Comparison of the efficiency of three techniques for labeling human adipose-derived stem cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the optimal methods for labeling human adipose-derived stem cells (ASCs).</p><p><b>METHODS</b>ASCs were isolated by collagenase digestion and density gradient centrifugation, and their cell surface markers and ability to differentiate into the adipogenic, chondrogenic, and osteogenic lineages were examined in vitro. Three different cell labeling methods, namely 5 µl DiI, 10 µg/ml BrdU and 50 MOI adenovirus carrying GFP, were used for ASC labeling, and the labeling efficiency were compared at different time points and in different passages using fluorescent microscope.</p><p><b>RESULTS</b>The isolated ASCs were capable of differentiating into adipogenic, osteogenic, chondrogenic lineages with positive stem cell marker expression. At 48 h after DiI staining, 100% of the ASCs emitted red fluorescence in the cytoplasm with fluorescent-negative nuclei, but the fluorescence intensity declined quickly after cell passaging. With 10 µg/ml BrdU, 90% of the cells showed green fluorescence in the cell nuclei at 48 h after the labeling, but the positivity rate also decreased gradually after cell passaging. Cell labeling with GFP adenovirus showed more stable labeling efficiency, and green fluorescence was detected at 24 h after labeling, and even till 5 days later more than 90% of the ASCs remained positive without an obvious attenuation of the fluorescent intensity even after cell passaging.</p><p><b>CONCLUSION</b>All the 3 techniques are applicable for labeling ASCs. Cell labeling with DiI and BrdU can be convenient and economic and well serve the purpose of short-term labeling. Adenovirus carrying GFP gene is the optimal choice for long-term ASC tracing.</p>

The roles of CD4+CD25+ T regulatory cells and Foxp3 mRNA expression in the subjects with and without responses to hepatitis B virus vaccination / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate the internal links between immune responses and Tregs and cytokine by the expression of T regulatory cells (Tregs), Foxp3 mRNA of different response groups and the detection of cytokine secretion after hepatitis B vaccination.</p><p><b>METHODS</b>Blood samples were collected in different response groups. Real-time fluorescence quantitative PCR was used to detect the expression of Foxp3 mRNA of peripheral blood mononuclear cells; The surface markers CD4 and CD25 in peripheral-blood mononuclear cells were determined by flow cytometry; ELISA tests were used to detect the production level of phytohemagglutinin (PHA) in peripheral blood mononuclear cells, IL -4, IL-12, IL-18 stimulated by HBsAg and (IFN) gamma.</p><p><b>RESULTS</b>(1) Foxp3 expressions in response group and non-response group were higher before or after PHA and HBsAg were stimulated. Differences were statistically significant (P value less than 0.05) ; (2) In peripheral blood, the percentage of CD4+CD25+ Treg of CD4+ T cells in response group (0.59%+/-0.46%) was obviously lower than those in control group (1.30%+/-1.44%) ; (3) Peripheral blood mononuclear cells stimulated by PHA and HbsAg in each group, the concentration of IFNgamma in non-response group [(11.00+/-9.03) IU/ml] was markedly lower than those in response group [(38.88+/-28.16) IU/ml],and differences were statistically significant (P value less than 0.01); (4) In PHA- or HBsAg-stimulated peripheral-blood mononuclear cells, the concentrations of IL-18, IL-4 and IL-12 had no significant difference.</p><p><b>CONCLUSIONS</b>To some extent, CD4+CD25+Foxp3+ Treg cells may be involved in negative regulation of the immune responses to HBV vaccination. Immune non-response to HBV vaccination may be connected to insufficient secretion of IFNgamma; There was no correlation between the titer of anti-HBs and the expressions of IFNgamma and CD4+CD25+ Foxp3.</p>