RESUMO
Objective To investigate the neuroprotective effects of neurotrophin-3 (NT-3) expression controlled by five copies of the hypoxia-responsive elements after focal cerebral ischemia .Methods Three groups of rats re-ceived RV-5H-NT3, RV-5H-EGFP or saline injection .Three days after gene transfer , the rats underwent 90 min of transient middle cerebral artery occlusion ( tMCAO) , followed by 1-28 days of reperfusion .Immunohistostaining and western blotting were performed to detect ischemia/hypoxia-regulated expression of NT-3 controlled by HRE . The volume of brain infarction and the apoptosis were analysised by TTC and TUNEL staining .The neurological scoring was determined by neurological behavior tests .Results Three days after tMCAO , brain NT-3 expression was significantly increased in the RV-5HNT3-transduced animals compared with the RV-5H-EGFP or saline group (P<0.05), and brain infarct volume was smaller in the RV-5H-NT3-transduced group than the RV-5H-EGFP or saline group ( P<0.05 ) .The percentage of TUNEL-positive cells was reduced in RV-5 H-NT3-transduced brains compared with the RV-5 HEGFP or saline group 3 and 7 days after tMCAO ( P<0.05 ) .Furthermore , the neurolog-ical status of RV-5H-NT3-transduced rats was better than that of RV-5H-EGFP-or saline-transduced animals from 1 day to 4 weeks after tMCAO ( P<0.05 ) .Conclusions HRE may modulate NT-3 expression in the ischemic brain tissue and that the up-regulated NT-3 may effectively improve neurological status following tMCAO due to de-creased initial damage .
RESUMO
To develope a 5 lipoxygenase inducible expression system for further understanding the function and signal transduction pathway of 5 lipoxygenase, providing information for drug screening.The 2 kb 5 LO cDNA sequence containing HindⅢ site was generated by PCR using pEGFP 5LO as a template. Full length 5LO cDNA was subsequently cloned into the corresponding Pst Ⅰ and Sal Ⅰ sites of the plasmid pBI G to generate the mammalian expression vector pBI 5LO. HeLa Tet On cell line was co transfected with pBI 5LO and pTK Hyg plasmids. Inducible expression of target gene was detected using X gal histochemical staining of cell monolayers for ? galactosidase after incubating the transfected cell line with 1 ?g/ml doxycycline for 48 h.The HindⅢ restriction digest site was inserted into polyclonal site of pBI G and recombinant vector pBI 5LO was constructed. The desired recombinant plasmid pBI 5LO was identified by restriction analysis, and orientation and junctions were confirmed by sequencing. Induction of the target gene expression by doxcycline treatment was demonstrated by X gal histochemical staining and nuclei of the cells that have expressed the pBI 5LO expression vector were brilliant blue.The 5LO inducible expression system is established successfully and it works well in cell model.