的观察口腔扁平苔藓（oral lichen planus, OLP）病损区口腔黏膜超微结构的变化,以及Bcl-2 、Bax和凋亡细胞的表达水平,探讨细胞凋亡在OLP发生、发展中的作用。方法选取35例OLP患者和10例健康志愿者（对照组）。采用免疫组织化学法检测0LP患者和正常对照组口腔黏膜组织中Bcl-2 、Bax的表达,应用TUNEL法及流式细胞术检测上皮细胞的凋亡情况,应用透射电镜技术观察口腔黏膜超微结构的变化。采用SPSS17.0软件包对数据进行统计学处理。结果免疫组织化学检测显示,Bcl-2在OLP中的阳性表达率与正常口腔黏膜相比无显著差异（P>0.05）,但在淋巴细胞浸润带处表达增强。Bax在OLP中的阳性表达率和表达强度与正常口腔黏膜相比显著增高（P<0.01）。OLP病变组织的上皮细胞凋亡指数显著高于对照组（P<0.01）,与流式细胞术测定结果一致（OLP组上皮细胞凋亡百分率显著高于对照组）。透射电镜观察发现,OLP组口腔黏膜凋亡细胞增多,染色质边聚,并见凋亡小体。结论凋亡细胞表达上调以及病损区超微结构改变,病损区淋巴细胞浸润带处Bcl-2 与上皮基底层角质形成细胞中Bax的表达增强,证实细胞凋亡异常与OLP 的发生、发展有密切关系。     

P53 and bcl-2 immunoexpression in patients with oral lichen planus and oral squamous cell carcinoma

Objective: The aim of this study was to determine by immunohistochemistry the presence and significance of p53 and bcl-2 proteins in oral lichen planus (OLP) and oral squamous cell carcinoma (OSCC). Study Design: We used 21 cases diagnosed as OLP 16 diagnosed as OSCC and four normal gingival biopsies taken from healthy patients were used as controls. Slides were processed for immunohistochemistry using anti-p53 and anti-bcl-2 monoclonal antibodies. Results: We found p53 immunoexpression in 71.4% OLP cases and 68.7% OSCC cases, with no immunoexpression in control cases. Bcl-2 was negative for all OLP and OSCC cases, and mild positivity was observed in normal tissue. We found significant correlation among p53 expression and OSCC malignancy. Conclusions: Our results suggest that TP53 system mainly promotes a hyperproliferative state by cell cycle arrest of the OLP epithelial cells for repairing damaged DNA nor apoptosis and that anti-apoptotic action of bcl-2 is not important in this disease. Key words:Oral lichen planus, oral squamous cell carcinoma, p53, Bcl-2, carcinogenesis, malignant transformation.     

口腔癌前病变细胞凋亡状况及Bcl-2、Bax表达的研究

目的 :通过对口腔白斑、扁平苔藓病变发展及癌变过程中细胞凋亡状况和凋亡相关蛋白表达水平的研究 ,探讨口腔白斑、扁平苔藓的癌变机理及病变机制。方法 :采用原位末端转移酶标记法及免疫组化法 ,观察分析 10例正常口腔黏膜上皮 ,18例扁平苔藓 ,2 3例白斑 ,2 2例口腔鳞癌上皮组织中细胞凋亡状况及凋亡相关蛋白Bcl- 2、Bax的表达水平。结果 :上皮 (轻 ,中 ,重 )不典型增生 ,鳞癌及糜烂型扁平苔藓的凋亡指数均高于正常 (P <0 .0 1)。Bcl- 2在白斑、扁平苔藓上皮细胞层无异常表达 ,但在扁平苔藓固有层淋巴细胞浸润处过度表达 ,在鳞癌组织中显高表达 (P <0 .0 1)。Bax在上皮单纯增生、轻、中度不典型增生和鳞癌及糜烂型扁平苔藓组织中显过度表达 (P <0 .0 5 )。结论 :白斑、扁平苔藓的病变发展及癌变过程中 ,上皮细胞凋亡状况发生了改变 ,Bax参与了白斑癌变的早期事件。扁平苔藓的发病机制与Bcl- 2、Bax在特定部位的过度表达有关     

Apoptosis-associated markers in oral lichen planus

Hypothesizing that loss of basal cells in oral lichen planus is due to apoptosis. we evaluated LP specimens for apoptosis-regulating proteins [positive regulators Bcl-x_s, Bax, Fas/Fas-ligand, p53. and negative regulators (anti-apoptotic) Bcl-2, Bcl-x_L] and compared results with reactions in normal mucosa and chronically inflamed gingiva. Also, sections were evaluated with an in situ TUNEL assay that identifies apoptotic DNA fragments. Basal keratinocytes in normal buccal mucosa, nonspecific gingivitis, and LP were negative for Bcl-2 protein, but meta-nocytes and iymphoid cells were positive. Keratinocyte staining for Bcl-x was negative to weak in normal buccal mucosa and gingivitis, and moderate in LP. Keratinocytes (especially upper prickle cells) in all tissues stained similarly for Bax at weak to moderate levels. Also, no differences in Fas and Fas-ligand staining were evident. Prominent p53-positive staining was seen in all LP biopsies (10–100% of basal keratinocytes) but not in normal buccal mucosa and gingivitis. Few basal keratinocytes in 5/10 LP cases exhibited a positive in situ signal for DNA fragment-associated apoptosis. That the Bcl-2 family of proteins and Fas/Fasligand were detected in normal and diseased tissues, and were occasionally expressed differently in oral LP, supports the notion that apoptosis is a potential mechanism of keratinocyte loss, especially in LP. The pattern of p53 staining in oral LP suggests over-expression of wild-type protein; a phenomenon that would arrest the cell cycle to allow repair of damaged DNA. or trigger apoptosis. While immunohistochemical evidence for apoptosis-associated basal keratinocyte death in LP was slight, it appeared that it may be p53 protein, and possibly Bcl-x. associated.     

The immunohistology of CD40 in human oral epithelium in health and disease

BACKGROUND: CD40 has a role in the regulation of immune responses, cell proliferation and migration, and apoptosis. Little is known of its distribution in oral mucosal pathology. METHODS: Oral keratinocyte lines were tested for CD40 protein by Western blotting. Immunohistochemistry was used to stain paraffin sections of oral mucosa in health and in inflammatory, reactive, dysplastic and malignant disease. RESULTS: Western blotting confirmed the presence of CD40 in oral keratinocytes. CD40 was generally expressed by keratinocytes in the basal layer, with variable parabasal expression. Langerhans cells also stained positively. Expression was lost in nine of 33 (27%) epithelial dysplasias, seven of which were severe. Eighty-one percent of well, 69% of moderately and 50% of poorly differentiated oral squamous cell carcinomas (OSCC) expressed CD40. Overall, 45 of 65 (69%) OSCC were positive. The pattern of expression was unrelated to tumour differentiation. CONCLUSION: CD40 expression by basal and parabasal oral keratinocytes is physiological. Expression is lost in approximately one-third of oral epithelial dysplasias and OSCC. The significance of such loss remains unknown, but may be related to immunological or other abnormalities of keratinocyte homeostasis.     

Inhibition of apoptotic signals in overgrowth of human gingival fibroblasts by cyclosporin A treatment

Cyclosporin A (CsA), an immunosuppressive drug, has overgrowth effects on human gingival fibroblasts (HGF) in vitro. However, the molecular mechanism responsible for the CsA-induced gingival overgrowth remains still unclear. The present study is aimed to investigate the correlation with the apoptotic signal pathway in CsA-induced overgrowth of HGF. CsA-treated HGF were assessed for cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, for reactive oxygen species (ROS) detection by flow cytometry, for proliferation ability using the 5-bromo-20-deoxyuridine (BrdU), for caspase activities biochemically, for expression of apoptotic signal molecules such as cytochrome c, Fas and Fas-L and Bcl-2 family by Western blotting and VDAC by RT-PCR. CsA increased the cell viability, but not the number of BrdU-positive HGF, indicating that CsA fails to induce the proliferation of HGF. CsA also decreased the intracellular reactive oxygen species level in HGF. This was accompanied by that the antiapoptotic protein Bcl-2 was upregulated whereas the proapoptotic protein Bax was downregulated. Moreover, CsA downregulated VDAC, a mitochondrial transition pore, and decreased the level of cytochrome c released from the mitochondria into the cytosol and activation of caspase-3 and -9 associated with mitochondria-mediated apoptosis. On the other hand, Fas-L level and caspase-8 activation, the major mediator of the death receptor-mediated apoptosis, were diminished in the CsA-treated HGF. CsA inhibits the apoptotic signal molecules such as cytochrome c, caspases and Fas-L with the regulation of Bcl-2 family whereas it has no effect on cell division, which can contribute to overgrowth of HGF. These findings suggest that the decreased apoptosis plays a more important role than the increased cell proliferation in the CsA-induced overgrowth of HGF.     

P53 and bcl-2 immunoexpression in patients with oral lichen planus and oral squamous cell carcinoma

Objective: The aim of this study was to determine by immunohistochemistry the presence and significance of p53 and bcl-2 proteins in oral lichen planus (OLP) and oral squamous cell carcinoma (OSCC). Study Design: We used 21 cases diagnosed as OLP 16 diagnosed as OSCC and four normal gingival biopsies taken from healthy patients were used as controls. Slides were processed for immunohistochemistry using anti-p53 and anti-bcl-2 monoclonal antibodies. Results: We found p53 immunoexpression in 71.4% OLP cases and 68.7% OSCC cases, with no immunoexpression in control cases. Bcl-2 was negative for all OLP and OSCC cases, and mild positivity was observed in normal tissue. We found significant correlation among p53 expression and OSCC malignancy. Conclusions: Our results suggest that TP53 system mainly promotes a hyperproliferative state by cell cycle arrest of the OLP epithelial cells for repairing damaged DNA nor apoptosis and that anti-apoptotic action of bcl-2 is not important in this disease.     

Effect of cyclosporin A on apoptosis and expression of p53 and bcl-2 proteins in the gingiva of renal transplant patients

BACKGROUND: Gingival overgrowth (GO) is a common side effect of cyclosporin A (CsA) therapy, but the exact mechanism for this is unknown. Apoptosis plays an important role in the maintenance of tissue homeostasis and mediators of this process may be involved in the pathogenesis of drug-induced GO. This study compared p53 expression, bcl-2 expression, and apoptosis in gingival samples from CsA-treated renal transplant recipients to findings in controls with gingivitis. METHODS: Twenty-two kidney recipients with CsA-induced GO and 15 systemically healthy subjects with gingivitis were included in the study. The 15 systemically and periodontally healthy volunteer control group were immunohistochemically analyzed for grades of p53 and bcl-2 expression, and were processed using terminal TdT-mediated dUTP-biotin nick-end labeling (TUNEL) technique to identify and grade levels of apoptosis. RESULTS: There were no differences between the CsA group and the control group with respect to grades of p53 and bcl-2 expression (P >0.05 for both). However, the CsA group showed a lower apoptosis grade than the control group (P <0.05). None of the clinical parameters was significantly correlated with any of the immunohistochemical findings for p53 or bcl-2 (P >0.05 for all). Similarly, grade of apoptosis was not correlated with any of the clinical parameters (P >0.05). There was a significant positive correlation between serum CsA level and level of bcl-2 expression, but serum CsA was not significantly correlated with level of apoptosis or level of p53 expression. CONCLUSION: The results indicate that the pathogenesis of CsA-induced GO might involve inhibition of apoptosis, and overexpression of bcl-2 in the setting of high serum CsA.     

Cyclosporin-induced gingival overgrowth: a clinical-epidemiological evaluation of 121 Italian renal transplant recipients

BACKGROUND: Although other immunosuppressive agents have been recently introduced (e.g., tacrolimus), it has been calculated that in the next decade about 1 million people will still be taking cyclosporin (CsA). The association between gingival overgrowth (GO) and the use of CsA is still not clear. In the present study we evaluated the prevalence and the degree of GO in a group of Italian renal transplant patients and the possible relationship between gingival lesions and demographic, oral, systemic, and pharmacological variables. METHODS: One hundred twenty-one renal transplant recipients receiving immunosuppressive therapy with CsA were evaluated in this study. Patients were classified in two groups. In the first (screening group), we included all those patients referred by the Parma University Renal Transplant Center for a general oral checkup, with no specific indications for GO. The second group (non-screening group) included all those patients who specifically had been referred to the Oral Pathology and Oral Medicine Unit because of GO. We considered the following variables: gender, daily CsA dose, duration of immunosuppressive treatment, CsA plasma concentration, concomitant use of another immunosuppressive agent (azathioprine), use of other GO inducers (calcium channel blockers, anti-epileptic drugs), oral hygiene scores, and other drugs taken at the time of oral examination. RESULTS: Fisher's exact test and chi square test demonstrated that in the screening group, duration of immunosuppressive treatment and oral hygiene scores were associated both with the prevalence and the high GO scores (P (1) (DIT) <0.0001; P (2) (DIT)=0.0023; P (1) (hyg)=0.0084; P (2) (hyg)=0.0068). In the screening group, concomitant use of CsA and azathioprine is related to a low development degree of GO (P=0.0088). In the non-screening group, we found a significant association between poor oral hygiene and high degree of GO (P=0.0349). CONCLUSION: In addition to a probable genetic predisposition, duration of immunosuppressive treatment and oral hygiene status are the most important variables related to development and degree of GO during the use of CsA in this study.     

环孢素A诱导牙龈增生动物模型的建立及其病理学表现

目的:建立环孢素A(CyclosporinACsA)诱导的牙龈增生小鼠动物模型,观察其病理学表现,并进行组织测量,确定其可靠性。方法:将CsA溶于橄榄油中,按 30mgCsA/kg体重 /d胃饲远交群ICR雄性小鼠,分别在给药 2、4、6、8周时,随机取实验组小鼠下颌前部标本,体视显微镜下测量其下颌前牙龈体积;制作切片,光镜下观察其牙龈病理表现,并利用图象分析系统测量舌侧牙龈缘上皮厚度。结果:小鼠经胃饲 30mg/kg体重 /d的CsA,①4周后,肉眼见部分下颌前牙舌侧牙龈球形肥大;②体视显微镜下牙龈体积测量显示下颌前牙舌侧牙龈 4周至 8周明显增大(P<0. 01);③此时光镜下开始出现明显的牙龈增生病理表现:上皮增厚,上皮钉突明显且变宽,牙龈结缔组织增厚,基质和成纤维细胞增生,血管扩张等。④龈缘上皮厚度测量 2周时上皮增厚(P<0. 01), 4周增厚更明显(P<0. 001),持续至 8周。结论:胃饲小鼠CsA可诱导牙龈增生,其病理表现与其他动物模型和临床病理一致,该模型有一定可靠性,可用于进一步研究CsA诱导的牙龈增生的发病机理。     

发夹状核酶阻断HPV16转化口腔上皮细胞的研究

目的:探讨发夹状核酶阻断人乳头状瘤病毒16(HPV16)转化口腔上皮细胞的能力及可能的机制。方法:设计合成HPV16E6特异性发夹状核酶基因;构建重组真核表达载体;脂质体介导转染HPV16诱导的永生化口腔上皮细胞(HIOEC);空白载体对照;潮霉素筛选;斑点杂交检测细胞中核酶的表达;PCR、RT-PCR和免疫细胞化学分别检测细胞中HPV16E6的DNA、RNA和蛋白表达;荧光染色、流式细胞仪检测、生长曲线绘制观察细胞生物学特性的改变。结果:成功构建含HPV16E6特异性发夹状核酶的重组真核表达载体pcDNA-RZE6;转导筛选后,获得阳性克隆HIOEC-RZE6和空白载体对照HIOEC-pcDNA;HIOEC-RZE6中核酶转录、HPV16E6RNA和蛋白表达下降,细胞增殖减缓,出现凋亡;HIOEC-pcDNA中未见同样变化。结论:HPV16E6特异性发夹状核酶可以抑制HPV16E6表达,并阻断HIOEC进一步恶变,诱导细胞凋亡。     

Levels of leukotriene B4 and platelet activating factor in gingival crevicular fluid in renal transplant patients receiving cyclosporine A

BACKGROUND: Cyclosporine A (CsA) is a potent immunosuppressant effectively used to prevent organ transplant rejection and also to treat several systemic diseases. CsA-induced gingival overgrowth (CsA GO) is the most widely seen side effect of this drug; its pathogenesis is not completely understood. The aim of the present study was to identify the role of leukotriene B4 (LTB4) and platelet activating factor (PAF) in the pathogenesis of CsA GO. METHODS: LTB4 and PAF levels were detected in gingival crevicular fluid (GCF) samples from renal transplant patients receiving CsA therapy and exhibiting CsA GO, from patients with gingivitis and from periodontally healthy subjects. Plaque index, papilla bleeding index, and hyperplastic index were recorded at each study site. GCF samples and clinical data were obtained from: 2 sites exhibiting CsA GO (CsA GO+) and 2 sites not exhibiting CsA GO (CsA GO-) in each CsA-treated patient; 2 diseased sites in each patient with gingivitis; and 2 healthy sites in each subject with clinically healthy periodontium. LTB4 was extracted from the samples by solid-phase method using C18 cartridge and purified by high-performance liquid chromatographic (HPLC) method and analyzed by radioimmunoassay (RIA). PAF was extracted from GCF samples passing through amberlit resin columns, purified by HPLC, and analyzed by RIA. RESULTS: Total amounts of LTB4 and PAF in GCF were higher in CsA GO+ sites compared to the healthy sites from healthy controls. However, the amount of LTB4 and PAF elevation in CsA GO+ sites was not significantly higher than those in diseased sites. Clinical degrees of gingival inflammation were also similar between CsA GO+ and diseased sites. LTB4 and PAF total amounts in GCF were higher in CsA GO+ sites compared to CsA GO- sites in the same subjects, but this difference just failed to reach significance. Similar findings were obtained with concentration data. CONCLUSIONS: The results of this study indicate that CsA therapy does not have a significant effect on GCF LTB4 and PAF levels and that gingival inflammation seems to be the main reason for their elevation. In CsA-treated patients, alterations in LTB4 and PAF levels might play a role in CsA GO through some asyet unknown mechanism. To our knowledge, this is the first report describing the levels of lipid mediators in GCF of CsA-treated patients. We assume that further studies will contribute to the understanding of the pathogenesis of CsA-induced gingival overgrowth.