Objective:To identify the role of transcription factors Sp1 and Sp3 in the expressional regulation of ezrin in human esophageal carcinoma cells.Methods:Esophageal carcinoma EC109 cells were transfected with expressing vectors CMV-Sp1 or CMV-Sp3,and the effect of Sp1 and Sp3 over-expression on ezrin mRNA and protein expression was determined by real time RT-PCR and Western blot analysis.Furthermore,EC109 cells were cotransfected with the ezrin promoter-directed luciferase reporter vector and control vector pRL-TK along with transcription factor expression vector.The roles of Sp1 and Sp3 in ezrin promoter activation and whether this activation occurred through the Sp1 binding site,-75/-69,were analyzed by dual-luciferase reporter assay system.Results:Over-expression of transcription factors Sp1 and Sp3 significantly increased the expression of ezrin mRNA and protein and the ezrin promoter activity in EC109 cells.Sp1 and Sp3 enhanced the promoter activity through different binding sites and only Sp1 did that through the-75/-69 site.Conclusion:Sp1 and Sp3 can regulate ezrin expression in EC109 cells.

Objectives:To establish hybridomas secreting monoclonal antibodies(McAbs) against O6-methylguanine-DNA methyltransferase(MGMT) and to observe the relationship between MGMT expression and clinical responses to ACNU and BCNU in human brain tumors.Methods:The hybridomas were established by cell fusion.MGMT expression in 60 glioma specimens was detected by means of immunohistochemical assay.Results: Seven hybridomas secreting McAbs against MGMT were obtained.Thirty tumor specimens had no detectable or low level of MGMT expression(Mer-), while 30 specimens had high level of MGMT expression(Mer+). The Mer- patients showed more sensitive to ACNU and BCNU than the Mer+ patients.Conclusions: The high specific hybridomas secreting monoclonal antibodies(McAbs) against MGMT were established.The preliminary study indicated that MGMT negative tumors were sensitive to ACNU and BCNU, whereas MGMT positive ones were more resistant to nitrosourea drugs.

Previous studies suggest that NGAL (neutro phil gelatinase-associated lipocalin) is involved in the transformation and development of esophageal carcinoma. Alteration of NGAL expression can trigger the change of cellular morphology in esophageal carcinoma cells. However, the mechanisms remain unclear. To get a better understanding of NGAL function in esophageal carcinoma, NGAL protein was expressed in methylotrophic yeast, Pichia pastoris, and purified by chromatography. EC1.71 cells expressed high levels of NGALR (NGAL receptor) and EC109 cells expressed low levels of NGALR were used as cells model. The trafficking and the possible function of NGAL protein were then analyzed in the esophageal carcinoma cells. The results showed that 5-FAM-labeled recombinant NGAL protein could internalize into the EC1.71 and EC109 cells. Furthermore, the internalized NGAL protein could induce the alteration of cellular morphology, resulting in generation of autophagosome, transcriptional up-regulation of genes associated with autophagy and increase of phospho-ERK1/2 (p-ERK1/2). Interestingly, the treatment with the NGAL protein did not affect the intracellular iron level. These data indicate that induced autophagy by exogenous NGAL protein is a mechanism that internalized NGAL plays important roles in esophageal carcinoma cells, independent with NGAL-mediated iron transport process, while ERK1/2 signal pathway is involved in activation of autophagy by exogenous NGAL protein.

Objective To investigate the isolation and culture of porcine bone marrow mesenchymal stem cell (BMSC) with α-1, 3-galactosyltransferase (GGTA1) gene knockout (GTKO), GTKO/ human CD46 (hCD46) insertion and cytidine monopho-N-acetylneuraminic acid hydroxylase (CMAH)/GGTA1 gene knockout (Neu5GC/Gal), and the protective effect of co-culture with porcine islets on islet cells. Methods Bone marrow was extracted from different transgenic pigs modified with GTKO, GTKO/hCD46 and Neu5GC/Gal. Porcine BMSC were isolated by the whole bone marrow adherent method and then cultured. The morphology of BMSC was observed and the surface markers of BMSC were identified by flow cytometry. Meantime, the multi-directional differentiation induced by BMSC was observed, and the labeling and tracing of BMSC were realized by green fluorescent protein (GFP) transfection. The porcine BMSC transfected with GFP were co-cultured with porcine islet cells. Morphological changes of porcine islet cells were observed, and compared with those in the porcine islet cell alone culture group. Results BMSC derived from pigs were spindle-shaped in vitro, expressing biomarkers of CD29, CD44, CD73, CD90, CD105 and CD166 rather than CD34 and CD45. These cells were able to differentiate into adipocytes, osteoblasts and chondrocytes. Porcine BMSC with GFP transfection could be labeled and traced, which could be stably expressed in the daughter cells after cell division. Porcine BMSC exerted certain protective effect on islet cells. Conclusions GFP-labeled porcine BMSC modified with GTKO, GTKO/hCD46 and Neu5GC/Gal are successfully established, which exert certain protective effect upon islet cells.

Objective To compare the acute effect of high-intensity interval training(HIIT)and moder-ate-intensity continuous training(MICT)on ectopic lipid levels in overweight and obese youth.Methods Twenty obese or overweight subjects,aged 24.15±1.98 years,participated in two crossover random-ized own-control trials of HIIT and MICT.In HIIT,participants performed high-intensity cycling at 85%VO2max for 9 sets of 2 minutes,interspersed with low-intensity cycling at 25%VO2max for 10 sets of 2 minutes each and the session started and ended with low-intensity cycling at 25%VO2max.Howev-er,in MICT,all participants cycled continuously at 50%VO2max for 60 minutes,maintaining a pedal-ing speed of 50-55 rpm,with a 7-day interval between the two interventions.Before,as well as im-mediately,60 and 120 minutes after exercise intervention,all subjects underwent magnetic resonance imaging(MRI)scans to observe the fat fraction(FF)and spin relaxation rate R2* of the rectus femo-ris,biceps femoris and liver.Results Rectus femoris FF decreased significantly immediately after HIIT and MICT(P<0.05),without significant differences.Moreover,sixty minutes after MICT,rectus femoris FF returned to pre-exercise levels,while 120 minutes after HIIT,the values restored to the pre-exer-cise levels.However,no significant changes were found in the biceps femoris and liver FF before and after the two exercise interventions(P>0.05).Meanwhile,the rectus femoris R2* was significantly lower in both the HIIT and MICT groups immediately after exercise(P<0.05)and remained significantly low-er in both groups 120 minutes after the exercise(P<0.05),with no significant differences between the two groups.Biceps femoris and liver R2* were significantly higher in both groups immediately after the exercise intervention(P<0.05).Liver R2* returned to pre-exercise levels 120 minutes after HIIT group,but remained significantly lower than pre-intervention levels after MICT(P<0.05).Conclusion Both acute HIIT and MICT are effective in reducing intramuscular fat in the working muscles of over-weight and obese adults but have no significant effect on liver fat.Acute HIIT and MICT show similar fat-burning effects,but a single bout of exercise proves more effective for fat loss in the active mus-cles compared to the antagonist muscles.