ABSTRACT
Objective To construct human acidic fibroblast growth factor (aFGF) recombinant eu karyotic expression vector and transfect it into muscle satellite cells(MSCs) of rat,in purpose of further study the method to set up cell bank.Methods The aFGF gene was cloned from human total RNA which was obtained from human skeletal muscle tissue by RT-PCR method.Human interleukin 2 (IL-2) signal peptide sequence (SPS) was obtained by direct chemosynthesis method.Then aFGF and SPS were fused to obtain SPS-aFGF.Finally,directional cloning SPS-aFGF into pEGFP-N1,the recombinant (pEGFP-N1-SPS-aFGF) was obtained.The recombinant was confirmed by endonuclease digestion and DNA sequencing.MSCs were purified by difference-speed adherence method and were ideontified by immunofluorescence assay.The correct cells were divided into 3 groups:Experimental group (aFGF +N 1),control group (N 1),blank group (blank).All the groups were transfected by Lipofectamine 2000TM Reagent,and pEGFP-N1-SPS-aFGF,pEGFP-N1 were respectively added in experimental group and control group while blank group was added none plasmid.Fluorescence microscope was employed to detect transfection efficiency tendency along with time changes.The expression of target gene was detected by fluorescent quantitation PCR and Western blot.Results (1) The sequencing of pEGFP-N1-SPS-aFGF was completely correct and the outcome of endonuclease was equal to actual ban s-ize.(2)The expression of GFP in transfected cells were observed by fluorescencemicroscope and transfection efficiency reached the peak at 72 h.(3)Real-time fluorescent quantitation PCR proved strong aFGF mRNA expression in transfected cells (the average relative expression of experimental group was 1464.95)with aFGF gene,while it was detected a little in the other groups (the average relative expression of control group was 1.016 and blank group was 1.000) (P ＜ 0.05).Western blot also proved strong expression in Experimental group then the other two groups.Conclusion aFGF eukaryotic expression vector was successfully constructed and transfected into MSCs.This study may be expected to obtain some specific functions cells.
ABSTRACT
The expression levels and changes of endogenous acid fibroblast growth factor (aFGF) in microwave bum wound tissues were detected in order to investigate how to get better therapeutic ef- fects by using the exogenous aFGF for repairing trauma. A burnt-wound animal model was estab-lished by NS-FⅡ multifunction spectrum therapeutics equipment, and reverse transcrip-tase-polymerase chain reaction (RT-PCR) and immunohistochemistry assay were applied to detect the expression levels of endogenous aFGF mRNA in microwave bum wound tissues. The expression level of endogenous aFGF mRNA was significantly increased in the bum wound tissues 12 h after bum, reached the peak at 48 h, and gradually deceased 96 h after bum. The expression of endogenous aFGF mRNA after tissue damage was reversible, and its intensity was in accordance with the repair process of tissue damage, suggesting endogenous aFGF may take part in the cell metabolism and pro-liferation, and then promote the repair of the bum wound.
ABSTRACT
Aim To study protective effects of aFGF on rats' renal tubular epithelial cells injured by gentamicin in vitro.Methods After renal cortice were grounded,filtered and digested with 0.25%trypsin,renal tubular epithelial cells were cultured.The injury models of epithelial cells of renal tubules were established by gentamicin and then the protective roles of aFGF in injured renal tubular epithelial cells were observed.Results ① By observing the morphology of the epithelial cells and comparing GM group with control group,CAT、SOD、Na~+,K~+-ATP enzyme activities decreased,while NAG activity and MDA level increased(P
ABSTRACT
Aim To explore effects of acidic fibroblast growth factor(aFGF)on the growth of renal tubular epithelial cells cultured in vitro. Methods Renal tubular epithelial cells in rats were gained through digestion by pamcreatin. aFGF was added into cultures of renal tubular epithelial cells, as experimental groups. The number of cells was counted and the shape of cells was observed at 12,24,48 and 72 h. Results Renal tubular epithelial cells were successfully obtained from kidneys. After treated for 72 h,the renal tubular epithelial cells showed different proliferation in experimental and control groups. There was no obvious difference at 12 h, but there was statistical difference between the two groups at 24 h,48h and 72 h(P