Objective To study the isolation method and the culture method of primary human endometrial cells and to iden‐tify the purity and biological activity .Methods We digested human endometrial tissue by collagenase at first ,and then to separate , purify and culture the human glandular stromal cells(ESC) and endometrial epithelial cells(EEC)by differential centrifugation ,dab‐ble screen filtration and adhesion purification technology .Ultimately ,we identified the isolated cells with cytokeratin and vimentin immunocytochemical staining and immunofluorescence method .Results Stromal cells showed a parallel growth .The cells were spindle or polygonal ,and the vimentin antibody showed a positive immunohistochemical staining ,the purity was more than 95% .At the same time ,glandular epithelial cells grew in whorls .The cells were polygonal or tadpole shaped ,and the cytokeratin antibody immunohistochemical staining ,the purity were up to 90% .Conclusion The successful isolation and culture of high quantity ,viabili‐ty and purity by collagenase digestion and dabble screen filter method of endometriall cells make a strong operability .The laboratory which has the basic cell culture conditions can develop the experiment .

Objective To investigate the influence of gestational weight gain (GWG) on the incidence of macrosomia, and to establish the reference ranges of GWG based on the incidence of macrosomia. Methods A multicenter, cross-sectional study was conducted. Totally, 112485 women were recruited from 39 hospitals in 14 provinces in China. Totally, 61149 cases were eligible with singleton pregnancies and non-premature deliveries. The associations of pre-pregnancy body mass index (BMI), GWG, newborn gender and gestational diabetes with macrosomia were analyzed with logistic regression. The normal GWG ranges were calculated in all maternal BMI subgroups, based on the normal incidence of macrosomia was set as the range of 5.0% to 10.0%. Results In this study, the incidence of macrosomia was 7.46%(4563/611149). The macrosociam was positive related with maternal height, delivery week,pre-pregnancy BMI, GWG, gestational diabetes, primipara, and male babies significantly (P<0.05), based on unadjusted and adjusted logestic regression. The normal range of GWG 20.0-25.0, 10.0-20.0, 0-10.0 and 0-5.0 kg in subgroups of underweight (pre-pregnancy BMI<18.5 kg/m2), normal (18.5-24.9 kg/m2), overweight (25.0-29.9 kg/m2) and obese (≥30.0 kg/m2), respectively. Conclusion The reference range of GWG in China based on the incidence of macrosomia is established.

Objective To explore the value of tricuspid annular displacement (TAD) in evaluating right ventricular systolic function by speckle tracking imaging.Methods Forty healthy people (RVEF ＞50%) and forty five patients(RVEF ＜50%)were enrolled in this study.The apical four-chamber view was obtained and tricuspid annular midpoint displacement (TADmid) were measured by speckle tracking imaging.Right ventricular ejection fraction (RVEF) was measured by three-dimensional echocardiography.The correlation of tricuspid annular midpoint displacement (TADmid) and right ventricular ejection fraction (RVEF) was analyzed.Results These was statistical significance of tricuspid annular midpoint displacement between healthy people (RVEF ＞ 50%) and patients (RVEF ＜ 50%) (t = 7.28,P ＜ 0.01).There was positive correlation between TADmid and RVEF (P ＜ 0.01).The correlation coefficient between TADmid and RVEF was 0.76 (P ＜ 0.0l).The cut-off value of TADmid for RVEF ＞ 50% was 9.7 with a sensitivity of 72% and a specificity of 86%.Conclusions TAD by speckle tracking imaging showed an excellent correlation with RVEF.Tricuspid annular displacement by speckle tracking imaging was a simple and easy method,and it might be a new index in assessing right ventricular systolic function in clinical practice.

Objective To investigate the expression and function of thymus and activation regulated chemokine (TARC) and its special receptor CCR4 at placenta villous in the first trimester placenta villous.Methods Placenta villous was collected from healthy women undergoing artificial abortion at 6 to 8 weeks of gestation,mRNA levels of TARC,CCR4 were analyzed using semi-quantitative reverse transcription (RT)-PCR methods.Immunohistochemistry assay was used to assess the protein localization and expression of TARC,CCR4.Additionally,extravillous cytotrophoblasts were isolated and cultured.Expression of TARC and CCR4 was measured by immunofluorescence assay.Invasion of cell line HTR8/SVneo was analyzed by transwell assay at concentration of 10,25,50 and 100 ng/ml of TARC matched with RPMI 1640 fetal bovine serum free eulture medium as control group.In the mean time,blocking experiment was also added to detect TARC regulating cell invasion,which were classified into four groups:control,100 ng/ml rhTARC,20 μg/ml anti-TARC + 100 ng/ml rhTARC,100 ng/ml rhTARC + 20 μg/ml IgG.The influence of 100 ng/ml TARC on expression level of integrin-α5 and integrin-β1 were measured by using western-blot assay.Results (1)In vivo assay:expression of TARC and CCR4 mRNA were detectable in first trimester placenta villous,TARC protein was localized in cytotrophoblasts,syncytiotrophoblasts and cell column especially on the distal portion,while CCR4 protein was localized on invading interstitial cytotrophobalsts.(2)In vitro assay:①TARC,CCR4 was also expressed in primary isolated extravillous cytotrophoblasts by immunofluorescence assay; ②Matrigel invasion assay demonstrated that TARC had specific dose dependent stimulatory effects on the cells invading through the matrigel precoated filter,the number of cells migration into the lower chamber were:142 ± 31 at 10 ng/ml group,161 ±46 at 25 ng/ml group,201 ±30 at 50 ng/ml group,312 ±48 at 100 ng/ml group,117 ± 33 at control group,the significant response observed from 25 ng/ml (P ＜ 0.05)and reached a peak effect at 100 ng/ml (P ＜ 0.01); ③Blocking experiment demonstrated that when trophoblast invasion was monitored in response to TARC neutralizing antibody (15 μg/ml) together with rhTARC 100 ng/ml.The stimulatory activity of rhTARC was completely overcome,with the cells invasion into the lower chambers were 100 ng/ml rhTARC,20 μg/ml anti-TARC + 100 ng/ml rhTARC,100 ng/ml rhTARC +20 μg/ml IgG,control:313 ±47,113 ±41,287 ±75 and 128 ±23,respectively;④Western-blot assay demonstrated that if cells were treated with 100 ng/ml rhTARC,the expression of integrin-α5 were significantly increased(P ＜0.01),integrin-β1 level also increased when compared with control(P ＜0.05).Conclusion TARC was expressed specifically at human fetal-maternal interface.Trophoblast invasion and migration mainly was regulated by up-regulation integrin-α5 and integrin-β1,which plays an role in trophoblasts differentiation and placentation.

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.

BACKGROUND:Knee joint function limitation often occurs after internal fixation of complex femoral condyle fractures, but the mechanism and its influencing factors are also unclear.
OBJECTIVE:To screen and analyze the relevant factors of knee joint function limitation after internal fixation of complex femoral condyle fractures.
METHODS:We retrospectively summarized postoperative fol ow-up data of 6 and 12 months from 121 patients with complex femoral condyle fractures. Knee joint function recovery was evaluated according to Merchan criteria. A multiple stepwise regression analysis was carried out in terms of gender, age, causes, concomitant injuries, skin and soft tissue injury, fracture type, fixed method, operation time, postoperative plaster fixed situation, healing of postoperative incision, bone healing and postoperative functional exercises, to summarize the relevant influencing factors for knee joint function limitation.
RESULTS AND CONCLUSION:Whether the knee joint function after internal fixation was limited acted as the dependent variable Y, and factors with statistical significance of the single factor analysis served as the independent variable X. We used the multiple stepwise regression analysis for multiple factors analysis. Results showed that the gender of patients (X1), with or without concomitant injuries (X3), soft tissue damage (X4) and operation time (X6), a total of four factors, could not be introduced into the model, suggesting that these four factors had no significant correlation with postoperative knee joint function limitation. Another eight factors could be introduced into the factor analysis model, showing that the cause of injury (X2), fracture type (X5), the choice of internal fixation (X7), with or without bone graft (X8), with or without postoperative plaster cast (X9), postoperative knee joint functional exercise or not (X10) and postoperative wound healing (X11), the degree of postoperative bone healing (X12) are closely related to postoperative knee joint function limitation in complex femoral condyles fractures.

objective To analyze the killer cell immunoglobulin-like receptors(KIR)gene polymorphism of pre-eclampsia patients and approach the correlation between KIR genes and pre-eclampsia.Methods The KIR gene polymorphisms of 71 pre-eclampsia patients and 100 healthy pregnant women were detected by PCR-SSP.The KIR2DL4 mRNA level in placentas from pre-eclampsia and gestational normal pregnancies were quantified by real time RT-PCR.Forty pre-eclampsia patients and 38 healthy pregnant women were detected for single nucleotide polymorphisms(SNP)in the gene coding and joint areas between introns and extrons by directly sequencing techniques of KIR2DL4 genomic DNA.Finally,all alleles and genotypes of KIR2DL4 gene were case-control studied.Result Distributions of some relatively activating KIR genotypes shewed a significant association with pre-eclampsia.Real-time RT-PCR showed that KIR2DL4 mRNA can be measured both in placenta of women with pre-eclampsia being of pre-eclampsia waft significantly lower than that of normal pregnancy.only as much as 0.276 times that of controls.We identified 18 polymorphisms,of which,7 were first reported.But no significant differences in genotype distributions or allele frequencies were observed in these SNPs between cases and controls.Conclusion The distributions of some relatively activating KIR genotypes showed a significant association with pre-eclampsia,which indicates that the polymorphism of KIR genes may be associated with the genetic predisposition to pre-eclampsia.And because the expression of KIR2DL4 mRNA in the placentas of cases wag significantly lower than control group,it is speculated that the decrease of KIR2DL4 expression in placenta may participate in the pathogenesis of pre-eclampsia.

OBJECTIVETo investigate the immunological characteristics of human tonsil mesenchymal stem cells (TMSCs).

METHODSHuman tonsil tissues were obtained from the children patients with chronic tonsillitis. TMSCs were separated, cultured, and were detected the expression profiles of HLA-I, HLA-II, CD80, CD86 by flow cytometry. The measurement of immunogenicity, the effect on phytohemagglutinin (PHA) induced peripheral blood mononuclear cell (PBMCs) proliferation and mixed lymphocytes reaction (MLR) were performed to identify the immunological characteristics of TMSCs. The co-cultures of TMSCs + PBMCs + PHA and TMSCs + MLR were established, respectively, and the concentration of kynurenine, which is the metabolin of indoleamine 2, 3-dioxygenase, in the culture supernatant were examined. Then we added 1-methyl-L-tryptophan into the co-culture of TMSCs + PBMCs + PHA and TMSCs + MLR, respectively, and tested the proliferation of PBMCs. Each experiment was repeated three times, and there were six samples in each group. Statistical significance was assessed by analysis of variance (ANOVA), and a P value less than 0.05 was considered statistically significant.

RESULTSTMSCs expressed HLA-I, were negative for HLA-II and co-stimulatory molecules CD80 and CD86. The stimulation index in the group of TMSCs + allogeneic PBMCs was 1.38 ± 0.26, whereas the stimulation index in the group of allogeneic PBMCs was 1.22 ± 0.28, and there was no significant difference between the two groups (P > 0.05), indicating that TMSCs could not initiate the proliferation of allogeneic PBMCs. The stimulation indexes in the group of TMSCs + allogeneic PBMCs + PHA were 1.49 ± 0.29 and 1.23 ± 0.22, respectively, whereas the stimulation index in the group of allogeneic PBMCs + PHA was 4.60 ± 0.81, and the difference between the two groups had a statistical significance (P < 0.05) suggesting that TMSCs could inhibit PHA-induced PBMCs proliferation. The stimulation indexes in the group of TMSCs + MLR were 1.29 ± 0.23 and 1.26 ± 0.27, respectively, however, the stimulation index in the group of MLR was 3.04 ± 0.66, and the difference between the two groups had a statistical significance (P < 0.05), demonstrating that TMSCs could suppress MLR-induced PBMCs proliferation. The levels of kynurenine were (26.0 ± 2.3) μmol/L and (23.5 ± 4.5) μmol/L in the culture of TMSCs + PBMCs + PHA and TMSCs + MLR, respectively, thus elevating significantly. After adding of 1-methyl-L-tryptophan, TMSCs-mediated-proliferation suppression of PBMCs restored to normal levels.

CONCLUSIONTMSCs possess low immunogenecity and immunosuppressive function, may be used in allogeneic transplantation.

Cell Proliferation ; Cells, Cultured ; Child ; Coculture Techniques ; Flow Cytometry ; Humans ; Immunosuppression ; Kynurenine ; analysis ; Leukocytes, Mononuclear ; Lymphocyte Culture Test, Mixed ; methods ; Mesenchymal Stromal Cells ; cytology ; immunology ; Palatine Tonsil ; cytology ; Tryptophan ; administration & dosage ; analogs & derivatives

Objective To study the coexpression of protein folding modulators,including chaperones(DnaK,DnaJ,and GroEL-GroES) and disulfide isomerase Dsb molecules(DsbA and DsbC) on the bioactivity of eukaryotic expressed urokinase(UK).Methods The gene sequences of DnaK,DnaJ,GroEL-GroES,DsbA and DsbC were isolated from the chromosome of E.coli by PCR.The obtained fragments mentioned above were cloned into the plasmid pBAD-1 under the control of araB promoter and then the plasmids were transformed into E.coli BW25113 and HB101 together with a compatible vector pQE-UK.The amount of expressed UK was measured by Bradford method and its activity was analyzed by fibrin plate method for its fibrinolysis.Results The activity of expressed UK was increased by co-expressing with DnaK,GroEL-GroES,DsbA,and DsbC in BW25113.Among them,the highest activity was achieved by co-expressing with GroEL-GroES.Conclusion Co-expressing of chaperones or disulfide isomerase can improve the bioactivity of UK expressed in E.coli.

Objective To investigate the respiratory pathogen distribution characteristics in children with acute respiratory disease(ARD).Methods Distribution of respiratory pathogen in 28 600 children with ARD,treated from January 2011 to December 2014 in this hospital,were analyzed.Results Among the 28 600 children,12 162 cases were pathogen positive,including 7 704 cases(63.34%) with single pathogen infection and 4 458 cases(36.66%) with more than two kinds of pathogens infection.Time,season,sex and age distribution of pathogen infection were with statistical difference(P<0.05).There was significant difference in infection rate of pathogens between different time(P<0.05).Infection rate of Mycoplasmal pneumonia(MP) was the highest,and the infection rate of MP and influenza B virus(IFB) increased year by year.Except Legionella pneumophila(LP),the season distribution of infection rate of MP,Coxiella burneti(COX),Chlamydia pneumoniae(CP),adenovirus(ADV),respiratory syncytial virus(RSV),influenza A virus(IFA),IFB and parainfluenza virus(PIVs) were with statistical difference(P<0.05).The infection rate of MP and IFB were higher in the whole year.Conclusion Distribution of respiratory pathogen in children with ARD might be related with the changes of gender,age and season.Detection of respiratory infection pathogen could be with guiding value for clinical diagnosis and drug selection.