摘要
目的 探讨牙髓卟啉单胞菌（Porphyromonas endodontalis,Pe）脂多糖对小鼠成骨细胞基质金属蛋白酶9（matrix metalloproteinase-9,MMP-9）表达的影响及核因子κB（nuclear factor-κB,NF-κB）信号通路是否参与此过程,从分子水平更好地了解根尖周疾病骨破坏的发生机制.方法 实验分为3部分,以不同质量浓度的Pe脂多糖[0（空白对照组）、1、5、10、15及20 mg/L]刺激MC3T3-E1细胞24 h;以10 mg/L Pe脂多糖作用于细胞不同时间（0、10、24、48 h）后,采用实时反转录PCR、酶联免疫吸附测定和明胶酶谱法检测MMP-9 mRNA和蛋白的表达以及酶活性变化;采用NF-κB信号通路特异性抑制剂BAY 11-7082预处理1 h,检测其对Pe脂多糖刺激MC3T3-E1细胞后MMP-9 mRNA表达的影响.对结果行单因素方差分析和Dunnett t检验.结果 不同质量浓度Pe脂多糖刺激MC3T3-E1细胞后,MMP-9 mRNA和蛋白的表达具有剂量依赖性,蛋白表达量从（5395±362）ng/L（空白对照组）增加到（12684±375）ng/L（20 mg/L组）.用10 mg/L Pe脂多糖作用于MC3T3-E1细胞24 h时,MMP-9 mRNA表达量最高,约为空白对照组的7倍,随着作用时间延长,MMP-9 mRNA表达量下降;当10 mg/L Pe脂多糖作用于MC3T3-E1细胞48 h时MMP-9的蛋白表达量最高[（35055±2346）ng/L];酶活性也最高.10 mmol/L的BAY 11-7082预处理细胞1 h后,显著降低了Pe脂多糖诱导的MMP-9 mRNA的表达水平（P＜0.01）.结论 Pe脂多糖可能通过激活NF-κB信号通路诱导小鼠成骨细胞表达MMP-9.
Objective To evaluate the effects of lipopolysaccharides （LPS） extracted from Porphyromonas endodontalis （Pe） on the expression of matrix metalloproteinase-9 （MMP-9） mRNA and protein as well as enzyme activity in MC3T3-E1 cells and the role of nuclear factor-κB （NF-κB）in the process, so as to investigate the expression of MMP-9 dependent signaling pathways in mouse osteoblasts induced by Pe LPS. Methods The experiment was conducted in 3 sessions： MC3T3-E1 cells were treated with various concentrations of Pe LPS （0-20 mg/L） and 10 mg/L Pe LPS for different time intervals （0-48 h）. The expression of MMP-9 mRNA and protein were detected by real-time reverse transcription-PCR （RT-PCR） and enzyme-linked immunosorbent assay （ELISA）, while the enzyme activity was detected by gelatin zymography method. The expression of MMP-9 mRNA was also detected in 10 mg/L Pe LPS treated MC3T3-El cells after pretreated with specific NF-κB inhibitor BAY 11-7082 for l h.Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. Results The levels of MMP-9 mRNA and protein increased significantly after the treatment with various concentrations of Pe LPS （0-20 mg/L）,which indicated that Pe LPS induced osteoblasts to express MMP-9 in dose dependent manners. The expression of MMP-9 protein increased from （5395 ± 362） ng/L （blank control group） to （12684 ± 375） ng/L （20 mg/L group）. Maximal induction of MMP-9 mRNA expression was found in the MC3T3-E1 cells treated with 10 mg/L Pe LPS for 24 h.The expression of MMP-9 mRNA in the 20 mg/L group was about 7 times than that in the blank control group. After 24 h, the expression of MMP-9 mRNA decreased.Maximal expression of MMP-9 protein was found in the MC3T3-E1 cells treated with 10 mg/L Pe LPS for 48 h （[35055±2346] ng/L） showing the highest enzyme activity. The mRNA of MMP-9 decreased significantly after pretreatment with 10 μmol/L BAY 11-7082 for 1 h.Conclusions Pe LPS might induce the expression of MMP-9 in MC3T3-E1 cells through the signaling of NF-κB.
出处
《中华口腔医学杂志》
CAS
CSCD
北大核心
2017年第8期499-503,共5页
Chinese Journal of Stomatology
基金
国家自然科学基金（81500843）
沈阳市科学技术项目计划（F15-199-1-56）
