Adipophilin increases lipid accumulation in human vascular smooth muscle cells / 中华内分泌代谢杂志

Objective To observe the effect of acetylated low-density lipoprotein (AcLDL) on the expression of adipophilin and the effect of adipophilin on AcLDL uptake and lipid accumulation in human vascular smooth muscle cells (HVSMCs)in order to approach the role played by adipophilin in genesis of macroangiopathy. Methodse HVSMCs were treated with various amount of AcLDL. Adipophilin expression levels were detected by Northern blot and Western blot. The effects of adipophilin on AcLDL uptake and lipid accumulation in HVSMCs were observed by the methods of siRNA, flow cytometry, enzymatic method and oil red stain. Results AcLDL dose-dependently increased adipophilin expression in HVSMCs. Silence adipophilin by siRNA decreased AcLDL uptake (decreasing by 38.7%, P<0. 05) and lipids accumulation (tfiglyceride and total cholesterol decreasing by 30.6% and 29.8% respectively, both P<0. 01) in HVSMCs, Conclusion Adipophilin is able to increase AcLDL uptake and lipid accumulation in HVSMCs, suggesting that it might play a role in enhancing atherosclerosis.

Evaluation of interferon-gamma producing-cells using enzyme linked immunospot assay in mice model of acute graft versus host disease / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate IFN-gamma producing-cells (IFN-gamma PCs) in allogeneic mixed lymphocyte reaction (MLR) and acute graft versus host disease (aGVHD) model of mice.</p><p><b>METHODS</b>Enzyme linked immunospot assay (ELISPOT) was applied to study IFN-gamma PCs in MHC mismatched mice spleen cell MLR and aGVHD model of mice.</p><p><b>RESULT</b>IFN-gamma PCs increased significantly in MLR after allogeneic mice spleen cell stimulation. In the experimental mice aGVHD model, IFN-gamma PCs were significantly higher in the severe aGVHD group than those in the moderate aGVHD. In the moderate aGVHD group, mice with GVHD prophylaxis regimen demonstrated significantly lower level of IFN-gamma PCs, compared with those without prophylaxis. IFN-gamma PCs were significantly correlated with the GVHD clinical scores in the group with moderate aGVHD and prophylaxis regimen.</p><p><b>CONCLUSION</b>ELISPOT is a fast, sensitive and specific approach to evaluate alloresponse in allogeneic mice MLR and IFN-gamma PCs are correlated closely with the severity of aGVHD and prophylaxis regimen in the MHC-mismatched mice model.</p>

Effects of transforming growth factor beta1 on dendritic cells function / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of transforming growth factor beta1 (TGF-beta1) on dendritic cells (DC).</p><p><b>METHODS</b>Murine bone marrow cells were cultured with different cytokine combinations to develop immature DC (imDC, GM-CSF only) and TGFbeta-DC (GM-CSF + TGF-beta1), and their responses to lipopolysaccharide (LPS) stimulation were observed. The cell ultrastructure was observed by transmission electron microscopy and their phenotypes were assessed by flow cytometry (FCM). The allogeneic stimulating capacity of DC was assayed by mixed lymphocyte reaction (MLR) with BrdU incorporation. IL-12p70 protein was detected by ELISA and the expressions of Toll-like receptor 4 (TLR4) on DCs were analyzed with semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>Compared to imDC, the TGFbeta-DC had no significant alterations in ultrastructure after LPS stimulation. The expressions of CD80, CD86 were lower on TGFbeta-DC than on imDC [(4.14 +/- 0.95)% vs (13.90 +/- 7.22)%; (8.60 +/- 0.75)% vs (20.63 +/- 5.03)%, P < 0.05, both]. The TGFbeta-DC kept their immature morphology after LPS stimulation, but the expressions of I-Ab and CD80 were slightly increased. After 96 h MLR, TGFbeta-DC had weaker stimulating capacity than imDC did, especially when DC/T cells ratios were 1:4 and 1:1 (P < 0.05, both). TGFbeta-DC showed impaired IL-12p70 production and down-regulation of TLR4 expression.</p><p><b>CONCLUSIONS</b>TGF-beta1 can inhibit the expression of co-stimulatory molecules on DC. The TGFbeta-DC is resistant to maturation stimulus (LPS) and might be linked with TLR4 down-regulation.</p>

Increased expression and possible roles of nicotinamide N-methyltransferase in pancreatic islets of STZ-induced diabetic monkeys / 中国病理生理杂志

AIM: To verify and localize the expression of nicotinamide N-methyltransferase (NNMT) in pancreas of streptozotocin(STZ)-induced diabetic monkeys and understand its important role in ?-cell destruction in the pathogenesis of diabetes. METHODS: Through an olig-microarray gene chip, NNMT was identified as the most obviously up-regulated genes in pancreas of STZ-induced diabetic monkeys versus controls. Semiquantitative RT-PCR and Western blotting were performed to verify the differential expression at mRNA and protein level respectively. Then the cellular localization of NNMT expression within pancreas was identified by immunohistochemical(IHC) staining.RESULTS: An obvious high expression of NNMT at both mRNA and protein levels was shown in pancreas of STZ-induced diabetic monkeys compared to that of controls. Further localization of the protein by IHC staining in pancreas specimens showed that its altered expression was restricted to central islets, most of which were ? cells.CONCLUSION: Expression of NNMT is increased in islets of STZ- induced diabetic monkeys, which infers that NNMT might participate in the process of ? cell damage in diabetes probably through the mechanism of energy metabolism disturbance.

Full-length cDNA clone and protein expression of a solute carrier family 24 (sodium/potassium/calcium exchanger) member 6,SLC24A6 / 中国病理生理杂志

AIM: To clone and express the gene of SLC24A6, which provide the basis to illuminate the relationship between the SLC24A6 and insulin release. METHODS: The gene expression of SLC24A6 was analyzed in the insulinoma and normal pancreatic tissues by RT-PCR. The full-length cDNA sequence was subcloned into pET32a vector, and induced expression and purified in ROSSET (DE3) strain. At the same time, the ORF of SLC24A6 was cloned into green fluorescence protein vector pEGFP-C3 to study the location of SLC24A6 in the mouse insulinoma ?-TC3 cells. RESULTS: The mRNA expression of SLC24A6 in the human insulinoma tissue was significantly higher than that in normal pancreatic tissue. The fusion protein of SLC24A6 was a 80 kD protein and was purified successfully by prokaryotic vector in ROSSET strain. The localization of SLC24A6 in the mouse insulinoma ?-TC3 cells was located in the membrane of the cells. CONCLUSION: SLC24A6 might be related with insulin release. The prokaryotic expression of SLC24A6 provides the basis for the study on biological function and protein structure, and the location of SLC24A6 in the insulinoma cell will throw light on the relationship with insulin release.

Resistin gene overexpression in adipose tissue of SD rats with high-fat diet and its correlation with leptin and insulin / 中华内分泌代谢杂志

Objective To observe expression of resistin gene in adipose tissue of SD rats fed with high-fat diet and its correlation to insulin resistance, diabetes mellitus and leptin. Methods Using RT-PCR to mRNA from adipose tissue, the products were obtained, purified, constructed and squenced. Once its squence was verified, semi-quantitative RT-PCR and Northern Blot were employed to study its expressions in adipose tissues of rats with different diets. Oral glucose tolerance test and insulin release test were performed in 2 groups of rats. Results (1) The resistin gene was successfully constructed and sequenced, which was in accordance with the data of genbank. (2) Compared with the control, the expression of resistin in adipose tissue of high-fat diet rats was significantly enhanced, and blood glucose, serum insulin and leptin levels were also elevated in high-fat diet rats (P