人釉原蛋白真核表达载体体外表达产物生物学活性的检测

目的探讨人釉原蛋白基因重组质粒PcDNA3．1-AMG的真核细胞转染表达产物是否具有促进牙周组织再生的作用。方法脂质体介导PcDNA3．1-AMG体外转染COS1细胞．ELISA法检测转染细胞内及其培养上清液中重组釉原蛋白的表达：建立犬牙周组织缺损模型．局部使用转染表达产物冻干粉．8周后通过组织学观察牙周组织再生的情况。结果PcDNA3．1-AMG转染的COS1细胞内重组釉原蛋白浓度为（0．253±0．075）μg／ml。培养液中浓度为（0．065±0．011）μg／ml；使用转染表达产物8周后．牙周缺损区牙骨质、牙槽骨均有显著再生，并且新生牙骨质为有胶原纤维穿通的无细胞牙骨质。结论重组质粒PcDNA3．1－AMG在体外转染哺乳动物细胞后能够表达重组人釉原蛋白．并且表达产物具有促进牙周组织再生的生物学活性。     

猪发育期牙胚釉原蛋白的分离纯化及其多克隆抗体的制备

目的　制备猪釉质蛋白多克隆抗体,为釉原蛋白的检测提供条件。方法　选择1月龄乳猪,分离埋于上下颌骨内的牙胚,刮取牙胚表面干酪样尚未完全矿化的釉质基质,通过盐酸胍抽提及SephadexG-200柱层析分离纯化猪发育期牙胚釉原蛋白,联合免疫家兔,抗血清经DE-52纤维素纯化,并经ELISA测定抗体效价。结果　 SDS-PAGE电泳结果发现采用SephadexG-200葡聚糖凝胶过滤能达到较为理想的釉原蛋白分离纯化效果,用所提纯的猪釉原蛋白免疫家兔,成功地制得兔抗猪釉原蛋白抗血清,抗血清稀释达1∶32 000时,采用ELISA法仍有明显的抗原抗体反应。结论　本实验成功制备得到抗釉原蛋白多克隆抗体,为釉原蛋白的检测提供了条件。     

侵袭性牙周炎患者外周血CD-4+CD-25+调节性T细胞的检测及功能分析

Objective To investigate the changes of proportion and suppression function of CD-4+ CD-25+ regulatory T cells in the peripheral blood of patients with aggressive periodontitis.Methods Flow cytometric analysis was used to detect the frequency of CD-4+ CD-25+ regulatory T cells in the peripheral blood of 16 patients with generalized aggressive periodontitis and 17 patients with chronic periodontitis,as well as 17 periodontal healthy controls.Furthermore,CD-4+ CD-25+ regulatory T cells and CD-4+ CD-25- T cells were separated from peripheral blood of each enrolling subject using magnetic cell sorting technology.The direct suppression effect of CD-4+ CD-25+ regulatory T cells on CD-4+ CD-25- T lymphocytes proliferation was performed by the mixed lymphocytes reaction and measured by 3H-thymidine radioactive assay.Results The patients with generalized aggressive periodontitis had a lower frequency of CD-4+ CD-25+ regulatory T cells(9.71±4.01)%in the peripheral blood than periodontal healthy controls [(14.72±3.51)%]and chronic periodontitis patients[(17.01±5.16 )%],P＜0.05.A significant decrease was found in the suppression function of CD-4+ CD-25+ regulatory T cells from peripheral blood of patients with generalized aggressive periodontitis when co-cultured with CD-4+ CD-25- T lymphocytes in the proportion of 2:1,1:1 and 1:2 as compared with chronic periodontitis patients and periodontal healthy controls(P＜0.05).Conclusions Diminished numbers and decreased suppression function of CD-4+ CD-25+ regulatory T cells were found in patients with generalized aggressive periodontitis.     

侵袭性牙周炎患者外周血CD-4+CD-25+调节性T细胞的检测及功能分析

Objective To investigate the changes of proportion and suppression function of CD-4+ CD-25+ regulatory T cells in the peripheral blood of patients with aggressive periodontitis.Methods Flow cytometric analysis was used to detect the frequency of CD-4+ CD-25+ regulatory T cells in the peripheral blood of 16 patients with generalized aggressive periodontitis and 17 patients with chronic periodontitis,as well as 17 periodontal healthy controls.Furthermore,CD-4+ CD-25+ regulatory T cells and CD-4+ CD-25- T cells were separated from peripheral blood of each enrolling subject using magnetic cell sorting technology.The direct suppression effect of CD-4+ CD-25+ regulatory T cells on CD-4+ CD-25- T lymphocytes proliferation was performed by the mixed lymphocytes reaction and measured by 3H-thymidine radioactive assay.Results The patients with generalized aggressive periodontitis had a lower frequency of CD-4+ CD-25+ regulatory T cells(9.71±4.01)%in the peripheral blood than periodontal healthy controls [(14.72±3.51)%]and chronic periodontitis patients[(17.01±5.16 )%],P＜0.05.A significant decrease was found in the suppression function of CD-4+ CD-25+ regulatory T cells from peripheral blood of patients with generalized aggressive periodontitis when co-cultured with CD-4+ CD-25- T lymphocytes in the proportion of 2:1,1:1 and 1:2 as compared with chronic periodontitis patients and periodontal healthy controls(P＜0.05).Conclusions Diminished numbers and decreased suppression function of CD-4+ CD-25+ regulatory T cells were found in patients with generalized aggressive periodontitis.     

非手术方法治疗侵袭性牙周炎临床疗效观察

目的探讨非手术方法治疗侵袭性牙周炎的临床效果。方法选择广泛型侵袭性牙周炎患者15例,进行口腔卫生宣教、龈上洁治、龈下刮治和根面平整,并蹲服罗红霉素和甲硝唑1周。分别于治疗前和治疗后3、6、12、18个月检查及记录出血指数、探诊深度和附着水平,并进行分析比较。结果出血指数、探诊深度和附着水平在治疗前分别为3．37±0．56、（5.83±1．68）mm．（6．78±1．50）mm,治疗后18个月下降为（0．69±0．48）mm、（2．15±0．45）mm、（4．60±0．78）mm,差异均有统计学意义（P=0.000）。结论广泛型侵袭性牙周炎患者经过牙周非手术治疗后可以取得良好的治疗效果,并且疗效较为稳定。     

釉原蛋白基因表达质粒PcDNA3.1-AMG在小鼠肌肉组织中表达的研究

目的研究釉原蛋白（amelogenin,AMG）重组质粒PcDNA3.1-AMG能否在小鼠肌肉组织中获得正确表达.方法采用裸质粒直接注射法将溶于200 μL磷酸缓冲液中的100 μg质粒 PcDNA3.1（对照组）或PcDNA3.1-AMG（实验组）分别导入Balb/C小鼠的胫前肌内,注射后第3、7、14天分批处死小鼠,采用免疫组织化学染色法检测肌肉组织内釉原蛋白的表达.结果注射PcDNA3.1-AMG的肌肉组织的肌细胞和细胞间质中均检测到釉原蛋白的表达;而注射PcDNA3.1的肌肉组织的肌细胞和细胞间质中均未检测到釉原蛋白的表达.结论 PcDNA3.1-AMG成功地转染了肌肉细胞,并且外源基因能够在肌细胞内转录、翻译并正确表达釉原蛋白.     

牙周炎的阻断治疗

菌斑细菌是牙周炎发生的始动因子。研究表明，除控制菌斑外，还可以通过调节宿主反应辅助治疗牙周炎。在宿主反应过程中，组织破坏性酶如MMPs、弹性蛋白酶等，炎症介质和细胞因子如PGE1、IL－1、TNFα等起着非常重要的作用，介导了牙周组织的破坏。四不素类药笺、NSAID、IL－1Ra、sIL－1R、抗TNFα单克隆抗体和sTNFR等可以选择性地阻断这些活性物质对牙周组织的破坏，起到良好的辅助治疗作用。     

人釉原蛋白编码区基因真核表达载体PsecTaq2A-AMG的构建

目的　构建人釉原蛋白(AMG)编码区基因真核表达载体PsecTaq2A-AMG。方法　采用PCR技术体外扩增AMG完整分泌肽编码区。将扩增产物与PsecTaq2A分别用BamHI和Xhol I行双酶切,将获取的AMG目的基因片段连接到双酶切后的PsecTaq2A,构建重组质粒PsecTaq2A-AMG,并对重组质粒进行鉴定。结果　①PCR扩增产物经1·5%琼脂糖凝胶电泳,可见大小约519 bp的特异性条带,与预期结果一致。②重组克隆PsecTaq2A-AMG酶谱分析与预期结果一致,序列测定结果与GenBank中的人釉原蛋白序列完全一致。结论　用此方法可成功构建AMG 编码区基因真核表达载体PsecTaq2A-AMG。     

人釉原蛋白重组质粒PsecTaq2A-AMG在COS-1细胞系中表达

目的研究人釉原蛋白重组质粒PsecTaq2A-AMG在COS-1细胞系中的表达，为基因工程制备釉原蛋白奠定基础。方法采用脂质体载体法将釉原蛋白重组质粒PsecTaq2A-AMG导人COS-1细胞系，用Zeoin筛选得到稳定转染克隆，并经免疫组化及酶联免疫吸附实验（ELISA）检测细胞内和细胞培养液中重组釉原蛋白的表达。结果在未经转染的对照组细胞内和细胞培养液中均未检测到重组釉原蛋白的表达，而经转染的实验组不论细胞内或细胞培养液中均检测到重组釉原蛋白的较高表达，重组质粒PseeTaq2A．AMG转染组ELISA定量检测细胞内重组釉原蛋白浓度达0.877μg／ml。结论PsecTaq2A-AMG具有较强的在真核细胞系中表达和分泌重组釉原蛋白的能力，适宜基因工程体外制备重组釉原蛋白。     

侵袭性牙周炎患者外周血CD-4+CD-25+调节性T细胞的检测及功能分析

Objective To investigate the changes of proportion and suppression function of CD-4+ CD-25+ regulatory T cells in the peripheral blood of patients with aggressive periodontitis.Methods Flow cytometric analysis was used to detect the frequency of CD-4+ CD-25+ regulatory T cells in the peripheral blood of 16 patients with generalized aggressive periodontitis and 17 patients with chronic periodontitis,as well as 17 periodontal healthy controls.Furthermore,CD-4+ CD-25+ regulatory T cells and CD-4+ CD-25- T cells were separated from peripheral blood of each enrolling subject using magnetic cell sorting technology.The direct suppression effect of CD-4+ CD-25+ regulatory T cells on CD-4+ CD-25- T lymphocytes proliferation was performed by the mixed lymphocytes reaction and measured by 3H-thymidine radioactive assay.Results The patients with generalized aggressive periodontitis had a lower frequency of CD-4+ CD-25+ regulatory T cells(9.71±4.01)%in the peripheral blood than periodontal healthy controls [(14.72±3.51)%]and chronic periodontitis patients[(17.01±5.16 )%],P＜0.05.A significant decrease was found in the suppression function of CD-4+ CD-25+ regulatory T cells from peripheral blood of patients with generalized aggressive periodontitis when co-cultured with CD-4+ CD-25- T lymphocytes in the proportion of 2:1,1:1 and 1:2 as compared with chronic periodontitis patients and periodontal healthy controls(P＜0.05).Conclusions Diminished numbers and decreased suppression function of CD-4+ CD-25+ regulatory T cells were found in patients with generalized aggressive periodontitis.