Construction of recombinant plasmid with Porphyromonas gingivalis FimA deficiency / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To construct the recombinant plasmid pPHU281_A_Spec_B, which knock out Porphyrmonas gingivalis (Pg) FimA gene.</p><p><b>METHODS</b>Genomic DNA was extracted from PgATCC33277 which was cultured in anaerobic condition. The upstream and downstream gene of FimA was cloned from Pg genenomic DNA with specific restriction sites by polymerase chain reaction. Suicide vector pPHU281 was inserted by three fragments: upstream, downstream of FimA gene and spectinomycin resistance gene. The recombinant plasmid was confirmed by electrophoresis and sequenced after amplification in compentent cells DH-5α.</p><p><b>RESULTS</b>The gene sequence was identified by DNA sequencing analysis. The recombinant plasmid pPHU281_A_Spec_B was successfully constructed.</p><p><b>CONCLUSIONS</b>The recombinant plasmid pPHU281_A_Spec_B was constructed, which may be used for the constructon of FimA deficient Pg.</p>

Effect of longterm lower Porphyromonas gingivalis infection on the progression of atherosclerosis in apolipoprotein E-knocked out mice / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To assess the effect of longterm and lower oral inoculation with Porphyromonas gingivalis (Pg) on the progression of atherosclerosis in apolipoprotein E-knocked out (ApoE(-/-)) mice.</p><p><b>METHODS</b>Six-week-old male ApoE(-/-) mice were inoculated orally with 0.1 ml live Pg(10(13)/L) or bouillon culture-medium quintic per week for 15 consecutive weeks, altogether 75 times of inoculations. The lesion area of atherosclerosis in the aortic tree was measured by en face quantification by red oil O staining method. The atherosclerotic lesion was examined by histopathology. The levels of total cholesterol and triglycerides were compared.</p><p><b>RESULTS</b>At 22 weeks after inoculation, the mean atherosclerotic lesion area in inoculated mice was (98 363.68 ± 12 043.00) µm(2), which was significantly greater than that in noninoculated mice, which was (62 985.06 ± 7419.64) µm(2) (P = 0.035).</p><p><b>CONCLUSIONS</b>Longterm lower oral inoculation of Pg can accelerate the progression of atherosclerosis in apolipoprotein E-knocked out mice.</p>

Effect of Porphyromonas gingivalis on nitric oxide in cultured human umbilical vein endothelial cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To observe the effect of Porphyromonas gingivalis (Pg) on the production of nitric oxide (NO) in cultured human umbilical vein endothelial cells (HUVEC), and to investigate the pathway of damaging endothelial function by Pg.</p><p><b>METHODS</b>Pg ATCC33277 was cultured in anaerobic jar, and HUVEC was treated with various concentrations of Pg ATCC33277 at multiplicity of infection (MOI) of 1:10, 1:100 and 1:1000 for 4, 8, 12, 24 h respectively. The cells supernatants were collected and stored at -70 degrees C and NO concentration in the cells supernatants was measured by nitrate reductase assay.</p><p><b>RESULTS</b>Within 24 h, Pg at MOI of 1:10 and 1:100 stimulated the release of nitric oxide in cultured HUVEC. Within 12 h, Pg at an MOI of 1:1000 group increased NO production, and NO decreased at 24 h.</p><p><b>CONCLUSIONS</b>Pg has an effect on the production of NO. Low concentrations of Pg stimulated release of nitric oxide in endothelial cells but high concentrations can decrease the release of NO.</p>

Preliminary study on herpes simplex virus type 1 infection of human oral epithelial cell in vitro / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the way of herpes simplex virus type 1 (HSV-1) infecting human oral epithelial cell in vitro.</p><p><b>METHODS</b>Abundance of HSV-1 strains were used to infect human oral epithelial cells. The culture supernatant was collected to infect Vero cells again. Morphology of HSV-1 was identified by inverted microscope and transmission electron microscope. Nucleic acid of the virus was detected by PCR.</p><p><b>RESULTS</b>The infected human oral epithelial cells didn't display obvious cytopathic effect (CPE) while Vero cells infected by the culture supernatant showed typical CPE. The virus particles were observed in Vero cells. The nucleic acid of HSV-1 could be detected in infected human oral epithelial cells by PCR.</p><p><b>CONCLUSIONS</b>HSV-1 could successfully infect human oral epithelial cells.</p>

The expression and activity of alkaline phosphatase in human periodontal ligament cells with nanometer hydroxyapatite / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of nanometer hydroxyapatite on the proliferation and the osteogenetic differentiation of periodontal ligament cells (PDLC).</p><p><b>METHODS</b>Nano-hydroxyapatite powders were fabricated with sol-gel method. The fourth passage periodontal ligament cells were cultured with nanometer hydroxyapatite powder (nano-HA), dense hydroxyapatite powder (dense-HA) and only medium as control respectively. On the 5th, 8th day of culture, the osteogenetic differentiation of human periodontal ligament cells was evaluated though alkaline phosphatase (ALP) activity, ALP immunohistochemical stain and ALP positive flow cytometry.</p><p><b>RESULTS</b>There were significant differences among nano-HA group, dense-HA group and control group on the 5th and 8th day of culture. A majority of nano-HA group and dense-HA group cells sample showed positive ALP stain. But the ALP positive stain of nano-HA group cells sample was denser than that of dense-HA group. In FCM, the distribution of ALP positive cells cultured with nanoparticles were significantly more than that of other groups.</p><p><b>CONCLUSIONS</b>The nano-HA, as a calcium phosphate biomaterial, has ability to promote the activity of osteogenetic differentiation for periodontal ligament cells compared with dense-HA.</p>

The biological changes of NIH3T3 cells co-cultured with human bone morphogenetic protein-2 gene transfecting cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the ultrastructure and the alkaline phosphatase (ALP) activity changes of NIH3T3 cells incubated with secretive human bone morphogenetic protein-2 (hBMP-2) that is induced by gene transfection through transwell system.</p><p><b>METHODS</b>Eukaryotic expression vector (pcDNA3.1-B2) was transduced into NIH3T3 cells by Sofast, a positive compound transfection agent. The positive cell clones were selected with G418. The cytoplasmic and extracellular expression of BMP-2 in the NIH3T3 cells were determined by immunohistochemical and enzyme-linked immunosorbent assay (ELISA). NIH3T3 cells were co-cultured with hBMP-2 gene transfecting cells through transwell system, and the ultrastructure and ALP activity (the markers of osteogenetic differentiation) changes were observed.</p><p><b>RESULTS</b>There were cytoplasmic and extracellular expression of BMP-2 in transfecting NIH3T3 cells. The ultrastructure changes and the high expression of ALP suggested the osteogenetic differentiation tendency of NIH3T3 cells co-cultured with transfecting NIH3T3 cells.</p><p><b>CONCLUSIONS</b>Secretive BMP-2 that is induced by gene transfection could promote the osteogenetic differentiation of fibroblast cells.</p>

Effects of secretive bone morphogenetic protein 2 induced by gene transfection on the biological changes of NIH3T3 cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor beta superfamily, are powerful regulators of cartilage and bone formation. This study investigated the biological changes of NIH3T3 cells incubated with secretive BMP2 that was induced by gene transfection through transwell.</p><p><b>METHODS</b>Eukaryonic expression vector (pcDNA3.1-B2) was transfered into NIH3T3 cells with Sofast, a positive compound transfection agent. The positive cell clones were selected with G418. The cytoplasmic and extracellular expressions of BMP2 were determined by immunohistochemical stain and enzyme-linked immunosorbent assay. NIH3T3 cells were co-cultured with hBMP2 gene transfecting cells through transwell, and the ultrastructure, alkaline phosphatase activity and the expression of osteocalcin (the marker of osteogenetic differentiation) changes were observed.</p><p><b>RESULTS</b>There were cytoplasmic and extracellular expressions of BMP2 in transfecting NIH3T3 cells. The ultrastructural changes, the high activity of alkaline phosphatase and the positive stain of osteocalcin suggested the osteogenetic differentiation tendency of NIH3T3 cells co-cultured with transfecting NIH3T3 cells.</p><p><b>CONCLUSION</b>Secretive BMP2 that is induced by gene transfection could promote the osteogenetic differentiation of fibroblast cells.</p>