BACKGROUND:The research of dental stem cells in the fields of regenerative medicine and tissue engineering has been deepening,bringing hope for the repair of tooth-related tissues and the treatment of systemic diseases.However,there is a lack of systematic research and analysis on the biological characteristics of dental stem cells in different age groups. OBJECTIVE:To explore the biological characteristics of the human deciduous tooth and permanent tooth pulp stem cells cultured in umbilical cord blood platelet lysate to provide a reliable basis for human platelet lysates to replace fetal bovine serum. METHODS:The pulp tissues of deciduous teeth,juvenile permanent teeth and adult permanent teeth were taken out and cultured in DMEM/F-12 medium supplemented with 10%fetal bovine serum or different concentrations(5%,10%and 15%)of human platelet lysates.Cell proliferation in the four groups was detected by cytometry.The optimal concentration of human platelet lysates was selected for subsequent experiments.Under the optimal concentration of human platelet lysates,human deciduous tooth and juvenile and adult permanent tooth pulp stem cells were cultured in vitro.The cell growth status was observed under the microscope.The specific antigen on the cell surface was detected by flow cytometry.The cell proliferation ability was tested by the cell counting method and CCK-8 assay.The cell differentiation ability in vitro was observed by a three-line differentiation assay. RESULTS AND CONCLUSION:(1)The cell proliferation rate of the 10%human platelet lysate group was the highest.(2)In all three groups,fusiform fibrous cells grew and expanded from around the tissue block.There was no significant difference between deciduous teeth and juvenile permanent tooth cells,but the adult permanent tooth cells were larger than the deciduous and juvenile permanent tooth cells of the same generation.(3)The results of flow cytometry showed that deciduous teeth,juvenile permanent teeth and adult permanent teeth conformed to the phenotypic characteristics of mesenchymal stem cells.(4)The proliferative capacity of adult permanent dental pulp stem cells was significantly lower than those of deciduous teeth and juvenile permanent dental pulp stem cells(P<0.01).(5)mRNA expressions of osteoblast-related genes alkaline phosphatase and bone morphogenetic protein 2,lipoprotein lipase and peroxisome proliferator-activated receptor γ2,mRNA expressions of chondroblast related gene type II collagen α1 and cartilage oligomeric matrix protein in adult pulp stem cells of permanent teeth were significantly lower than those of deciduous teeth and juvenile permanent teeth pulp stem cells(P<0.01).(6)Compared with adult dental pulp stem cells,human deciduous teeth and juvenile permanent teeth dental pulp stem cells have the stronger proliferative capacity and multidirectional differentiation potential,and are more suitable for clinical research and disease treatment.

Objective To investigate the influence of early growth response gene-1 (EGR1) on the autophagy of host cells following infection with human T cell leukemia virus type 1 (HTLV-1).MethodsA HTLV-1-positive cell line MT2 was co-cultured with HeLa cells for 24 h to construct the virus early infection model.Immunoblotting assay was used to detect the expression of HTLV-1 core protein p19 and EGR1.Luciferase reporter gene analysis was used to detect the transcriptional activity of 5′-regulatory sequence of EGR1 at different time points after co-culturing.An effective small interfering RNA (siRNA) targeting EGR1 was screened out and transfected into HeLa cells by Lipofectamine 2000.Then the transfected HeLa cells were co-cultured with the HTLV-1-positive cell line MT2 for 24 h.Immunoblotting assay was used to detect HTLV-1 core protein p19, EGR1 and autophagy-related protein LC3.Real-time PCR was performed to detect viral load.Autophagosome was analyzed by immunofluorescence after co-culturing.Results The expression of EGR1 and the transcriptional activity of pEGR1-luc gradually increased after co-culturing HeLa cells with MT2 cells for 8 h (P<0.01).The expression of EGR1 was positively correlated with host cell autophagy following HTLV-1 infection.The effective siRNA for silencing the expression of EGR1 was obtained and named as siE2.The viral load, the expression of HTLV-1 core protein p19 and the proportion of LC3B/LC3A in the co-culture model were markedly down-regulated by RNA interference with siE2, which was concomitant with a persistent decrease of intracellular autophagosome (P<0.01).Conclusion EGR1 is associated with host cell autophagy and viral replication in HTLV-1 infection.

Objective To explore the expression of early growth response gene-1 (EGR-1) in T cells that were positive for Tax protein of human T-cell leukemia virus type 1 (HTLV-1) and its possible reg-ulatory mechanism .Methods A series of expression structures carrying the regulatory elements of EGR-1 in different length and luciferase reporter genes were constructed .TaxP cells were transfected with the con-structs containing reporter genes and cultured with 5μmol/L of NF-κB inhibitor BAY 11-7082 or equal vol-ume of DMSO.After cultured for 24 hours the cells were collected to test the luciferase activity .BAY 11-7082 or equal volume of DMSO was added into the supernatant of TaxP cell culture to test the expression of EGR-1 protein by Western blot after 24 hours of culture .Tax and its mutants M22 and M47 were transfected into 293 T cells respectively to test the expression of EGR-1 protein by Western blot after 24 hours of culture . Results The expression structures carrying the regulatory elements of EGR-1 in different length and their mutants followed by luciferase reporter genes were successfully constructed .The luciferase activity in the cells transfected with the constructs containing the elements E 1 and E2 were higher than that transfected with E3, DelE and MutE, but the reporter gene expressions were decreased with the interference of BAY 11-7082 (P<0.01).However, there were no significant changes with the luciferase activity in the cells transfected by elements E3, DelE and MutE.Western blot analysis indicated that the expression of EGR-1 protein was significantly decreased with the interference of BAY 10-7082 .The expression of EGR-1 protein in M22 mu-tants-transfected 293 T cells were decreased significantly in comparison with those by wild type tax-and M47-transfected cells .Conclusion NF-κB was the key nuclear factor in regulating the expression of EGR-1 pro-tein in Tax-positive T cells .

Objective To analyze the relationship between fasting plasma glucose (FPG) level and complexity of coronary artery lesions in patients with coronary stenosis by angiography. Methods The data of clinic and coronary angiogram (CAG) were retrospectively collected in 929 patients with established coronary stenosis by coronary angiography at Peking University Third Hospital from January 2009 to January 2011. The patients were grouped according to SYNTAX score, and the relationship between FPG level and SYNTAX score were analyzed using bivariate, Multivariate stepwise regression and logistic regression analysis. Results ①929 patients were devided into three groups:47 cases into low risk group (score<22), 189 into moderate risk group (score≥22 and<33) and 639 into high risk group (score≥33). Intergroup analysis showed that age (P=0.000), FPG level [5.20 (4.70,6.30) mmol/L, 5.70 (4.90,7.15) mmol/L, 5.80 (5.30,7.60) mmol/L, P=0.000], proportions of FPG abnormality [283 (40.8%), 100(52.9%), 28(59.6%), P=0.001] and patients with diabetes history (P=0.003) were increased along with SYNTAX score elevated.②Correlation analysis showed correlativity (r=0.167, P=0.000) between SYNTAX score and FPG. In non-diabetes history subgroup, correlation between SYNTAX score and FPG remained signiifcant (r=0.149, P=0.000). However, in diabetes history subgroup, the correlation was not significant. ③ Multivariate stepwise regression analysis showed an independent correlation between FPG and SYNTAX score (β=0.452, P=0.002). In non-diabetes history subgroup, the correlation remained significant (β=1.039, P=0.000).④ When moderate-high risk group serve as dependent variable, and age, gender, CAD risk factors and FPG serve as independent variables, logistic regression analysis screened out two variables:age (whole group:OR 1.033, 95%CI 1.017 ~ 1.049, P=0.000;non-diabetes history subgroup:OR 1.039, 95%CI 1.020 ~ 1.059, P=0.000) and FPG (whole group: OR 1.114, 95% CI 1.038 ~ 1.195, P=0.003; non-diabetes history subgroup:OR 1.299, 95%CI 1.088 ~ 1.387, P=0.001). Conclusions FPG is likely to relfect complexity of coronary artery lesions and predict SYNTAX score in patients with coronary stenosis, especially in patients without diabetes history.

Objective:To discuss the effect and mechanism of autophagy inhibitor 3-MA on arsenic trioxide inducing apoptosis of acute T-cell leukemia cell line Jurkat cells.Methods:Proliferation inhibition of Jurkat cells treated with arsenic trioxide was detected by XTT.Morphological characteristics of Jurkat cells treated with different concentrations arsenic trioxide were observed by electron mi-croscope.Microtubule-associated protein 1 light chain 3B (LC-3B) protein expression was detected by Western blot and flow cytome-try.Apoptosis rates of Jurkat cells treated with 3-MA combining arsenic trioxide were detected by flow cytometry using AnnexinV-FITC/PI double staining.Results:Arsenic trioxide inhibited the growth of Jurkat cells in a dose and time dependence.We observed different morphological characteristics of autophagy , apoptosis and necrosis accompanying more autophagosomes in Jurkat cells which were treated with arsenic trioxide 2.5,5,10 μmol/L after 24 h.LC3B mean fluorescence intensity (MFI)relative multiples were(3.1±0.2) fold,(4.6±0.31)fold,(34.2±4.5)fold with 5 μmol/L arsenic trioxide treated Jurkat cells 0,24,48 h,and the P values between each of the two groups were less than 0.05,which increased depending time consistently with the growth inhibition rates.LC-3B protein expression gradually increased treated Jurkat cells with arsenic trioxide after 24 h,48 h.The growth inhibition rate (60.6±8.3)%was significantly different treated with arsenic trioxide combining 3-methyl adenine ( 3-MA ) while it was ( 33.4 ±9.1 )% treated with arsenic trioxide alone, however, LC-3B protein expression gradually decreased.Jurkat cell apoptosis rate ( 44.96 ±3.60 )% was significantly increased treated with arsenic trioxide combining autophagy inhibitor(3-MA) while it was (2.94±0.26)% treated with arsenic trioxide alone, and this difference was statistically significant.Conclusion: 3-MA increased apoptosis rates of Jurkat cells inducing by Arsenic trioxide and it may be related with inhibition of autophagy and induction of apoptosis.

Objective To explore the regulatory effects of HTLV-1 ( human T-cell leukemia virus type 1 ) Tax protein on the expression of HMGB 1 ( high mobility group box 1 ) gene in T cells .Methods Total RNA and protein were extracted from Tax +-T cells ( TaxP ) , Tax--T cells ( TaxN ) and Jurkat cells which were stably transfected with pCMV-Tax and pCMV-Neo, respectively.Then, the expression levels of HMGB1 mRNA and protein in different CD 4+T cells were analyzed by real-time PCR and Western blot (WB).By using liposome-mediated method, pGL3-HMGB1-luc reporter genes and pGL3-neo-luc were tran-siently transfected into TaxP and TaxN cells and the basal transcriptional activity was observed in different T cells.Additionally, pCMV-Tax and pGL3-HMGB1-luc reporter genes were also co-transfected into Jurkat cells and the regulatory effects of Tax protein on HMGB 1 gene was detected .The chromatin immunoprecipi-tation (ChIP) assay was used to identify HMGB1 genomic sites directly targeted by Tax .Results The ex-pression levels of HMGB1 mRNA and protein in Tax+-T cells ( TaxP) were higher than those in Tax--T cells (TaxN).The transcription regulation trends for HMGB1 gene in TaxN and TaxP cells were similar but not identical in diverse T cells.pHLuc3 (containing -504-+83 HMGB1) showed the highest transcriptional ac-tivity of HMGB1 gene in both TaxP and TaxN cells , but HMGB1 transcriptional activity of pHLuc 6 in TaxP cells was significantly stronger than that in TaxN cells .Luciferase assays also showed that Tax protein promo-ted the transcription of HMGB1 gene in a dose-dependent manner .The ChIP assay further confirmed that Tax protein enriched at the HMGB1 region of -1163--1043.Conclusion The region of nt -504--383 is essen-tial for the basal promoter activity of -1163-+83 HMGB1 gene originated from pHLuc 6 reporter plasmid , and Tax protein enriched probably at the HMGB 1 site of -1163--1043 enhances HMGB1 transcription.

Objective To discuss the experience of medical treatment and rescue of the ‘SHENZHOU' astronauts in the landing place in the past 10 years,and to establish more effective mobile ICU in medical helicopters to ensure ‘SHENJIU' astronauts safety.Methods The data collected from foreign nations and our country was reviewed.Formerly experience in first-aid and rescue astronauts of our team was summerized.The important reasons of accidental injuries of astronaut during aerospace flight were listed.More effective and reasonable prophylactic measurements and clinical treatments of the accidental injuries of astronauts were brought forward.Results we established three effective mobile ICU that could ensure the safety of the astronauts.The carriers of the ICU were helicopters,and damage control surgery can be performed there.Fine armature made us more effective.Conclusions Reasonable and effective prophylactic measurements and clinical treatments were the important aspect of the successful manned aerospace flight.The first-aid system in helicopters could realize the destination of swift response and first-aid.

Objective To investigate the influence of extracellular high mobility group box 1 (HMGB1) on viral replication in HTLV-1 infected T cells.Methods HMGB1 in culture supernatants of adult T-cell leukemia virus 1 (HTLV-1) virus-negative cell:Jurkat,MOLT4 cells and HTLV-1 virus-positive cells:MT2,MT4,was detected by ELISA;The HTLV-1 long terminal repeat reporter gene (pHTLV-1-LTR-luc) was transfected into MT2 cells by Tfx-50-mediated transfection,and 0.25,0.50,0.75 μg/ml of HMGB1 polyclonal antibody(HMGB1 PcAb) and its isotype control rabbit IgG antibodies,0.03,0.1,0.3 μg/ml rhHMGB1 and its control PBS,were added into culture supernatant respectively,then luciferase activity was detected after 48 h;Similarly,0.25 μg/ml HMGB1 PcAb and the isotype control antibody,0.3 μg/ml rhH-MGB1 and the control PBS were added to the culture supernatant of MT2 cell,the viral gene,pol1,pol2,gag,env,etc,were performed by real-time PCR.Results Culture supernatant HMGB1 levels has no significant difference between HTLV-1 positive cells MT2 and MT4 and the other two virus-negative T cell lines;Compared with isotype control antibody group,the culture supernatant,to which is added 0.25 μg/ml HMGB1 PcAb,can significantly inhibit the HTLV-1-LTR transcriptional activity and suppress the expressions of the viral gene pol1,pol2,gag,env.Compared with the control PBS,0.3 μg/ml rhHMGB1 significantly promotes the transcriptional activity of the HTLV-1-LTR and the expressions of the viral gene pol1,pol2,gag,env.Conclusion The extracellular HMGB1 can promote viral replication of HTLV-1 infected T cells.

The professional care by multi-disciplinary team and priority of prevention should be carried out in the treatment of diabetic foot disease to reduce diabetic amputation.This article describes the professional experience in the treatment of four complicated cases with diabetic foot disease and emphasizes the importance of the co-operation among different specialists,including diabetologists and wound,vascular,orthopedic surgeons,etc.as well as of varied therapies applied in staged management of the diabetic foot care,by treating these patients with diabetic foot disease as early as possible.

Objective To study the role of endothelial cells on the inflammatory cytokine release in septic shock through the septic shock serum stimulating human primary endothelial cells (HPAEC) and peripheral blood mononuclear cells(PBMC).Methods PBMC isolated from healthy people by density gradient centrifugation.HPAEC cell surface markers CD144 and von Willebrand factor(vWF) molecule expression by RT-PCR and Western blot.Serum levels of IL-6,TNF-α,MCP-1 from septic shock patients and healthy human detected by ELISA.HPAEC and PBMC were stimulated with the isolated serums and LPS,respectively.ELISA was used to detect the supernatant IL-6,TNF-α,MCP-1 levels.HPAEC membrane molecules ICAM-1 expression was detected by flow cytometry with serum shock and LPS stimulation.Supernatant levels of IL-6,TNF-α,MCP-1 of HPAEC with S1P1 receptor agonist CYM-5442 pretreatment was detected by ELISA after shock serum stimulation.Results Endothelial cell markers CD144 and vWF molecules could be detected in the HPAEC.Levels of inflammatory cytokines IL-6,TNF-α,MCP-1 in patients with septic shock serum were significantly higher than healthy people (P＜0.01).PBMC and HPAEC with LPS or shock serum treatment respectively,compared with normal group,levels of inflammatory cytokines in the culture supernatant were significantly higher(P＜0.01).For PBMC,the level of inflammatory cytokines between shock group and LPS group were not significantly different (P＞0.05).But for HPAEC,levels of inflammatory cytokines in the supernatant of the shock group compared to the LPS group was significantly higher (P＜0.01).Similarly,when two cells after LPS stimulation,IL-6,TNF-α levels of HPAEC's supernatant were significantly lower than PBMC' s (P＜0.01),MCP-1 levels was no difference (P＞ 0.05).But when the stimulation of shock serum,HPAEC of IL-6,TNF-α levels and PBMC no significant difference (P ＞0.05).MCP-1 was significantly increased (P＜0.01).Shock patients serum stimulation S1P1 receptorspecific agonist CYM-5442 pretreatment of HPAEC with pretreatment of S1P1 receptor specific agonist CYM-5442,the culture supernatant of inflammatory cytokines IL-6,TNF-α,MCP-1 levels were significantly lower (P＜0.01).Conclusion Endothelial cells may play a central role on the release of inflammatory cytokine during septic shock.