【Objective】 To sequence P1 gene(184～1946 bp) includ ing SBR gene in expressional plasmid of transgenic plant. 【Methods】 pROP1 plas mid was extracted from E. coli DH5α, P1 gene was sequenced by PCR sequence kit and DNA autosequencer and compared with surface protein gene of Streptococc us mutans in GenBank. 【Results】 616 base pairs from 5′ end and 466 base pairs from 3′ end were sequenced, its accuracy is up to 98%. 【Conclusion】 The seq uence of P1 gene's 5′ end and 3′ end is consistent with DNA sequence of SBR re gion of Streptococcus mutans surface protein pac gene and may provide useful in formations to construct the transgenic plant anticaries vaccine.

Objective:To culture human alveolar osteoblasts and study its osteogenic characteristics in vitro.Methods:Human alveolar bone was obtained from human donor and cultured in explants.Cells migrating from explants were observed by inverted phase-contrast microscope and cell proliferation was detected by MTT assay.After cultured in conditional medium containing dexamethasone,?-glycerphosphoric sodium and ascorbic acid,cells were tested by ALP assay,Alizarin red assay and von Kossa assay,3H-proline labeling with collagenase digestion method.Results:The alveolar osteoblasts migrated from explants after 5 d culture.Cells could be passaged after 14-16 d culture.ALP values were obviously positive and cells showed positive reaction by Alizarin red assay and von Kossa assay in conditional medium.The cultured human alveolar osteoblasts secreted type I Collagen.Conclusion:The human alveolar osteoblasts cultured in this experiment grow well,and the morphological and biological characteristics of the culture cells are similar to those of osteoblasts.

Objective To investigate the the multi-directional differentiation potential between pluripotent of human gingival fibroblasts (HGFs) and human periodontal ligament cells (HPDLCs). Methods HPDLCs and HGFs were obtained from the primary culture. HPDLCs and HGFs at 3rd-4th passage were cultured in osteogenic, adipogenic or chondrogenic me-dium. Cells without differentiation were taken as control. Alizarin red, Alcian blue and oil red O staining were performed to detect osteogenic differentiation, chondrogenic and adipogenic differentiation in vitro, respectively. Reverse transcription polymerase chain reaction (RT-PCR) was applied to examine the expression of osteocalcin (OCN), runt-related transcription factor 2 (RUNX2) and collagen 1 (Col 1), peroxisome proliferator-activated receptor gamma 2 (PPARγ2) and collagen 10 (Col 10). Results HPDLCs and HGFs cultured in osteogenic medium showed massive calicium nodulus at day 28, but HP-DLCs formed more calicium nodulus than those of HGFs. The expressions of OCN, RUNX2 and Col 1 were significantly high-er in HPDLCs than those in HGFs (P<0.05). In chondrogenic medium both cells were found blue deposit at day 14, and the expression of Col 10 was significantly higher in HGFs than that of HPDLCs (P<0.01). Furthermore, in adipogenic medium HGFs showed more lipid-filled droplets stained with oil red O than HPDLCs at day 21. The expression of PPARγ2 was sig-nificantly higher in HGFs than that of HPDLCs (P<0.01). Conclusion HPDLCs has the better potency of osteogenic differ-etiation than HGFs, however, HGFs has the better potency of adipogenic and chondrogenic differentiation.

Objective To study the correlation between the expression of activator protein-1 (c-Fos/c-Jun) mRNA and gingival inflammation,so as to discuss the pathogenesis of periodontitis.Methods The gingival tissues were divided into three groups according to the gingival index (GI),including GI=0 group (control group,14 cases),GI=1 group (15 cases) and GI=2 group (11 cases).The total RNA in each gingival tissue was extracted,and cDNA was synthesized by reverse transcription synthesis.The expressions of c-Fos and c-Jun mRNA in healthy gingival tissue (GI=0 group) were detected by reverse transcription-polymerase chain reaction.The levels of c-Fos and c-Jun mRNA in all the groups were detected by real-time quantitative PCR.Results Both c-Fos and c-Jun mRNA was expressed in healthy gingival tissues.The levels of c-Fos and c-Jun mRNA in GI=1 group was 15.58±9.19 and 3.47± 1.77,respectively,which was significantly higher than 1.31±1.03 and 1.32±0.94 in GI=0 group,and the differences were statistically significant (all P＜0.05).The level of c-Fos mRNA in GI=2 group was 3.01±1.48,which was lower than that in GI=1 group (P＜0.05) and higher than that in GI=0 group (P＜0.05).The level of c-Jun mRNA in GI=2 group was 1.48±0.65,which was lower than that in GI=1 group,and had no significant difference with GI=0 group (P＞ 0.05).Conclusions Activator protein-1 (c-Fos/c-Jun) is associated with the degree of gingival inflammation,suggesting that it is involved in the occurrence and development of gingival inflammation.

Objective To observe the effects of basic fibroblast growth factor (bFGF) on osteogenic differentiation abili?ty and cell proliferation of human gingival fibroblasts (HGFs), and to explore the role of bFGF on the process of osteogenic differencitiaion in vitro. Methods HGFs were cultured in vitro until the 3rd passage when they were divided into four groups:normal medium as group 1, normal medium with 10μg/L bFGF as group 2, osteogenic medium as group 3 and osteo?genic medium with 10μg/L bFGF as group 4. MTT assay was used to evaluate the proliferation of HGFs. Alkaline phospha?tase (ALP) staining and Alizarin red staining were applied to investigate osteogenic potential of HGFs under different culture conditions. Results bFGF at concentration of 10 μg/L could increase HGFs proliferation in both normal and osteogenic medium (P<0.01). HGFs could be induced towards osteogenic differentiation and form mineralized nodule in osteogenic me?dium. However, 10μg/L bFGF had no effects on ALP activity and mineralized nodule formation of HGFs during osteogenic differentiation. Conclusion bFGF could promote the proliferation of HGFs but show no effects on osteogenic differentiation of HGFs at concentration of 10μg/L.

Objective To study the HPLC fingerprints of isoflavones from different medicinal plants of G.max and establish a sensitive and specific method for controlling the quality of the soybean isoflavone.Methods The gradient elution mode was applied in chromatographic separation.A C_(18) column(250 mm?4.6 mm,5 ?m) was used with the mobile phase: ice acetic acid water solution(pH value 3.2)-methanol,flow rate: 0.6 mL/min,detecting wavelength: 261 nm,and the column temperature: room temperature.All 25 samples collected from different species were determined.The clustering analysis and the software of similarity analysis were applied for datum analysis.Results This method had a good repeatability and reproducibility.The ratio of peaks′ area from distinct samples were different.Conclusion The method can show the difference of chemical compositions among species completely and can be used as a quality control method for soybean.

Objective To investigate the pluripotency of human gingival fibroblasts (hGFs), and provide a novel cell source for tissue engineering. Methods With informed consent from volunteers, fresh and healthy gingiva were collected. The hGFs were obtained from the gingiva by tissue culture. The third passage of hGFs was cultured in osteogenic medium, chondrogenic medium and adipogenic medium. Cells without differentiation were taken as control. Cells were examined by al?kaline phosphatase (ALP) staining, Alizarin red staining, Alcian blue staining and oil red O staining for detecting of the abili?ty of differentiation pluripotency. Real-time polymerase chain reaction was applied to examine the expression of osteogenic marker genes ALP, runt-related transcript factor 2 (Runx2), chondrogenic marker aggrecan (AGR) and adipogenic marker peroxisome proliferator-activated receptor gamma 2 (PPARγ2). Results The hGFs cultured in osteogenic medium showed massive violet deposit at day 7 and calcium nodulus at day 28, meanwhile, the expressions of ALP and Runx2 were higher than those of control (P<0.01). In chondrogenic group cells were found blue deposit at day 14. In adipogenic group lipid-filled droplets stained with oil red O were found in cells at day 14. However, hGFs in control group had no any positive stain?ing. Furthermore, expressions of AGR and PPARγ2 were significantly higher than those of control (P<0.01). Conclusion Human gingival fibroblasts have the pluripotency of osteogenic, adipogenic and chondrogenic differentiation.

Objective To investigate the influence of high glucose on Porphyromonasgingivalis(Pg)lipopolysaccharide(LPS) stimulating human gingival fibroblasts(HGF) to secret the cytokines.Methods HGF were obtained from the primary culture of the tissue explants.Cells were divided into four groups,low glucose(5.5 mmol/L) + 1 mg/L Pg LPS(group A); low glucose+ 10 mg/L Pg LPS(group B); high glucose (25 mmol/L) + 1 mg/L Pg LPS(group C);high glucose+ 10 mg/L Pg LPS(group D).The levels of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) in cell supernatants were detected by enzyme-linked immunosorbent assay at 6 h and 12 h.The expressions of toll-like receptor 2,4(TLR-2,4) were examined by real-time polymerase chain reaction.After pretreatment with anti-TLR2 and anti-TLR4 monoclonal antibody in HGF,TNF-α and L-1β levels were detected.Results TNF-α concentration increased obviously in high glucose 6 h and 12 h after Pg LPS stimulation(P＜0.01).IL-1β secretion increased(P＜0.01).Meanwhile,TLR2,4 mRNA expression increased,especially in high glucose+ 10 mg/L Pg LPS(P＜0.01).After inhibition of the TLR2,4 in high glucose+ 10 mg/L Pg LPS respectively,TNF-α level[(297.16± 11.49),(390.01 ± 12.81) ng/L] decreased(F=166.02,P＜0.01),and IL-1β level[(49.90±4.08),(99.35±5.01) ng/L] also decreased (F=153.51,P＜0.01).Conclusions High glucose may promote Pg LPS to stimulate the secretion of TNF-α and IL-1β through regulating TLR2,4 expression,which suggests that the elevating blood glucose precipitate in aggravating the process of periodontal disease.

Objective:To explore the effects of microRNA-126 (miR-126) on the proliferation of human myeloid leukemia mononuclear cells (THP-1)-derived macrophages in high glucose environment and the regulatory role of miR-126 in periodontitis with diabetes.Methods:THP-1 cells were cultured in vitro and 5 μg/L phorbol-12-myristate-13-acetate was applied to induce THP-1 cells differentiating into macrophages for 48 h in low glucose culture medium (5.5 mmol/L). THP-1-derived macrophages were then cultured with low glucose, medium glucose (15 mmol/L) or high glucose (25 mmol/L) media respectively. The proliferation of THP-1-derived macrophages was detected by cell counting kit-8 (CCK-8) method and the expressions of miR-126 and proliferation-associated factors were detected by quantitative real time PCR (qRT-PCR). The miR-126 mimic or inhibitor was transfected into THP-1-derived macrophages for 72 h. The proliferation of cells was detected by CCK-8 method and the expressions of miR-126 or proliferation-associated factors were detected by qRT-PCR. Results:Increasing glucose concentration decreased the proliferation of THP-1-derived macrophages (day 7, A values in low, medium and high glucose groups were 0.369±0.014, 0.214±0.009 and 0.200±0.010, respectively, P<0.01) as well as the survival rate ( P<0.05), promoted the expression of miR-126, B-cell lymphoma-2 (Bcl-2)-associated X protein (BAX) and caspase-3 ( P<0.05), and suppressed Bcl-2, phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2) expression ( P<0.05). After the miR-126 mimic was transfected in cells in low glucose medium for 72 h, compared with negative control (1.005±0.118), the expression of miR-126 significantly increased (2 980.227±170.431, P<0.05), and the proliferation of THP-1 derived macrophages decreased (negative control: 1.816±0.013, mimic group: 1.310±0.048, P<0.01), the level of BAX and caspase-3 significantly increased ( P<0.01, P<0.05), PIK3R2 and Bcl-2 significantly decreased ( P<0.05, P<0.01). After the miR-126 inhibitor was transfected in cells cultured in high glucose medium for 72 h, compared with negative control (0.723±0.133), the proliferation of inhibitor group increased (0.984±0.049, P<0.05), the level of BAX and caspase-3 significantly decreased ( P<0.01, P<0.05), PIK3R2 and Bcl-2 significantly increased ( P<0.01, P<0.05). Conclusions:High glucose condition can inhibit the proliferation of THP-1-derived macrophages and increase the expression of miR-126. MiR-126 can inhibit the proliferation of THP-1-derived macrophages in high glucose environment through up-regulating the expression of BAX and caspase-3 and down-regulating the expression of PIK3R2 and Bcl-2.

Endo-periodontal lesions(EPLs)involve both the periodontium and pulp tissue and have complicated etiologies and pathogenic mechanisms,including unique anatomical and microbiological characteristics and multiple contributing factors.This etiological complexity leads to difficulties in determining patient prognosis,posing great challenges in clinical practice.Furthermore,EPL-affected teeth require multidisciplinary therapy,including periodontal therapy,endodontic therapy and others,but there is still much debate about the appropriate timing of periodontal therapy and root canal therapy.By compiling the most recent findings on the etiology,pathogenesis,clinical characteristics,diagnosis,therapy,and prognosis of EPL-affected teeth,this consensus sought to support clinicians in making the best possible treatment decisions based on both biological and clinical evidence.