The Study On The Effect Of Osteogenic Differentiation On MicroRNA-146a In Human Periodontal Ligament Cells After Porphyromonas Gingivalis Lipopolysaccharide Stimulation

Objective:After culturing human periodontal ligament fibroblasts in vitro, we observe the osteogenic differentiation capacity of HPDLCs after being transfected with miRNA-146 a under the inflammatory state, and to explore its mechanism, so as to provide a broader way of thinking about periodontal treatment, regeneration of alveolar bone inflammation.Methods:1. After informed consent, the double teeth were obtained from healthy patients(aged from 12 to 18) who need orthodontic treatment, then washed the teeth with PBS repeatedly, scraped the periodontal membrane from root and cut up into 1 mm3 small pieces.After the periodontal membrane were digested by type I collagen enzyme, HPDLCs were obtained from the primary culture of the tissue explants. The morphology and growth of HGFs were observed by the inverted phase contrast microscope.2. To detect the effect of P.g LPS on the osteogenic differentiation capacity of HPDLCs, in which the control group, osteogenic induced group and Pg LPS(1 μg/ml)/ osteogenic induction group were set up. Alkaline phosphatase was detected at 7.Alizarin red staining and Alizarin Red semi-quantitative detection were performed at21 days. Real-time PCR was applied to detect the bone-related gene expression on day 3, 7 and 14.3. To detect the effect of miRNA-146 a on the osteogenic differentiation capacity of HPDLCs, in which miRNA-146 a mimics transfected group, negative control group and the control group were set up. Cells were cultured in osteogenic induction medium. Alkaline phosphatase was detected at 7. Alizarin red staining and Alizarin Red semi-quantitative detection were performed at 21 days. Real-time PCR was applied to detect the bone-related gene expression on day 7.4. To detect the effect of miRNA-146 a on the osteogenic differentiation capacity of HPDLCs after stimulated with P.g LPS. miRNA-146 a mimics+1 μg/ml P.g LPSgroup, negative control+1 μg/ml P.g LPS group, 1 μg/ml P.g LPS group were set up.Cells were cultured in osteogenic induction medium. Alkaline phosphatase was detected at 7. Alizarin red staining and Alizarin Red semi-quantitative detection were performed at 21 days. Real-time PCR was applied to detect the bone-related gene expression on day 7.Results:1. A few spindle cells migrated from the edge of tissue during the 8-12 days of the culture. The HPDLCs growed fast and well after being passaged.2. HPDLCs had the ability to suppress osteogenic differentiation.3. After being stimulated with P.g LPS, the expression of osteoblast-related genes COL1, RUNX2, ALP and OCN m RNA significantly reduced(P<0.05).Alkaline phosphatase staining and alizarin red staining significantly weakened than osteogenic induction group.4. After being transfected with miRNA-146 a, alkaline phosphatase staining and alizarin red staining significantly enhanced than osteogenic induction group, and the expression of osteoblast-related genes COL1, RUNX2, ALP and OCN mRNA significantly increased and with statistically difference(P<0.05).5. The alkaline phosphatase staining and alizarin red staining were significantly enhanced in miRNA-146a/P.g LPS than in P.g LPS stimulation.Also, the expression of osteoblast-related genes COL1, RUNX2, ALP and OCN mRNA significantly increased in miRNA-146a/P.g LPS than in P.g LPS stimulation(P <0.05).Conclusion:1. P.g LPS inhibits the osteogenic differentiation of HPDLCs.2. miRNA-146 a promotes the osteogenic differentiation of HPDLCs.3. The miRNA- 146 a can reverse the osteogenic inhibitory effect of P.g LPS on HPDLCs, which, to some extent, confirmed that overexpression of miRNA-146 a is advantageous to periodontal tissue repair in inflammatory state.