微RNA-146a反转牙龈卟啉单胞菌脂多糖对人牙周膜细胞成骨的抑制作用

microRNA-146a reverses the inhibitory effects of Porphyromonas gingivalis lipopolysaccharide on osteogenesis of human periodontal ligament cells

目的探讨微RNA-146a（microRNA-146a，miR-146a）对牙龈卟啉单胞菌（Porphyromonas gingivalis，Pg）脂多糖（lipopolysaccharide，LPS）刺激下人牙周膜细胞（human periodontal ligament cells，hPDLC）成骨分化的影响及机制，为牙周组织工程学研究提供实验依据。方法体外培养hPDLC，诱导成骨分化，将细胞分为5组：含5%胎牛血清的α-微量伊格尔培养基培养的hPDLC（非成骨分化培养组）；成骨分化液培养的hPDLC（成骨分化培养组）；成骨分化液＋1 mg/L Pg LPS处理的hPDLC（LPS组）；成骨分化液＋1 mg/L Pg LPS＋miR-146a模拟物处理的hPDLC（LPS＋miR-146a模拟物组）；成骨分化液＋1 mg/L Pg LPS＋miR-146a阴性对照处理的hPDLC（LPS＋miR-146a阴性对照组），每组设5个复孔。通过实时荧光定量PCR和茜素红染色法分别检测成骨标志物和矿化，同时应用非放射性转录因子检测hPDLC的核提取物中核因子κB（nuclear factor kappa B，NF-κB）p65的核活性。结果与成骨分化培养组相比，LPS组hPDLC中Ⅰ型胶原、碱性磷酸酶（alkaline phosphatase，ALP）、Runt相关转录因子2（Runt-related transcription factor-2，RUNX2）和骨钙蛋白水平显著降低（P〈0.05）；Pg LPS可以抑制矿化能力，刺激NF-κB p65的核转录活性的表达（成骨分化培养组：1.023±0.217，LPS组：6.252±0.613，P=0.008）；与LPS＋miR-146a阴性对照组相比，LPS＋miR-146a模拟物组第3天检测到hPDLC中除ALP（P=0.167）和RUNX2（P=0.580）外，Ⅰ型胶原（P=0.007）和骨钙蛋白mRNA（P=0.049）的表达均显著增加；第7和14天时Ⅰ型胶原、ALP、RUNX2和骨钙蛋白mRNA水平均显著增加（P〈0.05），并可以增强矿化能力，同时miR-146a模拟物可显著降低Pg LPS诱导的hPDLC中NF-κB p65的核转录活性（LPS＋miR-146a模拟物组：2.427±0.354；LPS＋miR-146a阴性对照组：5.863±0.482，P=0.019）。结论miR-146a可以通过促进hPDLC中成骨标志物的表达和抑制炎症通路，逆转Pg LPS对hPDLC成骨分化的抑制作用。

Objective To investigate the effects and mechanisms of microRNA-146a （miR-146a） on osteogenic differentiation of human periodontal ligament cells （hPDLC） stimulated by lipopolysaccharide （LPS） of Porphyromonas gingivalis （Pg）.Methods hPDLC were cultured in vitro and induced to the phase of osteogenic differentiation. These cells were divided into five groups： non-osteogenic differentiation cells, osteogenic differentiation cells, osteogenic differentiation cells treated with Pg LPS, osteogenic differentiation cells treated with Pg LPS and miR-146a mimic, osteogenic differentiation cells treated with Pg LPS and miR-146a negative control. Osteogenic markers and mineralization were detected via quantitative real-time PCR （qPCR） and alizarin red staining, respectively. Meanwhile, non-radioactive transcription factor assay was applied to explore the nuclear activity of nuclear factor kappa B （NF-κB） p65 in nuclear extracts of hPDLC.Results Compared with cells of osteogenic differentiation in non-LPS-stimulated groups, Pg LPS could decrease the markers of osteogenic differentiation of hPDLC such as collagen Ⅰ （Col-Ⅰ）, alkaline phosphatase （ALP）, Runt-related transcription factor-2 （RUNX2） and osteocalcin （OCN） （P〈0.05）, inhibit mineralization, and stimulate NF-κB p65 nuclear activity expression （non-LPS stimulated group： 1.023±0.217, LPS stimulated group： 6.252±0.613, P=0.008）. However, compared with cells in Pg LPS/miR-146a negative control group, miR-146a increased Col-Ⅰ （P=0.007） and OCN （P=0.049） mRNA expression, rather than ALP （P=0.167） and RUNX2 （P=0.580） at day 3; miR-146a also upregulated mRNA levels of Col-Ⅰ, ALP, RUNX2 and OCN （P〈0.05） at day 7 and day 14, and enhance mineralization. Meanwhile, miR-146a mimic could decrease the nuclear activity of NF-κB p65 induced by Pg LPS in hPDLC （miR-146a： 2.427±0.354, negative control： 5.863±0.482, P=0.019）.Conclusions miR-146a could reverse the inhibitory effects of Pg LPS on osteogenic differentiation of hPDLC through enhancing the expression of osteogenic markers and decreasing inflammatory pathway in hPDLC.