Effect of icariin on the expression of receptor activator nuclear factor-κB ligand and osteoprotegerin of human periodontal ligament cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of icariin on the expression of receptor activator nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) of human periodontal ligament cells (hPDLC) inhibited by sonicated extracts of Porphyromonas gingivalis (Pg).</p><p><b>METHODS</b>hPDLC were extracted from 14 premolars extracted from 11 to 18-year-old patients for orthodontic purpose and cultured. The 7th passages of hPDLC were divided into six groups, each group was stimulated with 0, 0.001, 0.01, 0.1, 1 mg/L icariin and 50 mg/L sonicated extracts for 48 h. Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression of RANKL and OPG.</p><p><b>RESULTS</b>Pg extracts group expressed more RANKL than the control group (3.60 ± 0.11 vs 1.08 ± 0.19), OPG expression decreased (0.32 ± 0.08 vs 1.01 ± 0.08), and RANKL/OPG ratio significantly increased (11.20 ± 0.13 vs 1.00 ± 0.10). Compared with control group, RANKL and OPG expression changed in groups stimulated with 0.001, 0.01, 0.1, 1 mg/L icariin. OPG expression was higher in 0.1 mg/L group than in the control (36.84 ± 0.05 vs 1.01 ± 0.08), RANKL/OPG ratio was significantly lower than the control group (0.04 ± 0.03 vs 1.00 ± 0.10) (P < 0.01).</p><p><b>CONCLUSIONS</b>The concentration of 0.1 mg/L icariin can inhibit hPDLC expression of RANKL in sonicated extracts of Pg and promote OPG expression.</p>

Effect of sonicated extracts of Porphyromonas gingivalis on receptor activator of NF-κB ligand and osteoprotegerin expression in periodontal ligament cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To evaluate the effects of sonicated extracts of Porphyromonas gingivalis (Pg) on receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) expression in human periodontal ligament cells (HPDLC) and the effect of Pg on bone resorption in periodontitis.</p><p><b>METHODS</b>HPDLC were exposed to 25, 50 mg/L sonicated extracts of Pg for 6 h, HPDLC without treatment served as control. The expression of RANKL-OPG mRNA and protein were examined by real time polymerase chain reaction and Western blotting. OPG protein in the supernatant was examined by enzyme linked immunosorbent assay (ELISA). The data were statistically analyzed by SPSS 13.0 and one-way analysis of variance (ANOVA).</p><p><b>RESULTS</b>When HPDLC were exposed to sonicated extracts of Pg, the expression of RANKL mRNA and protein in 25 mg/L and 50 mg/L groups were higher than that of control group (P < 0.05), the expression of OPG mRNA in 50 mg/L group (0.087 ± 0.021) was lower than that of control group (0.240 ± 0.019) (P < 0.05), and OPG protein in 25 mg/L and 50 mg/L groups (0.813 ± 0.007, 0.398 ± 0.009) was lower than that of control group (1.131 ± 0.005) (P < 0.01). OPG protein expression in the supernatant was not significantly different between experimental group and control group.</p><p><b>CONCLUSIONS</b>Sonicated extracts of Pg exposed to HPDLC can up-regulate RANKL expression, down-regulate OPG expression and influence bone metabolism.</p>

Preliminary study on anti-periodontitis immunization with DNA vaccine / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To observe the protection against periodontal bone loss in the Sprague-Dawley (SD) rats periodontitis model, with the recombined plasmid pcDNA3.1+/kgpcd as DNA gene vaccine.</p><p><b>METHODS</b>PcDNA3.1+/kgpcd was delivered into rats by submandibular gland-targeted injection. The anti-KGPcd sIgA in saliva was measured by indirect ELISA method. Immunohistochemistry staining was used to assess the protection in the animal model.</p><p><b>RESULTS</b>The level of specific anti-KGPcd sIgA in saliva of the experimental group was significantly higher than that of control group. HE staining showed that immunization with recombined plasmid pcDNA3.1+/kgpcd could protect or minimize tissue destruction caused by subsequent P. gingivalis challenge in the rat model.</p><p><b>CONCLUSIONS</b>The results indicate that pcDNA3.1+/kgpcd was a good candidate for anti-periodontitis gene vaccine and could provide protection against Porphyromonas gingivalis-caused periodontitis in rat lesion model.</p>

Construction of eukaryotic expression vector for KGPcd gene from Porphyromonas gingivalis and expression in mammalian cells / 华西口腔医学杂志

<p><b>OBJECTIVE</b>This study aimed at constructing secretory eukaryotic expression vector of KGPcd gene encoding whole amino acid residues of mature KGPcd from Porphyromonas gingivalis and investigating the transcription and expression of recombined plasmid VR1020/KGPcd in mammalian cells.</p><p><b>METHODS</b>Eukaryotic expression plasmid VR1020/KCPcd was constructed by using molecular cloning methods. Then, the VR1020/KGPcd was transfected into mammalian cell COS7 with Lipofectamine 2000 according to the manufacturer's instruction. The transcription of VR1020/KGPcd was assayed by reverse transcription polymerase chain reaction (RT-PCR). The expression product of VR1020/KGPcd was analyzed by using indirect immunofluorescence. The protein secretion in cultural medium was detected by ELISA method.</p><p><b>RESULTS</b>It proved that the VR1020/KGPcd could be transcribed and translated into transfected COS7 cells. The expressed targeted protein could be secreted into cultural supernatant and could be detected by ELISA.</p><p><b>CONCLUSION</b>The eukaryotic expression plasmid of VR1020/KGPcd was constructed successfully and its product can be expressed in mammalian cells. The results indicated that the recombinant plasmid has antigenicity and may be acted as candidate gene vaccine. This laid a basis for its use as gene vaccine candidates in the development of anti-periodontitis and paved the way for further study.</p>

CLONING AND SEQUENCING ANALYSIS OF GINGIPAIN K OF PORPHYROMONAS GINGIVALIS / 微生物学通报

The desired DNA product of KGPcd and KGP-hag was obtained from the total DNA of Porphyromonas gingivalis by PCR with two pairs of gene specific primers. The segment of KGPcd and KGP-hag (about 1.5kb and 1.6kb) was inserted into pGEM-T easy Vector. The double-stranded DNA of the postitive clone was analyzed by restriction endonuclease mapping and DNA sequenceing. The sequences of KGPcd and KGP-hag were consistent with those of the references appeared. The proteins of KGPcd and KGP-hag will be obtained for further study.