OBJECTIVETo investigate the effect of Metformin on the proliferation and collagen synthesis of the human keloids fibroblasts as well as the effect on phosphorylation of Akt/FoxO1 signal transduction pathway.

METHODSFibroblasts of keloid were divided into control group treated with medium solution and experimental groups treated with different concentrations of Metformin. 48 h later CCK-8 assay was adopted to evaluate cell survival; Western blot was performed to detect the Akt and FoxO1 phosphorylation; and Hydroxyproline reagent kit was used to detect the collagen synthesis.

RESULTSWith different concentrations (30, 60, 90, 120 mmol/L) of Metformin, the absorbance of cultured keloid fibroblasts detected by CCK8 assay decreased by (13.30 ± 2.04)%, (22.64 ± 4.70)%, (54.00 ± 5.34)% and (63.12 ± 3.48)%. The growth of fibroblasts was suppressed by Metformin in a dose-dependent manner. It showed that the level of phoshpo-akt and phoshpo-foxOl in keloids fibroblasts in experimental groups was lower than that in the control group and the collagen synthesis were also decreased in experimental groups, all in a dose-dependent manner (P < 0.05, P < 0.01).

CONCLUSIONSMetformin can effectively inhibit the proliferation and collagen synthesis of the human keloids fibroblasts in vitro, which may be associated with the suppression of phosphorylation of Akt/FoxO1 signaling pathway

Cell Proliferation ; drug effects ; Collagen ; biosynthesis ; Dose-Response Relationship, Drug ; Fibroblasts ; cytology ; drug effects ; metabolism ; Forkhead Box Protein O1 ; Forkhead Transcription Factors ; metabolism ; Humans ; Keloid ; pathology ; Metformin ; pharmacology ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; drug effects

Objective To evaluate the effect of metformin on the proliferation of keratinocytes,and to investigate its possible mechanism.Methods HaCaT human keratinocytes were divided into several groups to remain untreated (control group) or be treated with different concentrations (25,50,75,100 mmol/L) of mefformin for 24 hours (intervention groups).Subsequently,CCK8 assay was conducted to evaluate the proliferation of HaCaT cells,real-time quantitative PCR to measure the mRNA expressions of miR-21-5p and its downstream target gene PDCD4,and Western blot to detect the expression of PDCD4 protein in HaCaT cells.Statistical analysis was done by using one-way analysis of variance for multiple group comparisons and SNK-q test for paired comparisons.Results After 24-hour treatment,the proliferation of HaCaT cells was inhibited by (5.43 ± 3.67)％,(19.61 ± 6.95)％,(45.93 ± 9.56)％ and (61.91 ± 6.93)％ by metformin of 25,50,75 and 100 mmol/L,respectively,with significant differences observed in cell proliferation inhibition rates among these intervention groups (F =246.90,P ＜ 0.05).Cellular proliferative activity was similar between the control cells (0.00 ± 3.00％) and those treated with 25 mmol/L metformin,but significantly higher in the control cells than in the other 3 metformin-treated groups (all P ＜ 0.05),and significantly different between the 4 metformin-treated groups (all P ＜ 0.05).The relative mRNA expression level (2-△△Q) of miR-21-5p was 0.90 ± 0.11,0.33 ± 0.05,0.21 ± 0.07 and 0.14 ± 0.04 (F =36.99,P ＜ 0.01),while that of PDCD4 was 2.11 ± 0.64,7.22 ± 1.13,11.16 ± 1.23 and 19.12 ± 3.16 (F =96.26,P ＜ 0.05),and the expression level of PDCD4 protein was 1.22 ± 0.08,2.09 ± 0.20,2.26 ± 0.1 1 and 2.37 ± 0.07 (F=75.37,P＜ 0.05),respectively,in HaCaT cells treated with metformin of 25,50,75 and 100 mmol/L.Similarly,no significant difference was observed between the control cells and those treated with 25 mmol/L metformin in the expression level of miR-21-5p mRNA,PDCD4 mRNA or protein,but decreased expression of miR-21-5p mRNA and increased expression of PDCD4 mRNA and protein were noted in cells treated with the other 3 concentrations of metformin compared with the control cells (all P＜ 0.05),and significant differences were also found in the expression levels of miR-21-5p mRNA as well as PDCD4 mRNA and protein among the 4 intervention groups (all P ＜ 0.05).Conclusion Metformin can markedly inhibit the proliferation of HaCaT cells in vitro,likely by downregulating miR-21-5p expression and upregulating PDCD4 expression.

Objective To investigate the association of eIF4E and MMP-9 gene polymorphisms with psoriasis vulgaris in Han population of Shandong province.Methods A population based case-control association study was carried out in 188 patients with psoriasis vulgaris and 280 healthy human controls of Han nationality from Shandong province.Taqman SNP genotyping assay was performed to assess three SNPs,including rs4810482 and rs3918254 in MMP-9 gene and rs11723037 in eIF4E gene.Pairwise linkage disequilibrium was evaluated by using Haploview 4.2 software,and the frequencies of alleles and genotypes were analyzed by using Plink 1.07 software.Results The frequency of rs4810482 T allele was significantly lower in patients with psoriasis vulgaris than in the normal human controls(OR =1.49,95％ CI:1.12-1.99,P ＜ 0.01),and the significant difference still remained under recessive and dominant model.Bioinformatic analysis revealed that the rs4810482 altered the binding site of transcription factor,while no association was observed between psoriasis and either of the other two SNPs.Conclusions The SNP rs4810482 located at the upstream regulatory region of MMP-9 gene is significantly associated with psoriasis,hence,MMP-9 gene may be a susceptibility gene for psoriasis in Han population of Shandong province.

Objective To study the biocompatibility of carboxymethyl chitosan (CMCS) membrane with melanocytes from healthy human skin,and to investigate the feasibility to transport and carry melanocytes by using CMCS membrane.Methods CMCS membrane was prepared by a casting method combined with a glutaraldehydebased cross-linking method.Melanocytes were isolated from the foreskin of healthy men,and subjected to primary culture and subculture.The third-passage melanocytes were classified into two groups to be cultured on the CMCS membrane (test group) or traditional culture plates (control group).Methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferative activity of melanocytes,and a sodium hydroxide-based lysis method to determine melanin content.HMB45 staining was conducted,and tyrosinase activity was estimated for melanocytes.Results Inverted microscopy showed that melanocytes were evenly distributed on the CMCS membrane with a normal shape.The melanocytes adherent to the CMCS membrane stained positive for anti-HMB45 monoclonal antibody.The growth curve of the melanocytes on the CMCS membrane,which was obtained from MTT assay,demonstrated that CMCS membrane could support the normal growth of melanocytes.No significant difference was observed between the test group and control group in melanin content (0.083 ± 0.015 vs.0.066 ± 0.008,t =2.38,P ＞ 0.01) or tyrosinase activity (0.234 ± 0.083 vs.0.241 ± 0.061,t =0.23,P ＞ 0.05).Conclusion CMCS membrane can maintain the normal biological activity of melanocytes and have good biocompatibility with skin melanocytes.

Objective To screen for differentially expressed proteins in sera from patients with common types of psoriasis,and to identify plasma protein markers for psoriasis.Methods Serum samples were collected from 6 patients with progressive psoriasis vulgaris,5 patients with erythroderma psoriaticum,and 6 healthy human controls,and then pooled into 3 pools:psoriasis vulgaris pool,erythroderma psoriaticum pool and control pool.After removal of high-abundance albumin and IgG,the pooled samples were analyzed by two-dimensional electrophoresis (2-DE).An electrophoretic gel image analysis software was used to locate differentially expressed protein spots followed by peptide mass fingerprinting with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS).The National Center for Biotechnology Information (NCBI) protein databases were then searched for the identification of differentially expressed proteins.Results All the three pooled serum samples were well seperated by 2-DE.As the gel image analysis software showed,there were 33 protein spots differentially expressed between the patients with psoriasis vulgaris and the healthy controls,17 between the patients with erythroderma psoriaticum and the healthy controls,and 26 between the patients with psoriasis vulgaris and those with erythroderma psoriaticum.Finally,14 proteins were identified as differentially expressed proteins.The patients with psoriasis vulgaris showed higher expression of complement component 3,interleukin-16,vitamin D-binding protein and α1-antitrypsin compared with the healthy controls; the patients with erythroderma psoriaticum showed increased expression of complement component 3,complement component H,α1-antitrypsin,hemopexin and haptoglobin,but decreased expression of serum amyloid protein compared with the healthy controls,as well as enhanced expression of α1-antitrypsin,complement component H,complement component 4 and haptoglobin compared with those with psoriasis vulgaris.Conclusion Differences exist in serum protein profiles between patients with psoriasis vulgaris and erythroderma psoriaticum,and healthy human controls.

Objective To detect the expression of the transcription factor CCAAT/enhancer-binding protein alpha (C/EBP-α) in the epidermis of psoriasis vulgaris lesions,and to investigate its correlation with abnormal keratinocyte proliferation and disease severity.Methods Biopsy specimens were obtained from the lesions of 30 patients with psoriasis vulgaris and normal skin of 30 healthy human controls.A two-step immunohistochemical procedure was performed to detect the expressions of C/EBP-αt and Ki-67 in these specimens,and Western blot to quantify the expression of C/EBP-α.The proliferation index of keratinocytes was calculated according to the expression intensity of Ki-67.Statistical analysis was carried out by using the SPSS 17.0 software,and Pearson correlation analysis was conducted to assess the relationship of C/EBP-α expression level with proliferation index of keratinocytes and psoriasis area and severity index (PASI) score.Results C/EBP-α was predominantly expressed in the cytoplasm of keratinocytes,while Ki-67 in the nuclei of keratinocytes.Compared with the normal skin,the psoriatic lesions showed a significantly lower expression of Ki-67 (t =7.82,P ＜ 0.05),but higher proliferation index of keratinocytes (t =4.54,P ＜ 0.05).The expression level of C/EBP-α was negatively correlated with the proliferation index of keratinocytes and PASI score in the patients (both P ＜ 0.05).Western blot also showed an obvious decrease in the expression of C/EBP-α in psoriatic lesions.Conclusions The expression of C/EBP-α is decreased in lesions of psoriasis vulgaris,which might be involved in the pathogenesis of psoriasis vulgaris.

Objective:To study the relationship between the expression of heat shock protein 27 (HSP27) in triple negative breast cancer (TNBC) and the clinico-pathological indexes of breast cancer, investigate the correlation between HSP27 and the fatty acid syn-thetase (FAS)/fatty acid synthetase ligand (FASL) of the cell apoptosis system in the Fas/Fasl system, and study the role of HSP27 in the invasion and metastasis of TNBC. Methods:The immunohistochemical S-P method was used to detect the expression of HSP27 and (FAS)/(FASL) in 100 TNBS tissue sampres, 100 non-TNBS tissue sampres, and 50 paraneoplastic tissues. This method was also used to analyze the correlations between the expression of HSP27 and the clinical and pathological indexes of TNBC, as well as be-tween the HSP27 expression and FAS/FASL expression. Results: HSP27 expression was significantly higher in TNBC than in the non-TNBC and paraneoplastic tissues (P<0.05). Statistically significant differences were observed in the FAS/FASL expression in the TNBC, non-TNBC, and paraneoplastic tissues (P<0.05). HSP27 expression had a negative correlation with FAS expression (P<0.05). HSP27 expression was positively correlated with FASL expression (P<0.05). FAS expression had a negative correlation with FASL ex-pression (P<0.05). HSP27 expression in TNBC was not correlated with age, staging, and tumor size (P>0.05), whereas HSP27 expres-sion was correlated with lymph node metastasis, number of nodal metastases, and P53 and Ki67 expression (P<0.05). Conclusion:The overexpression of HSP27 and the expression dysregulation of the FAS/FASL system may play a role in promoting TNBC transfer and invasion, cell proliferation, and poor prognosis.

OBJECTIVE To survey the distribution of class Ⅰ integron in extended-spectrum beta-lactamases(ESBLs)-producing Escherichia coli and Klebsiella pneumoniae and its contribution in horizontal transfer of ESBLs genes.METHODS The presence of class Ⅰ integron among 230 ESBLs-producers and 197 non-ESBLs-producers of E.coli and K.pneumoniae were detected by PCR.The correlation and co-transfer between integron and genes coding for SHV,CTX and TEM were studied. RESULTS One hundred and thirty-seven(59.6%) isolates were positive for intⅠ gene among ESBLs-producers,contrasted to 48(24.4%) in non-ESBLs-producers(P

BACKGROUND:Bilateral-sagitta-split-ramus-osteotomy (BSSRO) has become a conventional method to correct facial deformities, and the finite element method is a significant way to study biomechanics of the mandible and temporomandibular joint (TMJ) after BSSRO. OBJECTIVE: To establish a precise and high simulation model of mandible containing TMJ after BSSRO with internal fixation, which is the base to study the biomechanics of the mandible and TMJ after BSSRO. METHODS: Spiral CT scan was used to get the data of DICOM that were input into MIMICS to establish the three-dimensional model of the mandible. The three-dimensional model was wrapped into a single closed shel for mesh generation and conversion in ANSYS. Then, the model was input into the ANSYS software for temporomandibular joint reconstruction and simulation of BSSRO and internal fixation. RESULTS AND CONCLUSION: The three-dimensional finite element model of mandible containing TMJ after BSSRO was established using MIMICS and ANSYS. This model had biological similarity and geometric similarity in comparison with the human tissues. The model could undergo various internal fixations through antedisplacement, retroposition and rotational movement of the distal end. Based on different experimental purposes, the established model can apply a load to al parts to study changes in stress and displacement of different tissues after BSSRO and internal fixation, and it also can be used to study the effect of different fixation materials on the rear stability after internal fixation.

Objective To evaluate the trueness of 13 routine measurements for serum uric acid and the role of reference method in improving harmonization and trueness among routine measurement systems. Methods The research is related to the reagent evaluation.Usingisotope dilution liquid chromatography tandem mass spectrometry ( ID-LC/MS/MS) method as the comparison method, Wako, Sekisui, DiaSys, Maker,Dirui,Leadman,BSBE,Biosina,Mindray,MedicalSystem,LongMarch,and Kehua 13 kinds of uric acid kits were chosen as the evaluation methods with Hitachi 7170A as the analyzer.serum uric acid in 40 fresh frozen serawere collected from clinical laboratory of Beijing hospital in 2014,coveringboth physiological and pathological status ( 80 -940 μmol/L ) .19 kinds of prepared materials and the 40 fronzen sera were measuredby comparison method and evaluation methods and linear regression analysis was made for the results.The performance of evaluation methods was revealed and recalibration was performed on every evaluation methodby the linear regression equation.The variation of percent bias(%) of the uric acid values in 19 preparation materials was compared.Results All test methods demonstrated good precision ( CV<1.75) and good correlation (R2 >0.998, P<0.01) with the comparison method when measuring uric acid values in 40 fresh frozen sera The meanpercent bias was 0.17% ( -3.06% -7.31%).After recalibration, 4 of 19 samples with no matrix effect values percent bias reduced and met the demands of quality ( <4.8%) induced from biological variation.Conclusion All test methods demonstrated good trueness and their calibration traceability was verified.Recalibration using reference method or standard reference materials contributes to harmonization among methods.