Objective To construct an expression vector containing Ewing's sarcoma EWS FLI 1 specific binding sequence and diphtheria toxin A chain (DTA) gene sequence to investigate the killing effect of DTA on cultured Ewing's sarcoma cells so as to provide proof for the exploration of new methods for gene therapy for cancer. Methods Expression vector (pS2 DTA) containing EWS FLI 1 specific binding sequence and DTA gene sequence was constructed by replacing the luciferase gene in pS2 with DTA gene at the points of Nco Ⅰ and Xba Ⅰ by molecular technique. After transfection of pS2 DTA into Ewing's sarcoma cells and the control cells, DTA expression and its killing effect on cells were detected. Results pS2 DTA was highly expressed in Ewing's sarcoma cell line, and the killing effect of DTA was much higher than that in the control cells. Conclusion pS2 DTA has selective killing effect on Ewing's sarcoma cells and can inhibit the growth of Ewing's sarcoma cells. So pS2-DTA has potential value in the treatment of Ewing's sarcoma.

Objective To observe the specific expression of diphtheria toxin A chain gene(DTA) in Ewing's sarcoma nude mice models.Methods pS2DTA plasmid was constructed by the Lus gene in pS2 plasmid was replaced with the DTA gene in pIBI30 plasmid,and transformed into DH5?, then amplified.Five nude mice were injected with 2.4?10~(7) SKES1 cells,sacrificed on week 6,and the tumor tissues were implanted onto the back of another 20 nude mice.Till 6 weeks later the diameter of the tumor up to 0.8-1.0 cm,the Ewing's sarcoma nude mice models were successfully established and randomly assigned to receive the injection of 300 ?g lipidosome+100 ?l normal saline(control group) or 300 ?g lipidosome+100 ?l normal saline+100 ?g pS2DTA (experimental group) once a day for 5 d.On day 4 after the therapy finished,4 nude mice of each group were randomly sacrificed and the DTA expression in the tissue of tumor,liver and myocardium was detected by the immunohistochemistry and RTPCR.Results The immunochemical results showed DTA expression in the tissues of control group was all negative while the DTA expression in the experimental group was negative in the tissues of myocardium and liver,positive in tumor tissue.RTPCR showed DTA expression in control group was negative in all tissues while strongly positive in tumor tissue,weakly positive in liver tissue and negative in myocardiac tissue.Conclusion The pS2DTA regulated by the EWSFLI1 fusion gene has a specific expression in transplanted Ewing's sarcoma in mice.

Collagen (Coll), as the basic material of matrix scaffolds for cell growth, has been widely used in the field of tissue engineering and regenerative medicine. In this study, collagen protein was modified by L-lysine (Lys), and cross-linked by genipin (GN) to prepare the L-lysine-modified collagen (Lys-Coll-GN) scaffolds. Microstructure, pore size, porosity, stability and biocompatibility of Lys-Coll-GN scaffolds were observed. The results showed that the bond between L-lysine and collagen protein molecule was formed by generating amide linkage, and mouse embryo fibroblasts proliferation was not inhibited in the Lys-Coll-GN scaffolds. In the multiple comparisons of Coll-scaf- folds, Coll-GN scaffolds and Lys-Coll-GN scaffolds, Coll-scaffolds was the worst in mechanical characteristics while the highest in biodegradation rate. Compared to Coll-GN scaffolds, Lys-Coll-GN scaffolds had more fiber structure, higher interval porosity (P<0. 01). Although the tensile stress of Lys-Coll-GN scaffolds reduced significantly, its e- longation length extended when the scaffolds was fractured (P<0. 01). The percentage of Lys-Coll-GN scaffolds residual weight was lower than that of Coll-GN scaffolds after all the scaffolds were treated by collagenase for 5 days (P<0. 01). This study suggested that Lys-Coll-GN scaffold had good biocompatibility, and it improved the mechanical property and degradation velocity for collagen-based scaffold. This study gave a new predominant type of tissue engineering scaffold for the regenerative medicine.
Animals ; Biocompatible Materials ; chemistry ; Cell Proliferation ; Collagen ; chemistry ; Cross-Linking Reagents ; Fibroblasts ; cytology ; Iridoids ; chemistry ; Lysine ; chemistry ; Mice ; Porosity ; Tissue Engineering ; Tissue Scaffolds

A total of 60 infants with severe persistent pulmonary hypertension (PPHN) at our NICU from January 2006 to December 2012 were divided into research group[(n =32,high-frequency oscillatory ventilation(HFOV) plus sildenafil)]and control group (n =28,HFOV only).Mean pulmonary arterial pulmonary arterial (MPAP),blood gas analysis,oxygenation index (OI),PO2 to fraction of inspired oxygen ratio (PO2/FiO2) before and after treatment were compared between two groups.After 3-day treatment,MPAP (mm Hg)(32 ±6) vs.(43 ±9)mm Hg,PCO2(mm Hg)(36 ±9) vs.(43 ±9),OI(56 ±22) vs.(85 ±21) in research group were significantly lower than those in control group(P ＜ 0.05) ; in comparison with control group,PO2(mm Hg) (89 ±15) vs.(72 ±22),PO2/FiO2(mm Hg) (223 ± 18) vs.(196 ±24) in research group were significantly higher(P ＜ 0.05).The time of ventilation use (d) (5.4 ± 1.3) vs.(6.3 ± 1.6) in research group was shorter than that of control group(P ＜0.05) while research group showed a higher clinical efficiency rate(％) (81.2 vs.50.0) (P ＜0.05).A combination of HFOV and sildenafil for severe PPHN can significantly reduce MPAP,shorten the duration of ventilation use and improve cure rate.

Objective To Analyze CD4+ cell count which embodies curative effect in HIV and patients with AIDS, who have treated with TCM for half a year. Methods According to CD4+cell count, the patients were divided into 4 phases. Their CD4+cell count were analyzed before and after the treatment. Results 1.Rank sun test showed that CD4+cell count were significantly improved in people who used TCM treatment. On the whole, CD4+cell count was(317.76±175.61) in 1 cu mm before treatment, which was(350.60±175.92) in 1 cu mm after treatment, P<0.01. 2. Ridit showed that patients whose CD4+cell count more than 500 were in phase I. Their R=0.614, and the 95%confidence interval was 0.5702 to 0.6579. Patients whose CD4+cell count more than 350 and less than 500 were in phase II. Their R=0.575, and the 95%confidence interval was 0.5439 to 0.6062. Patients whose CD4+cell count more than 200 and less than 350 were inphase III. Their R=0.460 and the 95%confidence interval was 0.4347 to 0.4849. Patients whose CD4+cell count less than 200 were in phase IV. Their R=0.428, and the 95%confidence interval was 0.3971 to 0.4589. There was no overlap between phase III, phase IV and phase I, phase II in 95% confidence interval. Conclusion TCM has the advantages in strengthening vital qi, but that is worse than western medicine in effort of expelling pathogen.

Objective: To construct a liver targeting gene transfer vector using hepatitis B virus envelope particles. Methods: Hepatitis B viruses were obtained from the supernatant of HepG 2.2.15 cells by a PEG8000 system and were inactivated by ?-propiolactone to prepare hepatitis B virus envelope. The hepatitis B virus envelope was used to pack 5.3 kb pIRES_2-EGFP to assess their packing ability. Subsequently, the products were studied with ELISA, PCR, SDS-PAGE, and electron microscopy. Finally, the product was used to transfect HepG2 cells and the green fluorescent protein (GFP) expression was observed under a fluorescent microscope. The rate of GFP positive cells was determined by flow cytometer.Results: The acquired hepatitis B virus envelope retained the surface protein HBsAg+pre S_1+pre S_2, but with no virus DNA. The prepared envelope had high packing ability for GFP and the packed GFP had a high transfection rate in HepG2 cell. Conclusion: Hepatitis B virus envelope has been successfully obtained from the supernatant of HepG 2.2.15 cells with a PEG8000 system and ?-propiolactone.

Objective To re-evaluate the diagnoses of ankylosing spondylitis (AS) and nonradiographic axial spondyloarthritis (nr-axSpA) and analyze the incidence and reason of misdiagnosis.Methods Patients who were previously diagnosed as AS and nr-axSpA before referrals to Peking Union Medical College Hospital (PUMCH) were re-evaluated by three rheumatologists of PUMCH according to the modified New York criteria for AS and Assessment of SpondyloArthritis international Society (ASAS) axial SpA classification criteria for nr-axSpA.Results Totally 87 prior AS patients and 53 prior nr-axSpA patients were enrolled in this study.After re-evaluation,57 patients were still diagnosed as AS and 16 patients were still diagnosed as nr-axSpA.The misdiagnosis incidences were 34.48％ and 69.81％,respectively.The misdiagnosis incidence of nr-axSpA was higher than that of AS (P ＜ 0.01).Conclusions The misdiagnosis of AS were mainly due to the misjudgment of sacroiliac joints by CT.The misdiagnosis of nr-axSpA were mainly due to the misjudgment of sacroiliac joints by magnetic resonance imaging.Moreover,the misuse of ASAS axial SpA classification criteria contributed to the misdiagnosis also.

Objective To investigate the prevalence of HBV infection and the risk of hepatitis B virus (HBV) reactivation in patients with inflammatory arthritis receiving tumour hecrosis factor alpha (TNFα) inhibitors.Methods The liver function,serology of HBV and viral loads (HBV DNA) were tested before using TNFα inhibitors,at 3 months and 6 months.Patients with chronic hepatitis B (CHB) infection (HBV DNA ＞ 1 × 103copies/ml) were eliminated.Results A total of 162 patients were investigated including 156 patients who finished the study.Eleven (7.05％) patients were HBsAg-positive.Two patients with HBV DNA ＞ 1 × 103copies/ml were eliminated before starting anti-TNFα therapy.Among HBsAgpositive patients,HBV reactivation was documented in only one of the 11 patients.This patient with rheumatoid arthritis developed elevation of glutamic-pyruvic transaminase (ALT) and HBV DNA copies three months after infliximab therapy.Therefore lamivudine was given for three months,which translated into the fall of ALT and HBV DNA copies back to normal level.After follow-up for six months,the virology and serology remained stable.In contrast,none of the other 155 patients had demonstrated evidence of HBV infection or HBV reactivation.Conclusion The kinetics of HBV viral loads should be carefully monitored in patients with inflammatory arthritis and HBsAg-positive during anti-TNFα therapy.HBV reactivation should be treated with antiviral medicine through out the period of anti-TNFα therapy.

Objective To study the correlation between genetic polymorphisms of interleukin (IL)-1A/1B and susceptibility to tuberculosis (TB).Methods Genetic polymorphisms of IL-1A and IL1 B in 1032 TB patients and 1008 non-TB patients were analyzed using PCR-MassARRAY method.The correlation between genetic polymorphisms of IL-1A/1B and susceptibility to TB was statistically analyzed.Results Two tag SNPs of IL-1A and three tag SNPs of IL-1B were screened for the study.There were differences in the allele frequencies of rs2853550 and rs3783526 between TB group and non-TB group (P=0.047and P =0.034,respectively).IL-1 B SNP1 rs2853550 (P =0.025,OR =1.302,95 ％ CI =1.034-1.640,TC vs.CC) was found to be highly associated with TB,while the other SNPs showed no significant correlations with TB.Furthermore,IL-1B SNP1 rs2853550 [P=0.019,OR=1.308,95％ CI=1.045-1.638 for (TC+TT) vs.CC] in the dominant model conferred significant risk for TB,but IL-1A SNP2 rs3783526 [P=0.000,OR=0.764,95％ CI =0.591-0.988 for GG vs.(AA+GA)] in the recessive model showed protective effects against TB.The haplotype ‘TG’ in the IL-1B block showed a higher risk for TB compared with the common ‘ CA’ haplotype (P=0.032,OR=1.265,95％ CI=1.020-1.567).The diplotypes containing ‘ GA’ haplotype in IL-1A block and ‘ TG’ haplotype in IL-1B block were major risk factors for TB (for onecopy,adjusted P=0.014,OR=1.403 and 95％ CI=1.072-1.836; adjusted P=0.013,OR=1.339 and 95％ CI=1.063-1.688,respectively),but the diplotype with ‘CG’ in IL-1B block played a protective effect against TB (for two-copy,P=0.006,OR=0.664 and95％ CI=0.494-0.891).Conclusion The genetic polymorphisms of IL-1B rs2853550 might be closely associated with TB,but the GG genotype of IL1 A SNP rs3783526 might have the characteristic of anti-TB.