[show abstract][hide abstract] ABSTRACT: Objective: To investigate the periodontal status and associated risk factors among women of childbearing age to increase the awareness of oral health. Methods: The study was conducted on childbearing age women in Cixi, a city in Zhejiang Province in the southeast of China. A total of 754 women participated in periodontal examination while receiving prenatal care. Data of the women were collected from the Cixi Family Planning Commission and during an interview. Clinical periodontal indices, such as bleeding on probing (BOP), probing depth (PD), and clinical attachment level (CAL) were measured during the examination. Statistical analysis on subject-based data was performed. Results: The prevalence of periodontal disease among childbearing age women in Cixi was high (84.7%). A significant association was found between the disease and educational level, pregnancy, taking oral contraceptives, stress, alcohol consumption, overweight, dental visit, and teeth brushing (P<0.05). Women who suffered periodontal disease showed deep PD, obvious BOP, and clinical attachment loss. Among this population, pregnancy was closely associated with higher BOP percentage; teeth brushing no more than once per day or brushing for less than 1 min (P<0.001) after adjusting for age and stress. Conclusions: The periodontal status of childbearing age women in Cixi needs to be improved urgently. Attention towards the periodontal health should be warranted, especially for those in special statuses and with poor awareness.
Journal of Zhejiang University SCIENCE B 03/2013; 14(3):231-9. · 1.11 Impact Factor
[show abstract][hide abstract] ABSTRACT: Pregnancy gingivitis is an acute form of gingivitis that affects pregnant women, with a prevalence of 30%, possibly ranging up to 100%. Sometimes, pregnancy gingivitis shows a tendency toward a localized hyperplasia called gingival pyogenic granuloma. Pregnancy tumor is a benign gingival hyperplasia with the gingiva as the most commonly involved site, but rarely it involves almost the entire gingiva. A 22-year-old woman was referred to our clinic with a chief complaint of gingival swelling that had lasted for 2 days. The lesions progressed rapidly and extensively, and almost all the gingiva was involved a week later. Generalized erythema, edema, hyperplasia, a hemorrhagic tendency, and several typical hemangiomatous masses were noted. Pregnancy was denied by the patient at the first and second visits, but was confirmed 2 weeks after the primary visit. The patient was given oral hygiene instructions. She recovered well, and the mass gradually regressed and had disappeared completely at the end of 12 weeks of pregnancy, without recurrence. The gingival lesions were finally diagnosed as multiple gingival pregnancy tumors. The patient delivered a healthy infant. An extensive and rapid growth of gingival pregnancy tumors during the early first month of pregnancy is a rare occurrence that is not familiar to dentists, gynecologists, and obstetricians. Those practitioners engaged in oral medicine and periodontology, primary care obstetrics, and gynecology should be aware of such gingival lesions to avoid misdiagnosis and overtreatment.
[show abstract][hide abstract] ABSTRACT: To investigate the effect of osteoclast bone resorption supernatants on the osteogenic activity of mouse MC3T3-E1 cell line.
Mouse RAW264.7 cell line was induced to osteoclast which was identified with tartrate resistant acid phosphatase (TRAP) staining and osteoclast specific gene detection. The differentiated RAW264.7 osteoclast was co-cultured with bovine milling bone specimen followed by toluidine blue staining. Then mouse MC3T3-E1 cell was cultured with supernatant from the osteoclast bone absorbent model. Methyl thiazolyl tetrazolium (MTT) method, alizarin red S staining, enzyme-linked immunosorbent assay detection of osteocalcin, and reverse transcriptase polymerase chain reaction detection were adopted to investigate the proliferation, calcification and osteogenic activity of MC3T3-E1 cells.
TRAP staining, osteoclast specific gene detection and toluidine blue staining all indicated that RAW264.7 cell could be differentiated into functioning osteoclast. The supernatant from the osteoclast bone absorbent model could inhibit the proliferation of MC3T3-E1 cells, with the A value between 0.062 ± 0.004 and 0.405 ± 0.033 (P < 0.05). It could also increase the formation of calcification nods, promote the osteocalcin level which peaked with the tenth day's supernatant at a level of (2.965 ± 0.047) µg/L, as well as enhance the transcription of the alkaline phosphatase and Runt related transcription factor 2 gene.
RAW264.7 osteoclast bone absorbent supernatant might influence the osteogenic activity of osteoblast-like cell by inhibiting proliferation, promoting differentiation and calcification.
Zhonghua kou qiang yi xue za zhi = Zhonghua kouqiang yixue zazhi = Chinese journal of stomatology 01/2012; 47(1):32-7.
[show abstract][hide abstract] ABSTRACT: To investigate if Drynariae rhizoma (DR) and its main ingredient Naringin could reduce alveolar bone loss by stimulating the proliferation and differentiation of osteoblasts.
The effect of DR water (DRWE), ethanolic extract (DREE), and Naringin on MC3T3-E1 cells was evaluated respectively by MTT method and by measuring the activity of alkaline phosphatase (ALP activity) as well as the level of osteocalcin in medium. Bone mineral density (BMD) detection, osteoclast counting by tartrate resistant acid phosphatase staining, and histopathological analysis were performed in an induced rat model of alveolar bone resorption after gastric perfusion with DR extracts or Naringin.
DRWE and Naringin effectively increased the proliferation of MC3T3-E1 cells, whilst DREE and Naringin enhanced the differentiation of osteoblastic cells. The in vivo study indicated an elevated BMD value in the tooth-periodontal tissues from DRWE, DREE and Naringin treated groups after 10, 20 and 30 days of perfusion (P<0.05). In DRWE treated group, the number of osteoclasts at days 10, 20 and 30 decreased remarkably as compared to the corresponding negative controls (P<0.05), and no osteoclast could be found at day 30. New non-calcified bone-like matrix attached by osteoblasts at the root furcation was also shown.
DR could be a supplementary medicine for periodontal therapy as it could reduce bone resorption in rat model of alveolar bone resorption and exert osteogenic effect on osteoblasts.
Archives of oral biology 07/2011; 56(12):1655-62. · 1.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: To evaluate the effects of periodontal intervention on inflammatory cytokines, adiponectin, insulin resistance (IR), and metabolic control and to investigate the relationship between type 2 diabetes mellitus (T2DM) and moderately poor glycemic control and chronic periodontitis.
A total of 190 moderately poorly controlled (HbA1c between 7.5% and 9.5%) T2DM patients with periodontitis were randomly divided into two groups according to whether they underwent periodontal intervention: T2DM-NT and T2DM-T group. The levels of serum adiponectin, C-reactive protein (CRP), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), lipid profile, glucose, insulin, homeostasis model of assessment-insulin resistance (HOMA-IR) and homeostasis model assessment of β-cell function (HOMA-β) were measured at baseline and after 3 months.
The levels of clinical periodontal variables, the probing depth, attachment loss, bleeding index, and plaque index were improved significantly in T2DM-T group after 3 months compared to T2DM-NT group (all p<0.01). After 3 months, the serum levels of hsCRP, TNF-α, IL-6, fasting plasma glucose (FPG), glycosylated hemoglobin (HbA1c), fasting insulin (FINS) and HOMA-IR index decreased, and adiponectin was significantly increased in T2DM-T group compared to those in the T2DM-NT group (p<0.05 or p<0.01).
Periodontal intervention can improve glycemic control, lipid profile and IR, reduce serum inflammatory cytokine levels and increase serum adiponectin levels in moderately poorly controlled T2DM patients.
Internal Medicine 01/2011; 50(15):1569-74. · 0.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: To determine serum adiponectin, C-reactive protein (CRP), TNF-α and IL-6 levels in impaired glucose tolerance (IGT) and type 2 diabetes mellitus (T2DM) patients with periodontitis before and after periodontal intervention, and to investigate the relationship between T2DM and periodontitis.
A total of 50 IGT and 106 T2DM patients with periodontitis were enrolled. The T2DM patients were divided into two groups: T2DM without macrovascular disease (DM1) group and T2DM with macrovascular disease (DM2) group. Each group was randomly divided into two subgroups according to whether they performed periodontal intervention. The normal control group (NC group) consisted of 30 healthy adults. The serum adiponectin, CRP, TNF-α and IL-6 levels were measured at baseline and 3 months after periodontal intervention.
The serum adiponectin levels at baseline had decreased tendency with significant difference between each two groups, while CRP, TNF-α, and IL-6 levels had increased tendency with significant difference between each two groups among NC, IGT, DM1 and DM2 groups (all P<0.01). At 3 months after periodontal intervention, the serum adiponectin levels were increased than those without periodontal intervention (all P<0.01), while CRP, IL-6 and TNF-α significantly decreased (all P<0.05) in both IGT and DM1 groups. In DM2 group, only CRP levels at 3 months after periodontal intervention were significantly decreased (P<0.05). Moreover, the HbAlc levels in T2DM patients were improved at 3 months after periodontal invention (P<0.01).
Periodontal intervention is helpful for glucose control, which may be associated with increased serum adiponectin levels and decreased inflammatory cytokine levels.
Archives of oral biology 10/2010; 55(12):970-4. · 1.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: To determine the effect of Porphyromonas gingivalis lipopolysaccharide (LPS) on induced secretion of inflammatory cytokines by different cell lines.
LPS of P. gingivalis strain ATCC33277 (Pg-LPS) was extracted with phenol-water method, and identified by Limulus test and infrared spectrum analysis. KB, HGF-1 and THP-1 cells were treated with Pg-LPS of different concentrations and time duration, a commercial LPS of E.coli strain O111:B4 (E-LPS) was used as the control. TNF-alpha, IL-1beta, IL-6 and IL-8 levels in culture supernatants were measured by quantitative ELISA.
The minimal dosages of both Pg-LPS and E-LPS to solidify Limulus agents were 15 ng/ml and their infrared spectrums were similar. With the treatment of Pg-LPS or E-LPS, the TNF-alpha and IL-1beta levels secreted by HGF-1 cells were remarkably increased with a single perk (P<0.01) while a continuous enhancement of secretion by THP-1 cells was observed (P<0.01). Either Pg-LPS or E-LPS stimulated HGF-1 or THP-1 cells to continuously increase the secretion of IL-6 (P<0.01). Both Pg-LPS and E-LPS induced IL-8 secretion by THP-1 cells (P<0.01), but only Pg-LPS showed the similar effect on HGF-1 cells (P<0.01). Neither Pg-LPS nor E-LPS induced KB cells to secrete inflammatory cytokines.
Pg-LPS can promote target cells to increase their secretion of inflammatory cytokines. KB cells can not be used as the target cell to determine inflammation-causing effect of LPS.
Zhejiang da xue xue bao. Yi xue ban = Journal of Zhejiang University. Medical sciences 11/2008; 37(6):622-8.
[show abstract][hide abstract] ABSTRACT: Accumulating evidence indicates that herpesviruses may be putative pathogens in various types of periodontal diseases. The present study was performed to examine infections with different genotypes of human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) in subgingival samples from a Chinese population and to analyze the correlation with periodontal status. A nested PCR assay was used to identify the presence of HCMV, EBV type 1 (EBV-1), and EBV-2; and the amplicons were further analyzed by restriction fragment length polymorphism analysis. HCMV was detected in 79.0% of 143 chronic periodontitis (CP) patients, 78.5% of 65 gingivitis patients, and 76.3% of 76 periodontally healthy individuals, while EBV was found in 63.6%, 32.3%, and 30.3% of the three groups of subjects, respectively. The HCMV-positive PCR products from all the samples were identified as corresponding to gB genotype I (gB-I) or gB-II. HCMV gB-II (62.9%), EBV-1 (43.4%), and EBV-2 (18.2%) were associated with CP at higher frequencies (P < 0.05), whereas HCMV gB-I was more often observed in gingivitis patients (40.0%) and healthy individuals (40.8%) (P < 0.05). Furthermore, a higher rate of coinfection with HCMV and EBV was shown in CP patients (52.4%), especially dual infections with HCMV gB-II and EBV-1 (30.8%) or HCMV gB-II and EBV-2 (12.6%), compared with the rates of single infections with HCMV or EBV (P < 0.05). Infection with HCMV gB-II, EBV-1, or EBV-2 was correlated with higher rates of bleeding on probing (P < 0.05). In patients infected with HCMV gB-II or both HCMV and EBV, including HCMV gB-II and EBV-1, a deeper probing depth or more serious attachment loss was found (P < 0.05). These findings clearly indicate that HCMV gB-II is the dominant genotype detected in subgingival samples in CP. HCMV gB-II infection and HCMV gB-II coinfection with EBV-1 are closely associated with periodontal tissue inflammation and destruction.
Journal of Clinical Microbiology 11/2007; 45(11):3665-70. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: To investigate the effect of the collagenase gene (prtC) product of Porphyromonas gingivalis on inducing host cells to secrete inflammatory cytokines, and to discuss the correlation between the PrtC level in subgingival plaque samples and clinical parameters.
A prokaryotic expression system pET32a-prtC-Escheria coli BL21DE3 was constructed. Antigenicity and immunoreactivity of the recombinant PrtC protein (rPrtC) was identified by Western blotting. ELISA was applied to detect interleukin (IL)-1alpha, IL-8, and TNF-alpha levels in supernatants from rPrtC-induced human umbilical vein endothelial cells (HUVEC) originated ECV304 cells. Clinical parameters recorded at baseline and after treatment included bleeding on probing (BOP), probing depth (PD), and attachment loss (AL). ELISA was established to measure the PrtC level in 196 subgingival plaque samples from 49 patients with chronic periodontitis.
After coincubation with 1 microg/mL rPrtC for 24 h and with 5 or 10 microg/mL rPrtC for 12 h, the levels of IL-1 alpha, IL-8, and TNF-alpha secreted by the ECV304 cells increased significantly (P<0.05). The PrtC level in the BOP-positive or the > or =5 mm AL or > or = 6 mm PD sites was higher than that in the BOP-negative or the < or =2 mm AL or < or =6 mm PD sites (P<0.05), respectively. Compared with baseline, the PrtC levels in different AL sites or in the < or =6 mm PD pockets decreased remarkably after treatment (P<0.01), but in the BOP-positive or in the > 6 mm PD sites, the PrtC levels changed insignificantly (P>0.05).
rPrtC is able to directly induce host cells to synthesize and secrete IL-1 alpha, IL-8, and TNF-alpha. The PrtC level in subgingival samples is correlated with BOP, AL, and PD.
[show abstract][hide abstract] ABSTRACT: The aim of this study was to investigate subgingival infection frequencies of Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans strains with genetic variation in Chinese chronic periodontitis (CP) patients and to evaluate its correlation with clinical parameters.
Two multiplex polymerase chain reaction (PCR) assays were developed to detect the 16SrDNA, collagenase (prtC) and fimbria (fimA) genes of P. gingivalis and the 16SrDNA, leukotoxin (lktA) and fimbria-associated protein (fap) genes of A. actinomycetemcomitans in 60 sulcus samples from 30 periodontal healthy subjects and in 122 subgingival plaque samples from 61 patients with CP. The PCR products were further T-A cloned and sent for nucleotide sequence analysis.
The 16SrDNA, prtC and fimA genes of P. gingivalis were detected in 92.6%, 85.2% and 80.3% of the subgingival plaque samples respectively, while the 16SrDNA, lktA and fap genes of A. actinomycetemcomitans were in 84.4%, 75.4% and 50.0% respectively. Nucleotide sequence analysis showed 98.62%~100% homology of the PCR products in these genes with the reported sequences. P. gingivalis strains with prtC+/fimA+ and A. actinomycetemcomitans with lktA+ were predominant in deep pockets (>6 mm) or in sites with attachment loss > or =5 mm than in shallow pockets (3~4 mm) or in sites with attachment loss < or =2 mm (P<0.05). P. gingivalis strains with prtC+/fimA+ also showed higher frequency in gingival index (GI)=3 than in GI=1 group (P<0.05).
Infection of P. gingivalis with prtC+/fimA+ and A. actinomycetemcomitans with lktA+ correlates with periodontal destruction of CP in Chinese. Nonetheless P. gingivalis fimA, prtC genes and A. actinomycetemcomitans lktA gene are closely associated with periodontal destruction, while A. actinomycetemcomitans fap gene is not.
Journal of Zhejiang University SCIENCE B 02/2007; 8(2):121-31. · 1.11 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
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.
Journal of Zhejiang University SCIENCE B 12/2006; 7(11):876-83. · 1.11 Impact Factor
[show abstract][hide abstract] ABSTRACT: To investigate the correlation between infection of different human cytomegalovirus (HCMV) glycoprotein B (gB) genotypes and human chronic periodontitis.
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.
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).
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.
Zhonghua kou qiang yi xue za zhi = Zhonghua kouqiang yixue zazhi = Chinese journal of stomatology 04/2006; 41(4):212-5.
[show abstract][hide abstract] ABSTRACT: To demonstrate the effects of recombinant human insulin-like growth factor-I (rhIGF-I) and/or recombinant human bone morphogenetic protein-2 (rhBMP-2) on proliferation, differentiation and calcification of MC 3T3-E1 cells and NIH 3T3 cells.
Mouse osteoblast-like cell line MC 3T3-E1 and mouse fibroblast cell line NIH 3T3 were treated with different dosages of rhIGF-I or rhBMP-2 and rhIGF-I plus rhBMP-2. Cell proliferation was measured by methylthiazol tetrazolium (MTT)method and flow cytometry. Cell differentiation was examined by using alkaline phosphatase(ALP)measurement kit. Radioimmunoassay was applied to detect levels of osteocalcin (OC) secreted by cultured cells. Von kossa staining method was used to study the calcification effects.
MC 3T3-E1 cells treated with 1-50 ng/ml rhIGF-I and NIH 3T3 cells treated with 5-75 ng/ml rhIGF-I showed marked effects of promoting proliferation (P<0.01), increasing the percentages of S-phase cells, decreasing the percentages of G1-phase cells and increasing activities of cellular ALP and percentages of calcification area (P<0.05). 10-100 ng/ml rhBMP-2 was also able to promote proliferation (P<0.01), increase the percentages of S-phase cells, decrease the percentages of G1-phase cells and enhancing cellular ALP activities and percentages of calcification area for both the two cells (P>0.05).
rhIGF-I and rhBMP-2 have synergistical effects on promoting cell proliferation, early cell differentiation and calcification depending on the used dosages, but no significant effects on promoting advanced cell differentiation.
Zhejiang da xue xue bao. Yi xue ban = Journal of Zhejiang University. Medical sciences 01/2006; 35(1):55-63.
[show abstract][hide abstract] ABSTRACT: To detect the infection frequencies of different Epstein-Barr virus (EBV) genotypes in subgingival samples of chronic periodontitis, and the correlation among infection with different genotypes and the severity of periodontal lesion.
Nested PCR (nPCR) with EBV-1 or EBV-2 specific primers was used to detect EBV-1 and EBV-2 in the subgingival samples from 65 chronic periodontitis patients, 65 gingivitis patients and 24 periodontal healthy individuals. The amplicons were further identified by RFLP with endonucleases Afa I and Stu I. By using periodontal attachment loss (AL) and gingival index (GI) as the observing in, the correlation of infection with different EBV genotypes and the severity of periodontal lesion were analyzed.
0.01 ng of EBV-1 DNA could be detectable by the established nPCR. All the samples showed the same detection results by two separated nPCR. All the EBV-1 amplification products (497 bp) by using endonuclease Afa I digestion could be divided into two fragments with 355 bp and 142 bp respectively. After endonuclease Stu I digestion, all the EBV-2 amplification products (165 bp) displayed two fragments with 118 bp and 47 bp respectively. In the samples of chronic periodontitis patients, gingivitis patients, and healthy periodontal tissues, the positive rates were 28.5% (74/260), 16.9% (44/260), and 14.6% (14/96) for EBV-1; and were 8.1% (21/260), 3.1% (8/260), and 0% for EBV-2 respectively, and the total EBV positive rates were 36.5% (95/260), 20.0% (52/260) and 14.6% (14/96) respectively. None of the positive samples was detectable for both the EBV-1 and EBV-2. The positive rates of EBV-1, EBV-2 and the total EBV positive rates in the chronic periodontitis samples were all higher than those in the gingivitis samples (all P < 0.05) and healthy periodontal tissue samples (all P < 0.01), without a significant difference between the gingivitis samples and healthy periodontal tissue samples (P > 0.05). Infection of EBV or EBV-1 or EBV-2 in CP patients could not be associated with AL or GI.
Subgingival infection with either EBV-1 or EBV-2 is closely associated with chronic periodontitis. Infection of EBV may not correlate directly with severity of periodontitis.
[show abstract][hide abstract] ABSTRACT: 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.
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.
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).
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.
Journal of Zhejiang University SCIENCE B 08/2005; 6(7):699-704. · 1.11 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
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.
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).
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.
Chinese medical journal 06/2005; 118(11):915-21. · 0.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: To establish a 16S rDNA multiplex polymerase chain reaction (PCR) for simultaneously detecting P. gingivalis, A. actinomycetemcomitans and T. denticola in clinical specimens of chronic periodontitis and to study the correlation between different modes of infection and severity of the disease.
Periodontal pocket specimens from 152 patients with mild, moderate or advanced chronic periodontitis and gingival sulcus specimens from 30 periodontally healthy individuals were collected and placed in 200 microl lysis buffer. The specimens were incubated in 100 degrees C for 10 min and 10 microl of the supernatant was directly used as PCR template. DNAs from P. gingivalis strain ATCC33277, A. actinomycetemcomitans strain Y4, T. denticola strain FM and E. coli strain DH5alpha were used as positive and negative controls in PCR with all of which were prepared by routing phenol-chloroform method. A multiplex PCR assay, using three sets of primers specific to 16S rDNA genes of the three anaerobes, was developed to detect the specimens. The target amplification fragments from 3 cases of PCR products positive for all the three anaerobes were sequenced after T-A cloning. Chi-square test was applied to analyze the correlation between different coinfection of the three anaerobes and severity of the disease.
The established 16S rDNA multiplex PCR assay was able to detect P. gingivalis, A. actinomycetemcomitans and T. denticola at a minimum of 10, 20 and 20 cells, respectively. In comparison with the reported corresponding sequences, similarities of the nucleotide sequences from the three anaerobes 16S rDNA amplification fragments were as high as 99.45%, 97.08% and 96.59%, respectively. Of the 30 periodontally healthy gingival sulcus specimens, only one (3.3%) positive for P. gingivalis and two (6.7%) for A. actinomycetemcomitans could be identified but all of the rest were negative. In the 152 CP periodontal pocket specimens, 147 cases (96.7%) were positive for P. gingivalis, A. actinomycetemcomitans and/or T. denticola, respectively, and 5 cases (3.3%) were negative for all the three anaerobes. The positive rate of P. gingivalis detection (91.5%, 139/152) was significantly higher than those of A. actinomycetemcomitans (72.4%, 110/152) and T. denticola (80.9%, 123/152) (chi(2) = 7.07, 18.67; P < 0.01). 89.8% of the specimens from patients showed coinfections with two (26.5%) or three anaerobes (63.3%), and the coinfection rates in the specimens from moderate and advanced CP were remarkably higher than that from mild CP (chi(2) = 10.43, P < 0.01).
The 16S rDNA multiplex PCR established in this study showed high sensitivity and specificity, which could be used to detect P. gingivalis, A. actinomycetemcomitans and T. denticola in clinical specimens. CP was an disease caused by multiple pathogenic microbes while the synergistic pathopoiesis of the three microbes was closely related to the severity of the disease.
Zhonghua liu xing bing xue za zhi = Zhonghua liuxingbingxue zazhi 02/2005; 26(2):120-3.
[show abstract][hide abstract] ABSTRACT: To establish a rat model of experimental alveolar bone resorption and understand the therapeutic effect of a traditional Chinese medical herb Drynaria fortunei (DFS) on the rats suffering from alveolar bone resorption.
A SD rat model with experimental alveolar bone resorption was established by using injection with E. coli Lipopolysaccharide (E-LPS) in local tissue of the animals. A DFS preparation (DFS aqueous-extract) was extracted with distilled water. The modeled rats were administrated by perfusion with DFS aqueous-extract at a dosage of 15 g crude drug/kg once a day for 10, 20 and 30 d, respectively. The effects of DFS aqueous extract on experimental alveolar bone resorption were estimated by detections of serum alkali phosphate (ALP) activity, Ca2+ and osteocalcin (OC) levels, and TRAP stained osteoclast count, bone mineral density (BMD) measurement and HE stained histopathological examination of the tooth-periodontal samples.
In comparison with the controls, BMD values of the alveolar bones at experimental tooth sites of the tested rats with DFS aqueous-extract for 10 d administration were obviously increased (P < 0.05). In the tooth-periodontal samples from the rats for 10 d administration, disappearance of the osteoclast, decrease of Howship's lacuna numbers and formation of new non-calcified bone-like matrix attached by osteoblasts in alveolar bone at the root furcation from most of the samples occurred. Similar examining results in the tooth-periodontal samples from the rats for 20 and 30 d administrations were obtained, respectively. However, no statistically significant differences of the serum ALP activity, Ca2+ and OC levels among the tested rats for 10-30 d treatment and the controls (P > 0.05) were found.
The DFS aqueous extract has exact therapeutic effect on rat experimental alveolar bone resorption through suppressing bone resorption and promoting bone regeneration. Serum ALP activity, Ca2+ and osteocalcin (OC) levels can not be used as the effective index for examining alveolar bone resorption and regeneration in animal models.
Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica 07/2004; 29(6):549-53.
[show abstract][hide abstract] ABSTRACT: 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.
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.
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).
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.
Zhonghua kou qiang yi xue za zhi = Zhonghua kouqiang yixue zazhi = Chinese journal of stomatology 05/2004; 39(3):201-4.
[show abstract][hide abstract] ABSTRACT: To clone pgmA gene of Porphyromonas gingivalis, to construct the expression vector of the gene and to identify immunity of the fusion protein.
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.
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.
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.
Zhejiang da xue xue bao. Yi xue ban = Journal of Zhejiang University. Medical sciences 02/2004; 33(1):41-5.