AIM:To investigate the effect of activation of retinoid X receptor (RXR) on transforming growth factor β1 (TGF-β1) induced collagen synthesis under hypoxic environment in rat cardiac fibroblasts (CFs) and underlying molecular mechanisms .METHODS: CFs were cultured using myocardial tissue with dry method .Hypoxic environment was established for CFs by continuous nitrogen supplement .Type I and type III collagens in supernatants were detected by ELISA.Nuclear and cytoplasmic extractions were prepared using NE-PER nuclear and cytoplasmic extraction reagents .The protein levels of Smad2 and p-Smad2 were determined by Western blot and immunocytochemical staining .RESULTS:Un-der hypoxic condition , TGF-β1 (0.01~10 μg/L) increased the synthesis of type I and type III collagens in a dose-de-pendent manner in the CFs .At the concentration of 5μg/L, the synthesis of collagen I and III was significantly increased as compared with control group (P<0.01).RXR agonist 9-cis-retinoic acid (9-cis-RA;10 -9 ~10 -6 mol/L) decreased TGF-β1 (5μg/L)-induced synthesis of type I and III collagens in a dose-dependent manner in the CFs under hypoxic con-dition.The synthesis of type I and type III collagens was significantly inhibited by 9-cis-RA (P<0.01).Smad2 inhibitor ( 20 nmol/L) showed similar inhibitory effect on the synthesis of type I and III collagens induced by TGF -β1 under hypoxic condition.Compared with TGF-β1 intervention group, the cytoplasmic level of p-Smad2 in the CFs was significantly in-creased in TGF-β1+9-cis-RA group, but the nuclear p-Smad2 level was significantly decreased (P<0.05).CONCLU-SION:Retinoid X receptor agonist 9-cis-RA inhibits TGF-β1-induced synthesis of type I and type III collagens in the CFs by repressing p-Smad2 nuclear translocation under hypoxic condition .

BACKGROUND:The homing ability of mesenchymal stem cells is closely associated with the effects of celltransplantation. Clarifying the mechanism of chemotaxis and migration wil contribute to enhance the clinical application of mesenchymal stem cells. OBJECTIVE:To investigate the effect of Cdc42 in the homing of human umbilical cord mesenchymal stem cells. METHODS:First, mesenchymal stem cells were isolated from human umbilical cord, and co-cultured with tumor necrosis factorα, interleukin-1β, and transforming growth factorβ. Western blot assay was used to test the level of Cdc42. Besides, Cdc42 siRNA was synthesized by chemical method to transfect the cells, and cellmigration and adhesion were measured by Transwel and Matrigel separately. Meanwhile, the activity of signal molecule, extracellular regulated protein kinase 1/2, was evaluated by western blot. RESULTS AND CONCLUSION:The results indicated that the inflammation factors induced the highly expression of Cdc42 in human umbilical cord mesenchymal stem cells, almost double level to controls. siRNA notably inhibited the migration and adhesion of human umbilical cord mesenchymal stem cells through Cdc42 down-regulation, and the extracellular regulated protein kinase 1/2 and phosphorylation form were also decreased simultaneously. In a word, we speculate Cdc42 plays a role in the chemotaxis of human umbilical cord mesenchymal stem cells in vitro.

Objective To explore self-emulsifying particle size characterization methods and compare the regularity of various methods. Methods By setting the clarity level of turbidity standard solution, with two less soluble drugs-diterpene lactone compounds Chuanhuning and dihydropyridine drug nifedipine as model drugs, 10-12 clarity level prescriptions were selected from six different ternary phase diagram. Laser particle size scanner was used to determine the particle size, and UV-visible spectrophotometry to determine its absorbance. Three methods of particle size characterization rules were compared by drawing charts. Results There was a positive correlationship among droplet particle size, absorbance and clarity grade of emulsion formed by prescription in the same phase diagram. But, there was no regularity among droplet particle size, absorbance and clarity grade of emulsion formed by prescription in different phase diagram. Conclusion The droplet particle size of emulsion formed by prescription containing the same drugs and excipients in different proportions can be compared by clarity with visual method or absorbance with UV-visible spectrophotometer.

AIM: To investigate multi-potential of rat bo ne marrow mesenchymal stem cells (rBMMSC) and mutation inclination, the rBMMSC w ere long passaged in vitro. METHODS: Cellular cycles of diff erent passages were assayed by FA CSan flow cytometry and karyotypes of passage 6, passage 25 and passage 45 were compared by G-binding analysis. RESULTS: The early passages and long-term passages all showed st rong proliferation; passage 6, passage 25 and passage 45 all showed normal karyo type. CONCLUSION: Long-term culture and passage of rBMMSC still remain s strong proliferation. With this capability, the mutation inclination is not enhanced.

Objective To clarify the association between rs1050450 polymorphism in Glutathione peroxidase-1 (GPx-1) and the risk of cardi-ovascular diseases (CVD) by performing a meta-analysis of published studies. There is growing evidence from different study types for an association of the GPx-1 polymorphism and cardiovascular outcomes, but observational studies have so far shown inconsistent results. Me-thods Relevant publications were searched through PubMed, Embase database databases and the Cochrane Library. We used odds ratios (ORs) with 95%confidence intervals (CIs) to assess the strength of association under the best genetic model. Both Q statistic and the I2 were used to check heterogeneity. Meta-regression analysis was performed to explore heterogeneity source. Sensitivity analysis, cumulative me-ta-analysis analysis and publication bias were used to test the reliability of the results. Results Data were available from two cohort studies and 8 case-control studies involving 1,430 cases and 3,767 controls. The pooled ORs for overall CVD risk was 1.36 with 95%CI:1.08-1.70 under a co-dominant model, and that for East Asian subgroup was 1.84 (95%CI:1.39-2.43). Substantial heterogeneity for ORs were de-tected among all the included studies, mainly caused by ethnic differences between East Asian and non-East Asian populations. Although Egger’s regression test suggested no statistical significant publication bias, Begg’s funnel plot exhibited obvious asymmetry. The statistical significance disappeared after adjusting for potential publication bias in the overall studies. However, no substantial publication bias was found in the East Asian subgroup. Conclusions GPx-1 gene Pro198Leu and Pro197Leu polymorphisms considerably increased the risk of CVD in the East Asian population. Large-scale investigations are needed to confirm the results in different ethnicities.

Objective To analyze the setup errors of bone metastases patients by tomotherapy with megavoltage CT (MVCT) and calculate the CTV-PTV margins.Methods 30 patients with bone metastases were enrolled.All patients received tomotherapy,fixed with body net and received MVCT scanning before radiation.The MVCT images were registered with the kilovoltage CT (kVCT) images,the setup errors of X (lateral),Y (vertical),Z (longitudina) and Roll (transverse profile rotation) were obtained according to the formula M =2.5Σ+0.7σ calculated CTV-PTV margin.Results 30 patients were received 494 MVCT images.The errors of systemic±random were (2.85±0.77) mm,(3.11±0.95) mm,(2.21±0.55) mm,and (0,55±0.24)° on X,Y,Z and Roll directions,respectively.The CTV-PTV margins were 3.64 mm,4.17 mm,and 2.86 mm on X,Y,Z directions,respectively.Conclusion The application of image-guided technology for bone metastases can correct positioning in time,which greatly reduces setup errors of the fractionated treatment,further improves the treat accuracy and has a positive value in guiding clinical radiotherapy.

AIM: To investigate the biological characterics of human second-trimester fetal cord blood mesenchymal stem cells (MSC) and its application prospects in utero gene transfer/therapy (IUGT). METHODS: Nuclear cells separated from cord blood were cultured in DMEM medium. Surface antigens of the MSC were analyzed by the FACScan flow cytometry. Adipogenic and osteogenic mediums were used to assess the differentiation ability of the cells. Adenovirus vector deliver green fluorescent protein gene (Ad-GFP) was used to transfected the MSC and the expressing of GFP was detected by fluorescent microscope. The MSC were injected into the liver of newborn rat. The immunofluorescence analysis was conducted to determine the presence of double-positive CD105+/CD166+ cells in different organs of rats. MSC were subcutaneous injected into the human-nonobese diabetes/severe combined immunodeficiency disease (NOD/SCID) mice and carcinogenesises of the MSC in vivo were detected by pathological diagnosis. RESULTS: MSC could be separated from fetal cord blood. These cells were uniformly positive for CD29, CD44, CD59, CD105, CD166 and negative for CD34, CD45, CD80, CD86, HLA-DR. The cells had the abilities to differentiate into adipogenic and osteogenic cells in vitro, expressed the GFP at high levels (56 32%?3 28%). The MSC were located at different organs after injected into the newborn rats and didn't have carcinogenicity in vivo. CONCLUSION: Human second-trimester fetal cord blood MSC is an promising target cells in fetal IUGT.

Humanumbilicalveinendothelialcells(HUVECs)weretreatedwith3nmol/Lliraglutidefor10, 15, 30, 45, 60, 90, 120, 150, 180, 210, 240, and 270 minutes at the concentrations of 5. 5 or 30 mmol/L glucose. Western blot analysis was used to detected protein expression and phosphorylation of insulin receptor substrates-1 ( IRS-1 ) and endothelial nitric oxide synthase ( eNOS ) . The results showed that the baseline level of phosphorylated-eNOS/eNOS was lower in high glucose group than that in normal group(0. 239 ± 0. 016 vs 0. 400 ± 0. 02,P<0. 05). Liraglutide time-dependently increased phosphorylated-eNOS/eNOS and phosphorylated-IRS-1/IRS-1 levels at 5. 5 or 30 mmol/L glucose.

Objective Aiming at detecting Staphylococcus aureus、Pseudomonas aeruginosa and Klebsiella pneumoniae in laboratory animals,the paper provides a rapid,sensitive and simple test method.Methods According to Staphylococcus aureus nuc gene,Pseudomonas aeruginosa LasI gene,Klebsiella pneumonia PhoE gene and general 16S rRNA gene, designed specific primers;Through the optimization of multiplex PCR primer concentrations and annealing temperature, the specificity and sensitivity of detection, establishing multiplex PCR system.Application of the PCR system test specimens of artificial infections and experiment animal feces is compared with traditional test method.Results Multiplex PCR amplification of Staphylococcus aureus (153 bp), Pseudomonas aeruginosa (600 bp) with Klebsiella pneumoniae (368 bp) and general (520 bp).The multiplex sensitivity for the purpose of 10pg, specificity of detection was not detected from other pathogens.Application of establishing multiplex PCR system to detect the artificial positive samples, and detect 1 Pseudomonas aeruginosa positive case in 76 fecals.Conclusions This paper established the multiplex PCR method which has the advantages of specific,sensitive,simple and rapid, and provides a reliable way for rapid test in laboratory animals microbiology.

The aim of this study is to establish a multiplex polymerase chain raction (PCR) to identify of four kinds of laboratory animal pathogens: Pasteurella multocida, Bordetella bronchiseptica, Mycoplasma pneumoniae and Klebsiella pneumoniae.Methods Specific primers were designed based on GenBank data.The multiplex PCR system was established through optimization of multiple PCR and detection of its specificity and sensitivity.This technique was used to test artificially infected samples and tracheal secretions of experimental animals (rat, mouse, guinea pig, rabbit, hamster), and comparing the detection results by this method and traditional detection test.Results Target bands of Pasteurella multocida (356 bp), Bordetella bronchiseptica (237 bp), Mycoplasma pneumoniae (266 bp), and Klebsiella pneumoniae (142 bp) were obtained, with a detection sensitivity of Klebsiella pneumoniae of 10 pg, and that of Pasteurella multocida, Bordetella bronchiseptica and Mycoplasma pneumoniae of 1 pg by this newly developed multiplex PCR assay.No target bands were observed from the non-specific pathogens of artificially infected samples.The tracheal secretions taken from 45 experimental animals (mice and rabbits) were tested with this new PCR assay, among which 15 cases of Klebsiella pneumonia and 9 cases of Pasteurella multocida were detected as positive, while all the results of traditional method and serological test were negative.Conclusions A simple, rapid, specific and highly sensitive multiplex PCR system has been successfully established.It is valuable for detection of Pasteurella multocida, Bordetella bronchiseptica, Mycoplasma pneumoniae, and Klebsiella pneumoniae in laboratory animals.