Effect of lentivirus encoding acidic fibroblast growth factor on cycle and proliferation of adipose-derived stem cells / 中华医学美学美容杂志

Objective To study the effect of the lentivirus encoding acidic fibroblast growth factor transfecting human adipose-derived stem cells (ADSCs) on the cell cycle and proliferation of ADSCs.Methods ADSCs were isolated and extracted by enzymatic digestion from the liposuction aspirate.ADSCs were cultured,identified and osteogenic induced reagent was used to induce differentiation of ADSCs towards bone cells.To obtain lentivirus encoding FGF-1,the plasmid PWPXLd FGF-1 was co-transfected with plasmid psPAX2,pMD2.G in 293T cells.ADSCs were infected with lentivirus encoding FGF-1.Expression of green fluorescent protein (GFP) in infected FGF-1 was observed by fluorescence microscope and expression of FGF-1 in ADSCs was verified by Western blot analysis.Flow cytometry was used to detect the cell cycle of ADSCs infected with lentivirus encoding FGF-1.EDU assay was performed to examine cell viability.Results Lentivirus encoding FGF-1 was obtained.After ADSCs being infected green fluorescence was found in about 70％ ADSCs,and overexpression of FGF-1 protein was detected in infceted cells by Western blot analysis.The percentage of G2/M phase cells was significantly increased compared with the control group,and the proliferation of ADSCs infected with lentivirus encoding FGF-1 was promoted as compared with the control group.Conclusions FGF-1 can enhance G2/M phase of ADSCs and promote the proliferation of ADSCs.

Characteristics and in vitro cell compatibility of human acellular dermal matrix with improved method / 中华医学美学美容杂志

Objective To detect the characteristics and in vitro cell compatibility of human acellular dermal matrix (ADM) with the improved method.Methods Cell components of healthy human skins were removed by the improved method and the traditional method respectively.The porosity, degradation time in vitro of the ADM prepared by two methods and the cytotoxicity of the material infiltration liquid with the improved method on the adipose derived stem cells were detected.HE staining was used to detect the residual of the cells, the integrity of collagen and cell biocompatibility.Scanning electron microscopy (SEM) was used to detect the pore size.Results Both the two methods could completely remove the cells, and maintain the integrity of the collagen scaffold;The porosity of ADM with the improved method was higher (93.1±1.02)% than that of traditional method (74.27±2.04)% (P<0.05);There was no significant difference in the cytotoxicity and in vitro degradation time between the two kinds of ADM;While pore diameter of the improved method was significantly higher [(181.21±66.9) μm] than that [(102.38±15.63) μm] in dermal reticular surface with the traditonal method (P<0.05).Conclusions There is no obvious cytotoxicity of the ADM with the improved method, and therefore it is more suitable for cell adhesion growth with higher porosity and larger pore size.

Epigenetic regulatory effects of the methylation of CpG island shore on the expression of lag3 gene in various human T cell lines / 中华微生物学和免疫学杂志

Objective To investigate the effects of DNA methylation on the expression of lympho-cyte activation gene 3 (lag3) in different human T cell lines.Methods A quantitative PCR and a flow cy-tometry analysis were performed to measure the expression of lag3 gene in various T cell lines at mRNA and protein levels.The distribution of CpG sites within the promoter and body of lag3 gene was detected to locate the potential regulatory region(s) (CpG island and CpG island shore).The levels of DNA methylation in each cell line were analyzed.The T cell lines were demethylated with 5-Aza-2′-deoxycytidine (5-Aza-2′-dc) for further investigation on the changes of lag3 gene expression and DNA methylation.Results Jurkat E6-1 cells showed the highest expression level of lag3 gene as compared with J.CaM1.6 and CEM cells.Hyperm-ethylated CpG islands were detected in cells of each cell line.The methylation levels of CpG island shore in J.CaM1.6 and CEM cells were higher than that in Jurkat E6-1 cells.Treatment of J.CaM1.6 and CEM cells with 5-Aza-2′-dc significantly promoted the expression of lag3 gene at mRNA and protein levels as well as the demethylation of CpG island shore.No significant differences with the expression of lag3 gene and the methylation of CpG island were observed in Jurkat E6-1 cells with or without 5-Aza-2′-dc stimulation.Con-clusion Methylation and demethylation of CpG island shore played important roles in regulating the tran-scription of lag3 gene.

Simple and effective method for monitoring ambulatory electrocardio-gram in toads / 中国病理生理杂志

[ ABSTRACT] AIM:To screen out a suitable lead for monitoring the ambulatory electrocardiogram ( ECG) in un-restrained toad, and to investigate its practicability.METHODS:After subcutaneously implanting the electrodes in toads under anaesthesia, the ambulatory ECG of 5 leads were monitored with BL-420S data acquisition and analysis system, and the leads which could well express the waveform in ECG were screened out.The recovery process of the toads from the arti-ficial hibernation within 6 h, the day-to-day stability of the heart rate ( HR) and the heart rate variability ( HRV) in 5 suc-cessive days of hibernation, and the HR and HRV after freeze-thawing process were monitored to determine its practicabili-ty.RESULTS:Two out of 5 leads showed better ECG waveforms.Compared with 6 h post hibernation, lowered HR at 0 h and 1 h was observed, and the standard deviation of normal R-R intervals ( SDNN) was significantly increased ( P<0.05 or P<0.01), but the HR and SDNN from 2 to 5 h showed no significant difference, suggesting that the cardiac function reached the steady state after 2 h recovery.The HR at 2 h and 4 h on day 4 and day 5 decreased significantly compared with that on day 1 (P<0.05 or P<0.01), followed with a significant increase in SDNN (P<0.05 or P<0.01), sugges-ting that the ECG remained stable within 3 d.The HR increased, while SDNN decreased significantly at 1 h and 12 h post-thawing compared with that at pre-freeze (P<0.05), indicating the damaged cardiac function after freeze-thawing process. CONCLUSION:The method of subcutaneously implanting electrodes is suitable for effectively monitoring the ambulatory ECG in toads.

A Rapid and Simple Method for Cloning and Identification of cDNAs Related with IL-6 Effect / 现代生物医学进展

Cloning of differentially expressed genes is one of the hottest topics in biology.It is very important in cloning genes correlated with phenotype and diseases at molecular level.Here we utilized a simple and rapid PCR-based protocol to detect and isolate cDNA fragments from differentially expressed genes of IL-6-induccd and IL-6-uninduced U937 cells in tWO easy steps.To generate cDNAs from most mRNAs,the first step was reverse transcription using three fully degenerated 6-mer oligonucleotides as primers.The second step was PGR amplification of internal regions of the cDNAs with two or three longer primers with arbitrary but defined sequences.The PCR amplification was repeated on the same cDNA templates(first step) with different sets of primers.DNA fragments were easily displayed by 2% agarose gel electrophoresis and then the differential recovered fragments were used directly in cloning,sequencing,and RNA reverse Northern blot analysis.In this study,seven differential ESTs are obtained;two Sequences not found in GenBank,are novel ESTs.They were proved to be differentially expressed genes related with IL-6 effect by reverse Northern hybridization.