Effect of 15-Deoxy-Δ-prostaglandin JNanocapsules on Inflammation and Bone Regeneration in a Rat Bone Defect Model / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), one of the major metabolites from prostaglandin D2 in arachidonic acid metabolic pathway, has potential anti-inflammatory properties. The objective of this study was to explore the effects of 15d-PGJ2-loaded poly(D,L-lactide-co-glycolide) nanocapsules (15d-PGJ2-NC) on inflammatory responses and bone regeneration in local bone defect.</p><p><b>METHODS</b>The study was conducted on 96 Wistar rats from June 2014 to March 2016. Saline, unloaded nanoparticles, free 15d-PGJ2or 15d-PGJ2-NC, were delivered through a collagen vehicle inside surgically created transcortical defects in rat femurs. Interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) levels in the surrounding soft tissue were analyzed by Western blot and in the defect by quantitative real-time polymerase chain reaction over 14 days. Simultaneously, bone morphogenetic protein-6 (BMP-6) and platelet-derived growth factor-B (PDGF-B) messenger RNA (mRNA) in the defect were examined. New bone formation and EphrinB2 and osteoprotegerin (OPG) protein expression in the cortical defect were observed by Masson's Trichrome staining and immunohistochemistry over 28 days. Data were analyzed by one-way analysis of variance. Least-significant difference and Dunnett's T3 methods were used with a bilateral P< 0.05.</p><p><b>RESULTS</b>Application of l5d-PGJ2-NC (100 μg/ml) in the local bone defect significantly decreased IL-6, IL-1β, and TNF-α mRNA and protein, compared with saline-treated controls (P < 0.05). l5d-PGJ2-NC upregulated BMP-6 and PDGF-B mRNA (P < 0.05). New bone formation was observed in the cortical defect in l5d-PGJ2-NC-treated animals from 7th day onward (P < 0.001). Expression of EphrinB2 and OPG presented early on day 3 and persisted through day 28 in 15d-PGJ2-NC group (P < 0.05).</p><p><b>CONCLUSION</b>Stable l5d-PGJ2-NC complexes were prepared that could attenuate IL-6, IL-1β, and TNF-α expression, while increasing new bone formation and growth factors related to bone regeneration.</p>

Effect of lipopolysaccharide of Porphyromonas gingivalis on prostaglandin E2 biosynthetic pathway in human monocytic cell strain THP-1 / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of lipopolysaccharide of Porphyromonas gingivalis (Pg-LPS) on the bio-thythetic pathway of prostaglandin E2 (PGE2) and its difference from lipopolysaccharide of Escherichia coli (Ec-LPS).</p><p><b>METHODS</b>Purified Pg-LPS and Ec-LPS were used to stimulate a human monocytic cell strain THP-1. PGE2 concentration was determined by an enzyme immunoassay kit. The release of tritium labeled arachidonic acid (AA) was detected by a liquid scintillation counter. Reverse transcription polymerase chain reaction and western blot were used to analyse the expression of cytosolic phospholipase A2 (cPLA2) enzyme, cyclooxygenase-2 (COX-2), and microsomal prostaglandin E synthase-1 (mPGES-1).</p><p><b>RESULTS</b>The effect of Pg-LPS on induction of PGE2 and release of AA was significantly weaker than that of Ec-LPS (P < 0.05).Increased secretion of PGE2 was observed after stimulation with Pg-LPS for 6 h, which peak at 24 h at (221.40 +/- 29.46) ng/L; or with Ec-LPS for 1-48 h, at (161.80 +/- 17.31) approximately (379.80 +/- 37.35) ng/L. The highest levels of COX-2 and mPGES-1 were shown after 16 h treatment by Pg-LPS, or after 8 h and 16 h by Ec-LPS respectively.cPLA2 inhibitor AACOCF3 could lower the level of LPS-induced release of AA, while it did not influence the production of PGE2. COX-2 inhibitor NS-398 could remarkably reduce the concentration of PGE2.</p><p><b>CONCLUSIONS</b>Pg-LPS showed delayed and weaker effect on PGE2 biosynthetic pathway than Ec-LPS. Pg-LPS-induced PGE2 synthesis was mainly due to enhanced expression of COX-2 and mPGES-1, whereas cPLA2 played an insignificant role.</p>

Correlation between infection of different glycoprotein B genotypes of human cytomegalovirus and human chronic periodontitis / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the correlation between infection of different human cytomegalovirus (HCMV) glycoprotein B (gB) genotypes and human chronic periodontitis.</p><p><b>METHODS</b>A nested-polymerase chain reaction (nPCR) was employed to detect HCMV gB gene in the subgingival plaque samples from 65 chronic periodontitis patients and in the gingival crevicular fluid samples from 24 periodontally healthy control. The amplification fragments of gB gene were further genotyped by restriction fragment length polymorphism (RFLP). The correlation among infection with the different HCMV genotypes and the severity of periodontal lesion were evaluated.</p><p><b>RESULTS</b>In terms of teeth examined, the prevalence of HCMV (59.23%, 154/260) in the chronic periodontitis lesions was significantly higher than that of HCMV (32.29%, 31/96) in the periodontally healthy control (P < 0.01). Of the HCMV DNA positive samples from the chronic periodontitis lesions, 11.7% (18/154) was genotyped as gB I, 80.5% (124/154) as gB II, and 7.8% (12/154) as gB I and gB II co-infection, and of the HCMV DNA positive samples from the periodontal healthy control, 45.2% (14/31) was genotyped as gB I, 38.7% (12/31) as gB II, and 16.1% (5/31) as gB I and gB II co-infection. The gB II genotype was more dominant among the chronic periodontitis lesions compared with that among the periodontally healthy control (P < 0.01). In chronic periodontitis, no statistical significance could be found between infection of different HCMV gB genotypes and the different clinical parameters of CAL, PD and GI (P > 0.05).</p><p><b>CONCLUSIONS</b>Subgingival infection with HCMV is closely associated with chronic periodontitis. Infection of HCMV may not correlate directly with severity of periodontitis. However, gB II may be the dominant genotype of HCMV, which is associated with the chronic periodontitis.</p>

Infection frequency of Epstein-Barr virus in subgingival samples from patients with different periodontal status and its correlation with clinical parameters / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To detect the infection frequencies of different genotypes of Epstein-Barr virus (EBV) in subgingival samples from chronic periodontitis (CP) patients, and to discuss the correlation between infection with EBV and clinical parameters.</p><p><b>METHODS</b>Nested-PCR assay was used to detect EBV-1 and EBV-2 in subgingival samples from 65 CP patients, 65 gingivitis patients and 24 periodontally healthy individuals. The amplicons were further identified by restriction fragment length polymorphism analysis (RFLP) with endonucleases Afa I and Stu I. Clinical parameters mainly included bleeding on probing (BOP), probing depth (PD), attachment loss (AL) in six sites of the dentition.</p><p><b>RESULTS</b>In CP patients, gingivitis and periodontally healthy individuals, the infection frequencies were 47.7%, 24.6% and 16.7% for EBV-1, and 15.4%, 7.7% and 0% for EBV-2, respectively. In 2 out of the 65 CP patients co-infection of EBV-1 and EBV-2 was found. The positive rate of EBV-1 in chronic periodontitis patients was higher than that in gingivitis patients (P=0.01) and periodontally healthy individuals (P=0.01). But no significant difference was shown in EBV-1 frequency between gingivitis patients and healthy individuals (P>0.05) or in EBV-2 frequency among the three groups (P>0.05). In CP patients, higher mean BOP value was found in EBV-1 or EBV-2 positive patients than that in EBV negative ones (P<0.01), but with no statistical difference in the mean PD or AL value between EBV positive and negative patients (P>0.05). After initial periodontal treatment, 12 out of the 21 EBV-1 positive CP patients did not show detectable EBV-1 in subgingival samples.</p><p><b>CONCLUSION</b>nPCR plus RFLP analysis is a sensitive, specific and stable method to detect EBV-1 and EBV-2 in subgingival samples. Subgingival infection with EBV-1 is closely associated with chronic periodontitis. Infection of EBV in subgingival samples was correlated with BOP.</p>

Association between coinfection of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Treponema denticola and periodontal tissue destruction in chronic periodontitis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The association between the infection of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Treponema denticola in chronic periodontitis (CP) and the severity of periodontal disease remains to be elucidated. The aim of this study was to investigate the subgingival infection frequencies of three periodontopathic bacteria in Chinese CP patients and to evaluate the correlations between infection by these bacteria and periodontal destruction.</p><p><b>METHODS</b>A multiple PCR assay using primers derived from 16SrDNA genes of P. gingivalis, A. actinomycetemcomitans and T. denticola was established to measure simultaneously the presence of the three microbes in 162 subgingival samples from 81 Chinese CP patients.</p><p><b>RESULTS</b>The positive rates of P. gingivalis, A. actinomycetemcomitans and T. denticola in the subgingival samples were 84.6%, 83.3% and 88.3%, respectively. Of the subgingival samples, 68% revealed the coinfection of all the three microbes. The infection rates with P. gingivalis, A. actinomycetemcomitans or T. denticola alone was 5.9% (1/17), 17.6% (3/17) and 76.5% (13/17), respectively. A close association was present between the A. actinomycetemcomitans infection and gingival index (GI) (P < 0.01), but not between P. gingivalis or T. denticola infection and GI (P > 0.05). P. gingivalis and A. actinomycetemcomitans were more frequently detectable in middle and deep pockets than in shallow ones (P < 0.01), while T. denticola was found remarkably often in deep pockets (P < 0.05). The coinfection rate of the three microbes was significantly higher in sites with severe periodontitis than in those with mild periodontitis (P < 0.01).</p><p><b>CONCLUSIONS</b>The multiple PCR established in this study can be used as a sensitive and specific method to simultaneously detect all three microbes in subgingival samples. A. actinomycetemcomitans infection may be associated with CP and play an important role in the periodontal tissue destruction. The coinfection of P. gingivalis, A. actinomycetemcomitans and T. denticola can cause more serious periodontal destruction than infection of any one or two of the three microbes.</p>

Effects of IGF-II on promoting proliferation and regulating nitric oxide synthase gene expression in mouse osteoblast-like cell / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To investigate the effects of insulin-like growth factor II (IGF-II) on promoting cell proliferation, regulating levels of cellular nitric oxide (NO) and mRNA transcriptions of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) in mouse osteoblast-like cells.</p><p><b>METHODS</b>Mouse osteoblastic cell line MC3T3-E1 was selected as the effective cell of IGF-II. After the cells were treated with IGF-II at different concentrations for different time duration, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay was used to examine cell proliferation, and nitrate reductase method was applied to detect NO concentrations in cell culture supernatants and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to determine transcription levels of cellular iNOS and eNOS mRNAs.</p><p><b>RESULTS</b>After the MC3T3-E1 cells were treated with IGF-II at concentration of 1 ng/ml for 72 h, 10 and 100 ng/ml for 24, 48 and 72 h respectively, all the MTT values increased (P<0.05 or P<0.01) with obvious dosage-time dependent pattern. NO levels of the MC3T3-E1 cells treated with 100 ng/ml IGF-II for 48 h, and with 1, 10 and 100 ng/ml IGF-II for 72 h were remarkably lower than that of the normal control, respectively (P<0.05 or P<0.01). After the cells were treated with 100 ng/ml IGF-II for 48 h cellular iNOS mRNA levels were significantly decreased (P<0.01). But the levels of eNOS mRNA in the cells treated with each of the used IGF-II dosages for different time duration did not show any differences compared with the normal control (P>0.05).</p><p><b>CONCLUSION</b>IGF-II at different concentrations could promote proliferation of mouse MC3T3-E1 cell. This cell proliferation promotion was associated with the low NO levels maintained by IGF-II. Higher concentration of IGF-II could down-regulate iNOS gene expression at the level of transcription but not affect transcription of eNOS mRNA, which might be one of the mechanisms for IGF-II maintenance of the low NO levels in MC3T3-E1 cells.</p>

Cloning and expression of Porphyromonas gingivalis pgmA gene encoding outer membrane protein associated with fimbria / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To clone pgmA gene of Porphyromonas gingivalis, to construct the expression vector of the gene and to identify immunity of the fusion protein.</p><p><b>METHODS</b>The pgmA genes from ATCC 33277 and 47A-1 strains of P.gingivalis were amplified by high fidelity PCR. The nucleotide of the target DNA amplification fragments were sequenced after T-A cloning. The pET32a expression vectors inserted with pgmA gene fragments were constructed. PgmA fusion protein was expressed in E.coli strain BL21DE3 induced by IPTG with different dosages. Western blot test by using rabbit antiserum against the fusion protein was applied to determine immunity of the fusion protein. ELISA was applied to determine the immunoreaction of antibody against PgmA fusion protein and 65 strains of P.gingivalis isolates.</p><p><b>RESULT</b>The nucleotide sequence homology of the cloned pgmA gene fragments from ATCC 33277 and 47A-1 strains was 100%. In comparison with the reported corresponding sequences, the homologies of the nucleotide sequences of the cloned pgmA gene fragments were 98.98%, while the homologies of their putative amino acid sequences were 99.18%. The expression output of PgmA fusion protein in pET32a-pgmA-BL21DE3 system was approximately 50% of the total bacterial proteins. PgmA fusion protein was able to induce rabbit to produce specific antibody that could combine with PgmA protein. 92.3% of P. gingivalis isolates (60/65) were able to react with the antibody against PgmA fusion protein.</p><p><b>CONCLUSION</b>An expression system of P.gingivalis pgmA gene with high efficiency was established successfully. The expressed PgmA fusion protein possesse satisfied immunogenicity and immunoreactivity,which can be used as a candidate antigen in detection of P.gingivalis and possible development of corresponding vaccine.</p>

Effects of insulin-like growth factor II on regulating nitric oxide synthase gene expression in mouse osteoblast-like MC3T3-E1 cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To study the effects of insulin-like growth factor II (IGF-II) on regulating the levels of nitric oxide (NO) and the mRNA transcriptions of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) in mouse osteoblast-like cells.</p><p><b>METHODS</b>Mouse osteoblastic cell line MC3T3-E1 was selected as the effective cell of IGF-II. After the cells were treated with IGF-II at different concentrations for different intervals of time, MTT colorimetry was used for examining the cell proliferation. Nitrate reductase method was applied for detecting the NO concentrations in cell culture supernatants and RT-PCR employed for determining the levels of cellular iNOS and eNOS mRNAs.</p><p><b>RESULTS</b>After the MC3T3-E1 cells were treated with IGF-II at the dosages of 1 microg/L for 72 h, 10 and 100 microg/L for 24, 48 and 72 h respectively, all the MTT values increased markedly (P < 0.05 or P < 0.01). After the cells were treated for 48 and 72 h at the dosage of 100 microg/L IGF-II respectively, the levels of NO in the supernatants of cell cultures and cellular iNOS mRNA decreased significantly (P < 0.01). However, the levels of eNOS mRNA in the cells treated with any of the IGF-II dosages for the different times were stable (P > 0.05).</p><p><b>CONCLUSIONS</b>IGF-II at the dosages of 1 approximately 100 microg/L showed the effects on promoting proliferation, which as probably due to the maintenance of low NO levels. Inducible NOS gene expression at the level of transcription was down regulated in the MC3T3-E1 cell treated with higher dosage of IGF-II (100 microg/L) but eNOS mRNA was not, which might be one of the mechanisms for the maintenance of low NO levels.</p>