Objective: To constuct recombinant adenovirus vector carrying the human glutathione transferase ?(GST-?)gene and amplify the adenovirus vector in HEK293cells.Methods: GST-? gene with the suitable enzyme site are amplified from the original plasmid through PCR,then subcloned into the shuttle plsmid pAdtrackCMV after digested by the same enzyme.The recombinant plasmid pAdtrack-GST-? 1inearized by PmeI,plasmid pAdtrack-GST-? was electroporated into E.coli BJ5183 cells that had been electroporated adenovirus backbone plasmid pAdEasy-1.Picking up the homologous recombant plasmid AdEasy-GST-?,then it was digested with PacI and transfected into HEK293 cells to package the adenovirus,followed by identification of the recombinant adenovirus by means of observation of green fluorescence protein expression under fluorescent microscope.Recombinant virus supernatant infected HEK293 cells repeatedly,and the green fluorescence protein expression Was detected.Results: Through the restriction enzyme digestion and expression of GFP,recombinant adenoviral vector GST-? gene was constructed successfully.Conclusions: The recombinant adenoviral vector containing GST-? gene was successfuHy constructed,and based for the gene therapy of GST-? transferring the hematopoietic stem cells.

Objective: To investigate the expression of LRP,GST-?in the testicular germ cell tumor and evaluate their role in multidrug resistance of the testicular germ cell tumor.Methods: 41 samples of germ cell tumor without chemotherapy were analyzed immunohistochemically.Results: The total positive percentage of LRP in the testicular germ cell tumor was 41.5%,the frequence of LRP expression in Ⅰ stage,Ⅱ+Ⅲ stage,seminoma and non-seminoma germ cell tumor was 23.1%,73.3%,40.0% and 45.5% respectively.There was significant difference of the LRP expression between I stage and Ⅱ+Ⅲ stage in the testis germ cell tumor(P0.05).Conclusion: The expression of LRP was correlational with the clinical stage of the testicular germ cell tumor,and LRP expression may be an important prediction of patients with testicular germ cell tumor.【

BACKGROUND: Compared to those original viruses systems, adeasy adenovirus, a recombinant adenoviral system widely used in recent years, based on viruses with a deletion of both El and E3, reported by T.C. He in 1998, is an improved one. It simplifies the generation and production of such viruses and expedite the process of generating and testing recombinant adenoviruses using homologous recombination in bacteria rather than in eukaryotic cells. Moreover, it can be conveniently followed with the aid of green fluorescent protein encoded by EGFP gene incorporated into the viral backbone.OBJECTIVE: To construct the recombinant adenovirus and to evaluate them by transfect them to mesenchymal stem cells (MSCs)and detect the expression of target gene hlGF-I at gene and protein levels.DESIGN: Repetitive measurement wail.SETTING: The Institute of Pediatric Research, Chongqing University of Medical Science.METHODS: The study was performed at the Institute of Pediatric Research, Chongqing University of Medical Science from November 2004 to March 2005. After the amplification of truncated hlGF-1 gene from pcDNA3.l-hlGF-I by polymerase chain reaction (PCR), the gene fragment was inserted into the shuttle plasmid pAdtrack-CMV for homologous recombination with backbone plasmid pAdeasy-I in bacteria BJ5183 to get adenovirus.Ad-hlGF-1. The high titer adenovirus supernatant was obtained by repeated transducing of HEK 293 cells by adenovirus harvested after confirmation of the adenovirus structure. As target cells,MSCs were infected with adenovirus earned target gene, hIGF-1, to determine the expression of hlGF-1 gene.MAIN OUTCOME MEASURES: ① The construction of recombinant adenovirus vector;② the expression of target gene hIGF-1 in HEK 293 cells and the proper multiplicity of infection (MOI); ③ hIGF-1 gene expression in MSCs.RESULTS: The adenovirus vector based on adeasy system was constructed successfully and the Ad-hlGF transducing was successfully or efficiently expressed in MSCs cells. The ideal expression of harvested recombinant adenovirus in MSCs was detected by fluorescence microscope, RT-PCR, immunocytochemistry, and Western Blot.CONCLUSION: Adenovirus vector is an effective vector tools for gene expression and wansfection of MSCs. MSCs transduced with Ad-hIGF-1 maybe another option to gene-modified seed cells for articular cartilage tissue engineering.

The traditional teaching mode of "subject-centered" has many shortcomings such as knowledge separation and lack of integrity. In order to make up for the deficiencies of this mode, the organ-system integrated medical teaching mode has gradually become the trend of medical teaching reform. Taking "motor system" as an example, this paper discusses the practice of the organ-system integrated medical teaching mode from four aspects: teaching implementation, teaching quality supervision, supporting assessment system and teaching effectiveness.

Objective To characterize the viability and transgene expression of articular chon-drocytes cultured in 3-Dimensional scaffolds provided by four types of carriers.Methods Articular chondrocytes from rabbit knees were cultured and infected with adenovirus that could express green fluo-rescence protein (AdGFP) and GL3 luciferase (AdGL3-Luc).The viability and gene expression were determined with fluorescence microscopy and luciferase assays in four types of scaffolds;type I collagen sponge, fibrin glue, hyaluronan and open-cell polylactic acid (OPLA).Cartilage matrix production was assessed by Alcian blue staining.Results Articular chondrocytes of rabbits were effectively infected by AdGFP and exhibited sustained GFP expression.All the tested scaffolds supported the survival and gene expression of the infected chondrocytes.However, the highest transgene expression was observed in the OPLA carrier (P<0.01).Alcian blue-positive matrix materials were readily detected in OPLA cultures four weeks later.Conclusion OPLA supports the highest transgene expression and is the most conduc-tive scaffold for matrix production, suggesting that OPLA may be a suitable scaffold for cell-based gene therapy of articular cartilage repair.

[Abstract] Objective: To investigate the effect of alantolactone (ALT) on proliferation, migration, invasion and apoptosis of human osteosarcoma 143B cells and the underlying mechanism. Methods: Osteosarcoma 143B cells were treated with different concentrations of ALT (0, 4, 6, 8, 10 µmol/L). Then, the cell proliferation ability was detected by crystal violet staining and MTT assay, cell migration was determined by Wound-healing test, cell invasion was analyzed by Transwell assay and cell apoptosis rate was detected by Hoechst33258 staining. The mRNA and protein expressions of E-cadherin, N-cadherin, caspase-3, cleaved caspase-3 (c-caspase-3), poly ADP-ribose polymerase (PARP) and cleaved PARP (c-PARP) in 143B cells were detected by qPCR and Western blotting (WB), respectively. TCF/LEF (T cell lymphocyte factor/lymphoid enhancer factor) transcriptional activity was examined with Luciferase reporter gene assay. The mRNA and protein expressions of β-catenin as well as MMP-7 and c-Myc were detected by qPCR and WB, respectively. Results: ALT inhibited proliferation, migration and invasion of osteosarcoma143B cells and promoted apoptosis(P<0.05or P<0.01). After the treatment with ALT at 8, 10 µmol/L, the mRNA and protein expressions of E-cadherin and PARP, as well as the protein expressions of c-caspase-3 and c-PARP were up-regulated, while the mRNA and protein expressions of N-cadherin were downregulated (P<0.05 or P<0.01);At the sametime, theTCF/LEF transcriptional activity and the mRNA and protein expressions of β-catenin, MMP-7 and c-Myc were significantly down-regulated (P<0.05 or P<0.01). Conclusion:ALT may inhibit the proliferation, migration and invasion and promote cell apoptosis possibly through suppressing Wnt/β-catenin signaling pathway in osteosarcoma 143B cells.