[Lipopolysaccharide of Porphyromonas gingivalis promotes the autophagy of human gingival fibroblasts].

Objective To investigate the impact of lipopolysaccharide of Porphyromonas gingivalis (Pg-LPS) on the autophagy of human gingival fibroblasts (HGFs). Methods Firstly, HGFs was stimulated with 10 µg/mL Pg-LPS for 12 hours or 24 hours. Rapamycin was used as a positive control. The expression of LC3B was detected by Western blotting and the distribution of autophagosomes was observed by indirect immunofluorescence staining. At the same time, mitochondrial ROS (mtROS) was labeled by MitoSOX Red. The levels of mtROS and mitochondrial autophagy were measured in HGFs after treated with Pg-LPS. Then, T-butyl-4 (BHA), N-acetylcysteine (NAC) and coenzyme Q10 (CoQ10) were used separately to block the ROS and the expression of LC3B in Pg-LPS-treated HGFs was tested by Western blotting. Results After treatment with Pg-LPS, the ratio of LC3BII/LC3BI and the number of autophagic cells significantly increased, and the increase in the 24-hour treatment group was the more obvious than that in the 12-hour treatment group. The mtROS production and mitochondrial autophagy were significantly promoted after Pg-LPS treatment. In addition, CoQ10 effectively reduced Pg-LPS-induced autophagy of HGFs. Conclusion Pg-LPS can induce the autophagy of HGFs by raising mtROS production, and autophagy is involved in the degradation of damaged mitochondria to maintain cellular homeostasis.

[Establishment and identification of transiently expression system of bone marrow stromal cells modified by osteoprotegerin gene].

OBJECTIVE: In oder to treat periodontitis by using tissue engineering and gene engineering technology, the article established an transient expression system of bone marrow stromal cells (BMSC) modified by osteoprotegerin (OPG) gene and detected its expression using eukaryotic secreted expression pSecTag2/B-OPG plasmid. METHODS: By solation and culture of BMSC in vitro, the identified recombined plasmid was transiently transfected into BMSC by Lipofectamine 2000 and OPG expression in BMSC was determined by RT-PCR and Western blot in 6 weeks. RESULTS: The fragments of the recombinant plasmid digested with Hind III, EcoR I and BamH I and examined by 10 g/L agarose electrophoresis, were consistent with predicted size. The sequence of OPG was identical to the sequence provided by GeneBank [gi:33878056]. OPG transcribing in BMSC was confirmed by RT-PCR and OPG sustainable expressing in BMSC was detected by Western blot in 39 days. CONCLUSION: The transiently expression system of BMSC modified by OPG gene was successfully established.