AIM: To study the cytological characteristics and gene expression of normal cultured bEnd.3, a mouse brain microvascular endothelial cell strain. METHODS: The morphology of bEnd.3 was studied by light and electronic microscopy, its molecular markers were observed by immunocytochemistry. Cell proliferation kinetics and apoptosis were analyzed by flow cytometry and MTT assay, PGE_2 level was measured by ELISA, and expression of the genes that closely related with vascular endothelial functions was studied by gene micro-array. RESULTS: bEnd.3 had morphological characteristics of microvascular endothelial cells (MVEC) growing in a cobblestone pattern, forming tube-like structure or capillary network and having microvilli. Furthermore, bEnd.3 showed positive staining for vW factor and CD34 and secreted high level of PGE_2 (644.55?30.24 ng/L). Gene micro-array analysis showed CD31, CD36, CD105 expression, and other genes closely related to microvascular endothelial functions also expressed at relatively high level. In addition, bEnd.3 responsed sensitively to mitogen such as basic fibroblast growth factor. CONCLUSION: bEnd.3 is a kind of MVEC, and it can be utilized to study the mechanisms of some diseases such as cancers and cardio- cerebral vascular diseases.

Objective To investigate the value of lower limbs fixation methods for the treatment of intussusception in children with air enema with.Methods 2 1 1 pediatric patients with intussusception who had accepted the treatment of air enema with two dif-ferent fixation methods on lower limbs were enrolled.Comparisons of median treatment duration and therapeutic effect between the two methods were investigated.Results In 32 patients with knee-joint fixation method,27 were successful with median treatment duration 4.84 minutes.Meanwhile in other 179 ones with lower limbs fixation method,152 were successful with median duration 7.96 minutes.And the duration difference between two methods was found (P<0.05).Conclusion Knee-joint fixation may help significantly shorten the median treatment duration for the treatment of intussusception with air enema in children.

Objective To observe the surgical results of modified percutaneous endoscopic interlaminar decompression(PEID) and traditional PEID in the treatment of degenerative lumbar spinal stenosis (DLSS),and to explore the optimizations scheme of PEID for DLSS.Methods 60 patients (36 males and 24 females) were brought into the research for DLSS.According to the different operation ways,the patients were randomly divided into the modified PEID group(observation group) and traditional PEID group (control group) according to the digital table method,30 cases in each group.The surgical outcome,indicators included the change in spinal canal,visual analogue scale (VAS) score and Oswestry disability index (ODI) at pre-operation,postoperative 3 d,postoperative 3 months and postoperative 6 months,operation time,headache and painful stiff nape incidence in the operation,postoperative complications were compared between the two groups.Results The VAS scores (postoperative 3 days,postoperative 3 months and postoperative 6 months) of the observation group were (4.37 ± 1.20) points,(2.59 ± 1.30) points,(1.29 ± 1.21)points respectively,which of the control group were (4.45 ± 1.22)points,(2.67 ± 1.36)points,(1.17 ± 1.10)points respectively,which were significantly better than (7.93 ± 1.56)points of the observation group and (8.22 ± 1.70) points of the control group before operation,the differences were statistically significant (F =1 254.387,512.762,all P ＜ 0.05).The ODI scores (postoperative 3 days,postoperative 3 months and postoperative 6 months) of the observation group were (48.64 ± 19.59) points,(27.66 ± 10.22) points,(10.69 ± 8.87) points respectively,which of the control group were (47.22 ± 20.96) points,(25.17 ± 11.93) points,(10.16 ± 7.89) points respectively,which were significantly better than (75.20 ± 23.20) points of the observation group and (70.35 ± 28.66) points of the control group before operation,the differences were statistically significant(F =1 254.387,512.762,all P ＜ 0.05).The VAS and ODI scores (pre-operation,postoperative 3 days,postoperative 3 months andpostoperative 6 months) of the observation group and control group had no statistically significant differences (VAS:t =2.088,2.124,3.021,3.173;ODI:t =2.366,1.079,1.694,1.573,all P ＞ 0.05).The incidence of neck pain and operation time of the observation group were 20.69％,(63 ± 7) min,which were significantly lower than 87.50％ and (157 ± 8)rin of the control group,the differences were statistically significant(t =3.601,2.167,all P ＜ 0.05).The central sagittal diameter of the spinal canal and the central transverse diameter of the spinal canal between the observation group and the control group had no statistically significant differences (x2 =4.260,t =3.694,all P ＞ 0.05).Conclusion Modified PEID has advantages in surgical efficiency,operation time and headache and painful stiff nape incidence compared with traditional PEID,so it can be chosen for DLSS.

Objective To investigate the association between 2 single nucleotide polymorphisms( SNPs) [rs2243250 in interleukin(IL)- 4 gene and rs1800925 in IL - 13 gene]in Henoch - Schonlein purpura(HSP)and Henoch - Schonlein purpura nephritis(HSPN)in Han Chinese children. The level of IL - 4 and IL - 13 in serum were compared between HSP and HSPN. Methods A case - control study was adopted in this research,and 383 children with HSP or HSPN were recruited in this study,409 normal children served as controls. The genotypes of 2 SNPs were detected by using polymerase chain reaction - restriction fragment length polymorphism(PCR - RFLP),and the serum levels of IL - 4 and IL - 13 in HSP and HSPN patients were detected by using enzyme linked immunosorbent assay (ELISA). All the data were analyzed by SPSS 16. 0 software. Results (1)There was no association(polymorphism of SNP rs2243250 in IL - 4 gene)in HSP group and HSPN group compared with the healthy control group(P ﹥ 0. 05);and the polymorphism of another SNP(rs1800925 in IL - 13 gene)was associated with HSP(P = 0. 037),and the fre-quency of T allele of the study group was higher than that of the healthy control group(P = 0. 027). But there was no difference between the study group(HSP group and HSPN group)and the healthy control group in frequency of TT genotype(P = 0. 132),and there was no association between HSPN group and healthy control group(P = 0. 487);also there was no association between polymorphism of this SNP in HSP group and HSPN group(P = 0. 129).(2)There was an association between polymorphism of IL - 13 gene and IL - 13 level in serum,patients with TT genotype had a higher serum IL - 13 level than any other genotypes(P ﹤ 0. 05),but there was no statistically significant difference in IL - 4 level between TT genotype and other genotypes(P ﹥ 0. 05).(3)There was an association between polymorphism of IL - 13 gene and IL - 13 level in serum patients with HSPN who had a higher level of serum IL - 13 than the patients with HSP( P ﹤ 0. 05),but there was no difference in serum IL - 4 level between HSP group and HSPN group. Conclusions The polymorphism of SNP(rs1800925 in IL - 13 gene)was associated with HSP group,and the poly-morphism of this SNP might affect the level of IL - 13 in serum. The patients with HSPN have a higher level of serum IL -13 than the patients with HSP,and high level of IL -13 may be a risk factor in the progression from HSP to HSPN.

OBJECTIVETo establish a real-time quantitative PCR method for detecting the levels of the signal joint T cell receptor excision circles (sjTRECs) in murine thymocytes and spleen lymphocytes for determining the amount of naive T cells and evaluating the thymic function.

METHODSThe genomic DNA was extracted from murine thymocytes and splenocytes for PCR amplification of the target fragments. After purification of the PCR product, the recombination-activating gene 2 (RAG(2)) fragment was cloned into pGEMT-Easy vector to construct the standard plasmid. After PCR optimization, the standard curve was obtained and the samples (thymocytes and splenocytes of BALB/c and C(57)BL/6 mice) were detected for sjTRECs by real-time quantitative PCR.

RESULTSThe standard plasmid was correctly constructed, and the standard curve with high reliability was obtained. No statistical difference was observed in sjTREC contents in the T lymphocytes between the two mouse strains.

CONCLUSIONSReal-time quantitative PCR for sjTREC analysis is established successfully, which offers an important means for thymic function analysis and a reliable model establishment for study the thymus.

Animals ; Gene Rearrangement, T-Lymphocyte ; Lymphocyte Count ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Polymerase Chain Reaction ; Receptors, Antigen, T-Cell ; genetics ; metabolism ; T-Lymphocytes ; cytology ; Thymus Gland ; cytology ; immunology

Objective :To explore changes of serum levels of galectin-3 and pentraxin-3 (PTX-3) in patients with chro-nic heart failure (CHF) and their correlation with prognosis .Methods : A total of 150 CHF patients (CHF group) treated in our hospital and 150 healthy volunteers undergoing physical examination simultaneously (healthy group ) from Jan 2015 to Dec 2015 were enrolled .Serum levels of galectin-3 ,PTX-3 and N-terminal pro brain natriuretic peptide (NT-proBNP) ,left atrial diameter (LAD) ,left ventricular end-diastolic dimension (LVEDd) and left ven-tricular mass index (LVMI) were compared between two groups at enrollment .According to one-year follow-up out-come ,CHF group (12 cases lost ,another 138 cases) was divided into poor prognosis group (n＝36) and good prog-nosis group (n＝102).Spearman linear correlation analysis was used to analyze correlation among serum galectin-3 , PTX-3 levels and poor prognosis of CHF patients .Results :Compared with healthy group ,there were significant rise in serum levels of galectin-3 [ (2-23 ± 0-25) ng／ml vs .(16-61 ± 1-48) ng／ml] ,PTX-3 [ (1-28 ± 0-54) μg／L vs. (3-58 ± 0-52) μg／L] ,NT-proBNP [(223-23 ± 76-28) pg／ml vs.(952-75 ± 85-43) pg／ml] ,LAD ,LVEDd and LV-MI in CHF group , P＝0-001 all.Compared with good prognosis group at enrollment ,there were significant rise in serum levels of galectin-3 [ (18-52 ± 1-91) ng／ml vs.(24-63 ± 2-26) ng／ml] and PTX-3 [ (2-65 ± 0-74) μg／L vs. (3-95 ± 1-05) μg／L] in poor prognosis group , P＝0-001 both .Spearman linear correlation analysis indicated that serum galectin-3 and PTX-3 levels were significant positively correlated with major adverse cardiovascular events in CHF patients ( r＝0-608 ,0-558 , P＝0-001 both).Conclusion :Serum levels of galectin-3 ,PTX-3 and NT-proBNP levels are significant rise and closely related to prognosis of CHF patients ,can be used as auxiliary indexes predicting prognosis and may provide the basis for formulation of target therapy .

OBJECTIVETo isolate and characterize indigenous algicidal bacteria and their algae-lysing compounds active against Microcystis aeruginosa, strains TH1, TH2, and FACHB 905.

METHODSThe bacteria were identified using the Biolog automated microbial identification system and 16S rDNA sequence analysis. The algae-lysing compounds were isolated and purified by silica gel column chromatography and reverse-phase high performance liquid chromatography. Their structures were confirmed by Nuclear Magnetic Resonance (NMR) and Fourier Transform Infrared (FT-IR) spectroscopy. Algae-lysing activity was observed using microscopy.

RESULTSThe algae-lysing bacterium LTH-2 isolated from Lake Taihu was identified as Serratia marcescens. Strain LTH-2 secreted a red pigment identified as prodigiosin (C20H25N3O), which showed strong lytic activity with algal strains M. aeruginosa TH1, TH2, and FACHB 905 in a concentration-dependent manner. The 50% inhibitory concentration (IC50) of prodigiosin with the algal strains was 4.8 (± 0.4)× 10⁻² μg/mL, 8.9 (± 1.1)× 10⁻² μg/mL, and 1.7 (± 0.1)× 10⁻¹ μg/mL in 24 h, respectively.

CONCLUSIONThe bacterium LTH-2 and its pigment had strong Microcystis-lysing activity probably related to damage of cell membranes. The bacterium LTH-2 and its red pigment are potentially useful for regulating blooms of harmful M. aeruginosa.

Anti-Bacterial Agents ; pharmacology ; Bacteria ; classification ; genetics ; metabolism ; Lakes ; Microcystis ; growth & development ; Phylogeny

HPV16 E7 fusion protein was expressed in E. coli BL21, and its applied value for HPV was evaluated. HPV16 E7 gene was amplified by PCR, and cloned into prokaryotic expression vector pGEX6p-1. The recombinant plasmid was transformed into E. coli BL21, and HPV16 E7 fusion was expressed through IPTG induction. The expressed product was analyzed by SDS-PAGE and Western blot, subsequently purified according to Glutathione Sepharose 4B purification procedure. An indirect ELISA with the purified fusion protein as the coating antigen was then established to detect E7 serum antibodies from mice immunized with recombinant Listeria monocytogenes delivering HPV16 E7. The results demonstrated that the soluble fusion protein was highly expressed at 25 degrees C after induction with 0.5 mM IPTG. Furthermore, the result of Western blot analysis showed that the fusion protein had good specific reaction with an anti-E7 monoclonal antibody. Indirect ELISA result confirmed that the fusion protein could detect the serum antibodies against E7 with a titer of 1:200. The expressed GST-E7 fusion protein was immunocompetent, which was useful in the research of E7 biological function and therapeutic vaccine.
Animals ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Female ; Mice ; Mice, Inbred C57BL ; Papillomavirus E7 Proteins ; biosynthesis ; genetics ; isolation & purification ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; isolation & purification

OBJECTIVETo construct RNA interfering retrovirus vector targeting CXCR4 gene driven by human prostate-specific antigen promoter and investigate its targeted inhibition effects in androgen-responsive prostate cancer cells LNCaP.

METHODSTo clone the CXCR4 targeting siRNA gene by PCR. The PCR products were inserted into the pGensil-1 plasmid containing U6 promoter and EGFP. U6 promoter was replaced by hPSA promoter. Then, the recombinant EGFP-hPSA-siCXCR4 fragment was sub-cloned into pLXSN, which was evaluated by restriction enzyme. The pLXSN-EGFP-hPSA-siCXCR4 was transfected into PA317 cells with Lipofectamine 2000. The virus obtained from transfected PA317 cells was transfected into PC-3m, LNCaP and MCF-7 cells, respectively. The expression of CXCR4 mRNA and protein was detected by RT-PCR and Western blot. The invasion ability of prostate carcinoma cells was detected by Transwell experiment.

RESULTSThe recombinant pLXSN-hPSA-siCXCR4 was successfully constructed. The expression of CXCR4 mRNA and protein in LNCaP cells was blocked by pLXSN-hPSA-siCXCR4. The expression inhibition rate was (81.53 +/- 10.22)% at mRNA level detected by semi-quantitive RT-PCR and (90.52 +/- 9.31)% at protein level detected by Western blot, respectively, in LNCaP cells at 48 h. The expression of CXCR4 mRNA and protein was effectively inhibited by sequence-specific hPSA-siCXCR4 in LNCaP cells, but not in PC-3m and MCF-7 cells. The results of Transwell experiment showed that the number of cells in down-pore of micro-membrane was 139.9 +/- 14. 2 in the treated group, significantly less in comparison with 348.4 +/- 36. 4 in the controlled group (P < 0.05). However, the number of PC-3m and MCF-7 cells in down-pore of micro-membrane was not significantly different among the control and treated groups (P > 0.05).

CONCLUSIONThe downstream siRNA controlled by hPSA promoter in retrovirus system can be expressed selectively in androgen-responsive prostate carcinoma cells, showing an apparent targeting character. RNAi targeted to CXCR4 driven by hPSA promoter has a potential value in gene therapy of androgen-responsive prostate cancer.

Animals ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Humans ; Male ; Mice ; NIH 3T3 Cells ; Neoplasm Invasiveness ; Plasmids ; Promoter Regions, Genetic ; Prostate-Specific Antigen ; genetics ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Receptors, CXCR4 ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Retroviridae ; genetics ; Transfection