OBJECTIVETo evaluate the time course of granulocyte-colony-stimulating-factor (G-CSF), estrogen and various doses of atorvastatin on endothelial progenitor cells (EPCs) mobilization.

METHODA total of 48 male New Zealand White rabbits were treated with placebo, estrogen (0.25 mg.k(-1).d(-1)), Atorvastatin (2.5, 5, or 10 mg) and G-CSF (50 microg/rabbit/d), respectively. Peripheral EPCs number was surveyed weekly for 4 weeks by FACS analysis (double-positive for PE-CD34/FITC-CD133) and under fluorescent microscope (double-positive for FITC-UEA-1/Dil-acLDL). Serum nitric oxide (NO) and lipids were also measured at the third week.

RESULTSPeripheral EPCs was significantly increased in G-CSF treated animals and remained constant for 4 weeks compared to placebo treated animals. Atorvastatin increased peripheral EPCs dose-dependently from 2.5 to 5 mg and peaked at the third week while peripheral EPCs number was not affected by 10 mg.k(-1).d(-1) atorvastatin during the first 3 weeks and was significantly higher only in the fourth week compared to placebo group. Estrogen also significantly increased peripheral EPCs at the third and fourth week compared to placebo group. At the third week, serum NO was similar in G-CSF group, significantly higher in atorvastatin 5 mg.k(-1).d(-1) and estrogen groups while significantly lower in atorvastatin 10 mg.k(-1).d(-1) group compared to placebo group. Serum lipids were similar among various groups.

CONCLUSIONAtorvastatin, estrogen and G-CSF could mobilize EPCs. The mobilization efficacy is as follows: G-CSF > atorvastatin 5 mg.k(-1).d(-1) > estrogen > atorvastatin 2.5 mg.k(-1).d(-1) > atorvastatin 10 mg.k(-1).d(-1). NO might partly contribute to the mobilizing effect of estrogen and atorvastatin.

Animals ; Atorvastatin Calcium ; Endothelial Cells ; cytology ; drug effects ; Estrogens ; pharmacology ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Heptanoic Acids ; pharmacology ; Hypolipidemic Agents ; pharmacology ; Lipids ; blood ; Male ; Nitric Oxide ; blood ; Pyrroles ; pharmacology ; Rabbits ; Recombinant Proteins ; Stem Cells ; drug effects

Plasma obestatin level was determined in patients with impaired glucose regulation and type 2 diabetes mellitus.The plasma obestatin levels in patients of both groups were significantly decreased as compared with that in controls.Plasma obestatin level was negatively correlated with body mass index,HbA_(1C),waist-to-hip ratio,plasma insulin and HOMA-IR.Obestatin level seems to be related with metabolic disorder.

By using the cell density, cell viability, size distribution of cell aggregates,specific consumption rate of glucose (q_ glc ), specific production rate of lactate (q_ lac ), lactate transform rate (Y_ lac/glc ) and amino acids utilization as the evaluation indexes, the growth and metabolism of HEK293 cells under carrier-free immobilization culture mode were examined and compared with those of HEK293 cells cultured in static tissue flasks. It was found that HEK293 cells grown as suspended cell aggregates in spinner flasks maintained the basic growth and metabolism characteristics of HEK293 cells in stationary anchored culture, and HEK293 cells under carrier-free immobilization culture mode as suspended aggregates in stirred bioreactor facilitate perfusion performance and increase unit productivity. Cultivation of HEK293 cells in carrier-free immobilization culture mode has potential for further improving mammalian cells culture technique.

OBJECTIVETo investigate the efficacy and mechanism of Bushen Huoxue Recipe (, BHR) in the treatment of murine autoimmune premature ovarian failure (POF).

METHODSThe recombinant porcine zona pellucida 4 (pZP4) was expressed in E. coli BL21 (DE3) strain within prokaryotic plasmid pET28a (+), purified by Ni-affinity chromatography and verified by Western blot. Murine autoimmune POF model was established by immunization with pZP4 of female BALB/c mice. Fifty POF mice were randomly divided into 5 groups, which were respectively given low (3.75 mg/kg), moderate (7.5 mg/kg), and high dose (15.0 mg/kg) of BHR by gastrogavage once daily for 20 days, with 17-β-estradiol (0.13 mg/kg) and normal saline as positive and negative control. Estrous cycles were analyzed through vaginal smears, serum estradiol (E) levels, and anti-pZP4 antibody titers were detected by ELISA. The proliferative responses in vitro of spleen lymphocytes to pZP4 antigen restimulation were measured by [(3)H]-thymidine incorporation, and the histomorphology changes of ovary were evaluated by optical microscope.

RESULTSThe purified pZP4 was visible as a single lane with 14.4 kD in SDS-PAGE and Western blot. The murine POF model with lengthening estrous cycles, decreased levels of serum E2, high titers of serum anti-pZP4 antibody, and reduced ovarian follicles and corpus lutea were established by immunization with recombinant pZP4. Treatment with moderate and high dosage BHR significantly increased ovarian follicles and reduced the proliferation of spleen lymphocytes to the pZP4 antigen of POF mice (P <0.05). However, only the high dosage BHR administration significantly improved the estrous cycles, elevated the serum E levels (P <0.01), and decreased the serum anti-pZP4 antibody titers of model mice P<0.05).

CONCLUSIONSThe recombinant pZP4 could evoke the antigen-specific immune response in mice and induce the autoimmune ovarian injury. It has been demonstrated that BHR was able to increase the serum E levels and protect ovarian functions from the autoimmune injury in murine POF model.

Animals ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Egg Proteins ; immunology ; isolation & purification ; Female ; Immunization ; Immunocompetence ; drug effects ; Membrane Glycoproteins ; immunology ; isolation & purification ; Mice ; Mice, Inbred BALB C ; Ovary ; drug effects ; immunology ; pathology ; Primary Ovarian Insufficiency ; drug therapy ; immunology ; pathology ; Receptors, Cell Surface ; immunology ; isolation & purification ; Recombinant Proteins ; immunology ; isolation & purification ; Sus scrofa ; Zona Pellucida Glycoproteins

Tuberculosis(TB),a chronic infectious disease caused by Mycobacterium tuberculosis(MTB), is the outcome of the interaction between polygenes and environmental factors,which has been shown in lots of genetic studies. In recent years,the research of TB susceptibility genes and their single nucleotide polymorphism(SNP)loci has become a hot topic,but genes and SNPs exactly associated with TB susceptibility are rarely reported. What's more,some susceptibility genes are different among races, populations and geographical distributions, resulting in inconsistency, or even contrary conclusions drawn from local studies of different populations or areas. In order to obtain susceptibility genes that are widely applicable and exactly associated with TB,it is necessary to screen the genes preliminarily in the laboratory animals with gene diversity, and thereafter, verify those genes in animals or humans. The Collabrative Cross(CC)mice have the advantages of diverse and clear genetic background,which can provide a new tool for the research of TB susceptibility. This review summarizes the advances in the studies of TB susceptibility genes and their SNPs, and looking forward to the application of CC mice in these studies and its significance.

Objective In order to provide a scientific basis for influenza prevention and control,analyzing the epidemic characteristics and laws of influenza outbreaks in Xuzhou area during 2005-2011.Method Using fluorescent-PCR method to detect influenza virus nucleic acid on Nasopharyngeal swab specimens collected from influenza outbreak cases during 2005-2011 and fast classifying influenza virus A1 (H1N1),A3 (H3N2),new H1N1 BV (Victoria) and BY (Yamagate) on subtypes.At the same time,isolating the influenza virus with MDCK cells,and sending them to the National Influenza Center for review,after the preliminary identification of the isolated influenza virus.Results During 2005-2011,there are 53 influenza outbreaks in Xuzhou area,which caused by influenza virus subtype BV accounting for 26.42％ (14/53),A3 accounting for 49.1％ (26/53),A3 and A 1 mixture accounting for 3.77％ (2/53) and the new H1N1 accounting for 20.75％ (11/53).The outbreaks in 2007 and 2009 mainly caused by A3,and show that the winter spring (January) and summer autumn (September) as two popular peaks during 2005-2011 ; BV mainly causes the outbreaks from Feb.to Jun.Conclusion In Xuzhou area,since the winter of 2005,influenza virus subtype BV,the A3,and new H1N1 has alternately as mainly predominant strain,caused local influenza outbreaks.In which BV has increased trend year by year during 2005-2011.The students in primary and secondary schools are the major crowd of influenza outbreaks.Fluorescent-PCR detection methods could be a preferred method for reliable and rapid diagnostic of epidemic influenza outbreaks.

By using the size distribution of cell aggregates, viable cell density, cell viability, specific consumption rate of glucose (q(glc)), specific production rate of lactate (q(lac)) and lactate transform rate (Y(lac/glc)) as the evaluation indexes, the effects of hydrodynamic on aggregates formation, growth and metabolism of HEK293 cells in suspension culture were examined in 250mL spinner-flasks by setting the agitation rates at 25, 50, 75 and 100r/min, respectively. It was found that agitation plays an important role in HEK293 cell aggregates formation and cell aggregates size distribution. After 7d cultivation in spinner-flasks operated at 50r/min and 75r/min, the average diameter of HEK293 cell aggregates was 201 microm and 175 microm, respectively, with the fraction of aggregates larger than 225 microm less than 10%. The cell viability was kept above 90% with the metabolic indexes, including q(glc), q(lac) and Y(lac/glc) kept constant. These results demonstrated that hydrodynamic derived from the proper agitation play a decisive role in controlling the formation and size distribution of HEK293 cell aggregates, and provided sufficient mass transfer to support the normal growth and metabolism of HEK293 cells in suspended aggregates.
Bioreactors ; Cell Aggregation ; Cell Culture Techniques ; instrumentation ; Cell Line ; Cell Proliferation ; Humans ; Kinetics

Using the amino acids 1-147 of the yeast transcriptional activator GAL4 as the DNA-binding domain and four tandem repeats of the 12-aa peptide (DALDDFDLDMLG) of the herpesvirus as the activation domain, an artificial transcription factor, GVP4,was constructed via the linkage of the nuclear localization signal sequence of SV40. And then, GVP4 was cloned into expression vector pcDNA3 . 1/Hygro ( + ) . Various amounts of targeting sites of artificial transcription factor were linked to the upstream of promoter CMV in exogenous gene expression vector pcDNA3.1 ( + ) that separately harbored EGFP cDNA and t-PA cDNA.The CHO cells were then co-transfected with GVP4 expression vector and EGFP or t-PA expression vector. The effect of GVP4 on exogenous gene expression was evaluated by measuring the fluorescence intensity of EGFP in CHO cells and the concentration of t-PA in the supernatant. GVP4 showed positive effect on the enhancement of exogenous gene expression in CHO cells integrated with targeting sites of artificial transcription factor. And, CHO cells integrated with 10 targeting sites of GVP4 was more favorable to foreign gene expression, which resulted in 2-3-fold increase in both EGFP and t-PA expressions. These results indicated that artificial transcription factor is potent in the enhancement of exogenous gene expression in mammalian cells.
Amino Acid Sequence ; Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Flow Cytometry ; Gene Expression Regulation ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Tissue Plasminogen Activator ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism ; Transcriptional Activation ; Transfection

OBJECTIVE: To explore the genetic etiology of a child with Pitt-Hopkins syndrome. METHODS: A child who had presented at the Medical Genetics Center of Gansu Provincial Maternal and Child Health Care Hospital on February 24, 2021 and his parents were selected as the study subjects. Clinical data of the child was collected. Genomic DNA was extracted from peripheral blood samples of the child and his parents and subjected to trio-whole exome sequencing (trio-WES). Candidate variant was verified by Sanger sequencing. Karyotype analysis was also carried out for the child, and her mother was subjected to ultra-deep sequencing and prenatal diagnosis upon her subsequent pregnancy. RESULTS: The clinical manifestations of the proband included facial dysmorphism, Simian crease, and mental retardation. Genetic testing revealed that he has carried a heterozygous c.1762C>T (p.Arg588Cys) variant of the TCF4 gene, for which both parents had a wild-type. The variant was unreported previously and was rated as likely pathogenic based on the guidelines of the American College of Medical Genetics and Genomics (ACMG). Ultra-deep sequencing indicated that the variant has a proportion of 2.63% in the mother, suggesting the presence of low percentage mosaicism. Prenatal diagnosis of amniotic fluid sample suggested that the fetus did not carry the same variant. CONCLUSION The heterozygous c.1762C>T variant of the TCF4 gene probably underlay the disease in this child and has derived from the low percentage mosaicism in his mother.
Child ; Female ; Humans ; Male ; Pregnancy ; Intellectual Disability/genetics* ; Mosaicism ; Mothers ; Mutation ; Parents ; Transcription Factor 4/genetics*

OBJECTIVETo further study the characteristics of drugs with neutral property in two-way application and conditioned dominance by observing the action characteristic of 10 traditional Chinese medicines with neutral property in hemorheological indicators of heat stagnation and blood stasis syndrome and cold stagnation and blood stasis syndrome rats.

METHODThe model of heat stagnation and blood stasis syndrome rats was established by injecting carrageenan and dry yeast, while the model of cold stagnation and blood stasis syndrome rats was established by body freezing. Subsequently, 10 traditional Chinese medicines with neutral property, 5 traditional Chinese medicines with heat property and 5 traditional Chinese medicines with cold property were selected for intervention to observe the changes in such indicators as whole blood viscosity, plasma viscosity and hematocrit and analyze the action characteristics of drugs with neutral property.

RESULTANOVA showed that among six of the 10 traditional Chinese medicines with neutral property, including Typhae Pollen, Sarcandrae Herba and Sappan lignum, could obviously increase the hemorheological indicators of both heat stagnation and blood stasis syndrome and cold stagnation and blood stasis syndrome rats; five traditional Chinese medicines with cold property, such as Salviae Miltiorrhizae Radix et Rhizoma, Leonuri Herba, Rhei Radix et Rhizoma, could significantly ameliorate the hemorheological indicators of heat stagnation and blood stasis syndrome rats (P < 0.01 or P < 0.05), and Salvia Miltiorrhiza Radix et Rhizoma alone could ameliorate the hemorheological indicators of cold stagnation and blood stasis syndrome rats (P < 0.05); all of the five traditional Chinese medicines with heat property could significantly ameliorate the hemorheological indicators of cold stagnation and blood stasis syndrome rats (P < 0.01), among which Carthami Flos and Notoginseng Radix et Rhizoma could significantly ameliorate the hemorheological indicators of cold stagnation and blood stasis syndrome rats. According to the average high-shear blood viscosity analysis, drugs with neutral property showed similar action characteristics to those with cold property in ameliorating hemorheology indicators of heat stagnation and blood stasis syndrome rat and better effect than those with heat property in reducing whole blood viscosity; and traditional Chinese medicines with neutral property have the similar action characteristics to those with heat property in improving the hemorheology indicators of cold stagnation and blood stasis syndrome rat and better effect than those with heat property in reducing whole blood viscosity.

CONCLUSIONUnder the condition of heat stagnation and blood stasis syndrome, traditional Chinese medicines with neutral property show the similar action characteristics to those with cold property; but under the condition of cold stagnation and blood stasis syndrome, traditional Chinese medicines with neutral property show the similar action characteristics to those with heat property. This indicates that traditional Chinese medicines with neutral property show both heat and cold properties under he conditions of heat stagnation and blood stasis syndrome and cold stagnation and blood stasis syndrome.

Analysis of Variance ; Animals ; Blood Circulation ; drug effects ; physiology ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; therapeutic use ; Hemorheology ; drug effects ; Homeostasis ; drug effects ; Male ; Medicine, Chinese Traditional ; methods ; Rats ; Rats, Sprague-Dawley ; Thrombosis ; drug therapy ; physiopathology