纤维粘连蛋白与流体剪切力对体外培养的牙周膜成纤维细胞COX-2表达的影响

A study on the influence of fluid shear stress on the expression of COX-2 in HPDLF

目的:在体外利用流体剪切力模拟咬合创伤,对牙周膜成纤维细胞(HPDLF)给予不同浓度的纤维粘连蛋白(FN)处理,在不同时段测量HPDLF的COX-2mRNA的表达量,以期模拟HPDLF在口腔存在咬合创伤的情况下FN对其生物学行为的影响。方法:收集因正畸矫治需要拔除的健康年轻恒牙,以第3代细胞作为研究对象。样本分为A、B2组,A组为利用水平摇床施加剪切力培养,转速为35r/min;B组为静态培养。A、B2组中分别分为加入FN0μg/ml(对照)、20μg/ml、40μg/ml、60μg/ml等4组,每组4孔。无血清培养6h、12h。采用SPSS13.0软件包对结果进行配对t检验。结果:HPDLF原代培养,24h后80%组织块贴壁。7～10d有细胞从组织块周围游出。14d左右长满培养瓶。免疫组化染色显示抗波丝蛋白阳性,抗角蛋白阴性。RT-PCR结果显示,A组6h后COX-2表达即有体现;12h后,相同FN用量的样本的COX-2表达量较加力6h时高,且表达量随着FN用量的增加而减少。B组中,各样本电泳结果为阴性。光密度扫描分析,配对t检验结果t=13.45,P<0.01。结论:流体剪切力可以引起HPDLF的炎性反应。人血浆FN可以减少HPDLF的COX-2mRNA表达,呈时间、剂量依赖性。

PURPOSE： To utilize the fluid shear stress to simulate occlusion trauma in vitro. Different density of FN was given to HPDLF and the amount of COX-2 mRNA of HPDLF was measured at different time. METHODS： Young healthy permanent teeth which were extracted for orthodontic treatment were collected. The HPDLF was primarily cultured via the method for tissue block. The third generation cells were taken as the study objects. There were two groups in this study. Group A were cultured with the fluid shear stress given by a special swing bed （35r/min）. Group B were cultured quietly. Different density of FN （0,20,40,60μg/ml）was added into Group A and B. The cells were cultured for 6 and 12 hours without serum. RESULTS： After 24 hours, 80% tissue block anchoraged. After 7-10 days,there were cells moving from the tissue block,After about 14 days, the cell overgrew the culture flask.Immuocytochemistry showed that antifilamin was positive while anticeratin was negative. The expression of COX-2 mRNA was positive after 6 hours in group A,and the amount of the expression decreased with the increase of the amount of FN . The expression of COX-2 mRNA after 12 hours in group A was higher than that after 6 hours. The result of electrophoresis for group B was negative. The electrophoresis optical density of β-actin amplification fragment was uniform. CONCLUSIONS： The fluid shear stress can revoke the inflammatory reaction of HPDLF. Human plasma FN can decreased the expression of COX-2 mRNA of HPDLF,which is dose and time dependent.Supported by Research Fund of Education Bureau of Liaoning Province（Grant No.OSL481）,