ObjectiveTo investigate the effect of SalB on bone marrow-derived mesenchymal stem cells (BMSCs) apoptosis induced by hypoxia and serum deprivation (hypoxia/SD) in the vitro. Methods BMSCs were cultured in the vitro and randomly divided into control group, hypoxia/SD group and SalB group.SalB group was composed by four groups and were pretreated by complete medium with 0.1、 1、 10、 100 mg/L SalB for 1 hour. And after that they were washed with phosphate buffer for 2 times, added by IMDM with 0.1、1、 10、 100 mg/L SalB and cultured with hypoxia/SD group together in the same condition of hypoxia/SD for 6hours. The control group was cultured for 6 hours in the condition of aerobic and enough serum. Apoptosis was detected by Hoechst33342 staining with inverted phase contrast, fluorescence microscope and Annexin V/PI dual-color flow cytometry. Results Significant apoptosis of BMSCs was induced by hypoxia/SD in the vitro.The early apoptosis of BMSCs induced by hypoxia/SD was significantly decreased by SalB of 0.1、 1、 10 mg/L(P＜0.05) . Conclusion0.1、 1、 10 mg/L SalB can decrease the early apoptosis of BMSCs induced by hypoxia/SD.

Aim:To obtain recombinant human proinsulin C-peptide,a novel expression vector pEDCC was constructed to facilitate the expression and purification of C-peptide. Methods:Gene fragments encoding a truncated asparaginase fragment mutant,native C-peptide,a hinge fragment of human IgG1,an extra acid-labile dipeptide and a basic-amino-acid-riched octopeptide were introduced in turn into plasmid pET28a. The fusion protein ansB-C-hinge-DPKRKRKKSRNGSGR-C-peptide was expressed effectively as inclusion bodies after induced by lactose and partially purified by means of washing and ethanol fractionation. After being hydrolyzed,the polypeptide PKRKRKKSRNGSGR-C-peptide was liberated from the fusion partner. The N-terminal tetradecapeptide extension of C-peptide was subsequently cleaved by trypsin and removed by DE52 column. Results:The nucleotides sequence of plasmid pEDCC was confirmed to be identical with that of designed fusion protein. Recombinant human proinsulin C-peptide was obtained with high purity after purification. Conclusion:Employing truncated asparaginase as the fusion partner and basic-amino-acid-riched octopeptide to modulate isoelectric point is an effective approach to produce recombinant human proinsulin C-peptide.

Abstract: Objective　To investigate the distribution and drug resistance of pathogenic bacteria separated from ascites of patients in Children’s Hospital Affiliated to Zhengzhou University from 2015 to 2021, and to provide a basis for rational clinical antimicrobial agents. Methods　Bacterial culture, bacterial identification and drug sensitivity analysis were performed on 1 058 non-duplicate ascites culture specimens from January 2015 to December 2021. The clinica1 and microbiologica1 data were ana1yzed by WHONET 5.6 and SAS 9.4 Results　Of the 1 058 specimens, 586 (55.39%) were positive for pathogenic bacteria, with a total of 781 strains isolated. There was no significant trend of increase or decrease in the positivity rate over different years. Male children (63.99%) were more prevalent than female children. Appendicitis (59.22%) was the most common disease and Escherichia coli was the most common causative bacteria. Among neonates (≤28 d), the bacteria with the highest detection rate were Klebsiella pneumoniae (23.50%) and Enterococcus faecium (23.50%), while among children (>28 d), the highest detection rate was Escherichia coli (35.98%). Gram-negative bacteria accounted for 64.79% of the 781 strains, mainly Escherichia coli (38.28%), Klebsiella pneumoniae (8.58%), and Pseudomonas aeruginosa (5.89%); Gram-positive bacteria accounted for 29.45%, mainly Enterococcus faecium (8.58%), Streptococcus constellatus (2.69%), and Enterococcus avium (2.43%); fungi accounted for 1.66% and anaerobic bacteria accounted for 4.10%. The resistance rates of Escherichia coli to cefoperazone/sulbactam, piperacillin/tazobactam, imipenem and meropenem were 6.02%, 4.35%, 4.35%, and 3.68%, respectively. The resistance rates of Klebsiella pneumoniae to these drugs were 59.70%, 59.70%, 50.75% and 53.73% respectively. Linezolid-resistant strains of Enterococcus faecium were found. Conclusion　Appendicitis is the most common abdominal infection in children, and the distribution of ascites pathogens varies with ages and diseases. The pathogenic bacteria are mainly Gram-negative bacteria, and the drug resistance of Klebsiella pneumoniae was more serious. It is particularly important to use antibiotics correctly and rationally to reduce the emergence of drug resistant bacteria.

Objective To evaluate the risk factors of patients with acute kidney injury (AKI) in patients with chronic kidney disease (CKD) for the early detection and early treatment of CKD.Methods One hundred and twenty-seven CKD patients were divided into groups according to AKI existing, 60 cases with out AKI (CKD group), 67 cases with AKI (A/C group) and then A/C group patients were divided into non-older age group (35 cases, ＜60 years old) and older age group (32 cases, ≥60 years old).The protopathy, causative factors and so on were analyzed.Results There was different causative factors in different age group.Logistic regression model indicated that the major risk flactors of AKI in CKD were severe infection (OR = 5.236),hypovolemia (OR = 5.083 ),heart failure (OR = 8.283) and using renal toxicity medicine (OR = 5.246),P ＜ 0.05.Conclusion The major risk factors of AKI in CKD patients include severe infection, hypovolemia, heart failure and renal toxicity medicine.

Objective To explore the feasibility and efficacy of constructing tissue engineered cartilage in vitro(using) a perfusion-hydrodynamic pressure bioreactor. Methods Chondrocytes isolated from swine's auricular cartilage were seeded onto polyglycolic acid(PGA) to be cultured in a three dimensional environment for 1 week.Then the chondrocyte-polymer constructs were divided into two groups: the experimental group and control group(8 constructs in each group).The experimental group was put into the perfusion-hydrodynamic pressure bioreactor to be cultured for another 3 weeks.The parameters of bioreactor were set as follows: flow rate of 100 mL/min,clockwise and anticlockwise 30 min respectively,on/off 8 h/16h,hydrodynamic pressure of 100 kpa with 0.5 Hz for 4 h/d.The control group was cultured with the routine method.Specimens were harvested and analyzed by gross observation,histology,typeⅡcollagen immunohistochemistry and biochemistry after 4 weeks. Results After 4 weeks,gross observation showed cartilage-like tissue was formed in both groups,and tissue wet weight of experimental group and control group were(191.03?18.55) mg and(130.78?10.33) mg,respectively(P

Recently, gene therapy has been become a promising approach to cure hemophilia A, a most common recessive bleeding disease. The aim of this study was to determine the perspective of lentiviral vector in hemophilia A gene therapy in vitro and in NOD/SCID mice. Lentivirus transfer vector pXZ9/BDDFVIII containing human B-domain-deleted Factor VIII-IRES-eGFP coding sequence and mock control pXZ9 were constructed. Lentivirus was prepared by co-transfecting 3 plasmids into 293FT cells. 293FT, HLF, human bone marrow mesenchymal stem cells and Chang-liver cells were transfected with the prepared virus. Coagulant activity of human FVIII, human FVIII antigen, human FVIII mRNA transcription and genomic integration were assayed by ELISA, one-step method, RT-PCR and PCR after infection. Lentiviral particles were concentrated by ultracentrifugation and NOD/SCID mice were transfected via portal vein injection. Human FVIII antigen in mouse blood plasma was analyzed by ELISA. eGFP expression was observed by fluorescent microscopy and human FVIII transcription in mouse liver was analyzed by RT-PCR at one month after transduction. The results showed that the high titer of recombinant virus was prepared and used to efficiently transduce the target cells in vitro. At 72 h after transfection, high levels of FVIII activity and FVIII antigen were detected. Human FVIII gene transcription could be detected in the liver of NOD/SCID mice received lentiviral particles carrying FVIII gene. Mouse hepatocytes were transfected with recombinant lentivirus efficiently in vivo. Human FVIII level in mouse blood plasma reached to (49 ± 6) mU, (54 ± 8) mU and (23 ± 4) mU at 72 h, one week and one month after transfection respectively. It is concluded that the lentiviral particles carrying BDDhFVIII gene can high efficiently transfect the target cells both in vitro and in vivo, and the transfected target cells can secrete hFVIII efficiently. The sustained expression of human FVIII in NOD/SCID mice is observed after lentivirus transfection via portal vein injection.
Animals ; Cell Line ; Factor VIII ; genetics ; Gene Expression ; Genetic Therapy ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Peptide Fragments ; genetics ; Plasmids

OBJECTIVETo explore the feasibility of in vitro chondrogenesis by co-culture of chondrocytes and adipose-derived stromal cells (ADSCs) so as to confirm the hypothesis that chondrocytes can provide chondrogenic microenvironment to induce chondrogenic differentiation of ADSCs.

METHODSHuman ADSCs and porcine auricular chondrocytes were in vitro expanded respectively and then were mixed at the ratio of 7:3 (ADSCs: chondrocytes). 200 microl mixed cells (5.0 x 10(7)/ml) were seeded onto a polyglycolic acid/polylactic acid (PGA/PLA) scaffold, 8 mm in diameter and 2 mm in thickness, as co-culture group. Chondrocytes and ADSCs with the same cell number were seeded respectively onto the scaffold as positive control group and negative control group. 200 microl chondrocytes (1.5 x 10(7)/ml) were seeded as low concentration chondrocyte group. There were 6 specimens in each group. All specimens were harvested after in vitro culture for 8 weeks in DMEM plus 10% FBS. Gross observation, histology, immunohistochemistry, wet weight measurement and glycosaminoglycan (GAG) quantification were used to evaluate the results. Multiple-sample t-test statistics analysis was done to compare the difference of wet weight and glycosaminoglycan(GAG) content between the groups.

RESULTSCells in all groups had fine adhesion to the scaffold and could secrete extracellular matrix. In co-culture group and positive control group, cell-scaffold constructs could maintain the original size and shape during in vitro culture. At 8 weeks, cartilage-like tissue formed in gross appearance and histological features, and abundant type II collagen could be detected by immunohistochemistry. Wet weight and glycosaminoglycan(GAG) content of co-culture group were respectively (174 +/- 12) mg and (7.6 +/- 0.4) mg. There were respectively 75% (P < 0.01) and 79% (P<0.01) of those of positive control group. In negative control group, however, constructs shrunk gradually without mature cartilage lacuna in histology. In low concentration chondrocyte group, constructs also shrunk obviously with small amount of cartilage formation at the edge area of the construct, and wet weight was (85 +/- 5) mg, which was 37% (P<0.01) of that of positive control group.

CONCLUSIONSChondrocytes can provide chondrogenic microenvironment to induce chondrogenic differentiation of ADSCs and thus promote the in vitro chondrogenesis of ADSCs.

Adipocytes ; cytology ; Animals ; Cell Differentiation ; Cells, Cultured ; Chondrocytes ; cytology ; Coculture Techniques ; Humans ; Swine ; Tissue Engineering ; methods ; Tissue Scaffolds

OBJECTIVETo study the effect of peptide-conjugated near-infrared quantum dots (QDs) on growth, invasion and metastasis of human buccal squamous cell carcinoma cell line (BcaCD885 cell).

METHODS(1) BcaCD885 cells were labeled by cell-penetrating peptide-conjugated QDs with a maximum emission wavelength of 800 nm (QD800), then labeling efficiency was detected by flow cytometry, and laser-scanning confocal microscope was used to observe the distribution of QD800 within the cells. (2) Different concentrations of QD800 was applied to BcaCD885 cells, and the cell growth of control and three test groups were compared respectively. (3) BcaCD885 cells were labeled by QD800 (BcaCD885/QD800), then transwell chambers and wash way were used to detect the difference of invasion and metastasis ability between BcaCD885/QD800 and BcaCD885 cells.

RESULTS(1) The labeling rate of BcaCD885 cells after 6h was 94.07%, and QD800 distributes in the BcaCD885 cytoplasm. (2) Different concentrations of QD800 showed no negative effects on growth of BcaCD885 cells. (3) The ability of invasion, attachment and motion of BcaCD885 cells were not significantly different between test and control group (P > 0.05).

CONCLUSIONQDs showed no effects on growth, invasion and metastasis ability of BcaCD885 cells. Our results provide science foundation for QDs as a new fluorescence probes to real-time monitor cells and cells imaging in a living.

Animals ; Carcinoma, Squamous Cell ; Cell Line ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Mice ; Mice, Nude ; Peptides ; Quantum Dots

OBJECTIVETo evaluate the association between high sensitivity C-reactive protein (hsCRP) and breast cancer incidence among the non-diabetic females in a large-scale cohort study in Kailuan group.

METHODSThe Kailuan cohort was established on May 1, 2006. Baseline information on demography, lifestyle, medical history, and anthropometry, i.e., body height and weight, were collected during the baseline interview, and breast cancer incidence, mortality and other related outcome information were obtained by follow-up every two years and the related health condition database information were collected every year. Multivariable Cox proportional-hazards regression model was used to calculate the hazard ratios (HRs) and 95%CI (confidence interval) between the level of hsCRP at baseline interview and breast cancer incidence adjusted for age group, body mass index (BMI), marital status (married and single) and tobacco smoking (smokers and non-smokers) when appropriate.

RESULTSBy Dec 31, 2011, a total of 17 402 females were enrolled in the cohort. There were 85 286 person-years of follow-up with a mean follow-up period of (58.81 ± 4.52) months. A total of 75 incident breast cancer cases were collected. Subjects with the highest level (>3 mg/L) of hsCRP at baseline interview were associated with a significantly increased risk of breast cancer (adjusted HR = 1.80, 95%CI = 1.03-3.15) compared with those with the lowest level (<1 mg/L).

CONCLUSIONSElevated levels of hsCRP at baseline interview may be associated with an increased risk of breast cancer among non-diabetic females. Further follow-up and etiological exploration will help to evaluate the association between the hsCRP level and the risk of breast cancer more reliably.

Body Mass Index ; Breast Neoplasms ; diagnosis ; epidemiology ; metabolism ; C-Reactive Protein ; metabolism ; Cohort Studies ; Diabetes Mellitus ; Female ; Humans ; Incidence ; Middle Aged ; Proportional Hazards Models ; Prospective Studies ; Risk ; Risk Factors ; Smoking

BACKGROUND AND OBJECTIVEEarly diagnosis of nasopharyngeal carcinoma (NPC) is difficult due to the insufficient specificity of the conventional examination method. This study was to investigate potential and consistent biomarkers for NPC, particularly for early detection of NPC.

METHODSA proteomic pattern was identified in a training set (134 NPC patients and 73 control individuals) using the surface-enhanced laser desorption and ionization-mass spectrometry (SELDI-MS), and used to screen the test set (44 NPC patients and 25 control individuals) to determine the screening accuracy. To confirm the accuracy, it was used to test another group of 52 NPC patients and 32 healthy individuals at 6 months later.

RESULTSEight proteomic biomarkers with top-scored peak mass/charge ratios (m/z) of 8605 Da, 5320 Da, 5355 Da, 5380 Da, 5336 Da, 2791 Da, 7154 Da, and 9366 Da were selected as the potential biomarkers of NPC with a sensitivity of 90.9% (40/44) and a specificity of 92.0% (23/25). The performance was better than the current diagnostic method by using the Epstein-Barr virus (EBV) capsid antigen IgA antibodies (VCA/IgA). Similar sensitivity (88.5%) and specificity (90.6%) were achieved in another group of 84 samples.

CONCLUSIONSELDI-MS profiling might be a potential tool to identify patients with NPC, particularly at early clinical stages.

Adult ; Aged ; Algorithms ; Antibodies, Viral ; blood ; Antigens, Viral ; blood ; Biomarkers, Tumor ; blood ; Capsid Proteins ; blood ; Female ; Humans ; Male ; Middle Aged ; Nasopharyngeal Neoplasms ; blood ; diagnosis ; Neoplasm Proteins ; blood ; Proteomics ; methods ; Reproducibility of Results ; Sensitivity and Specificity ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; methods