Objective: To obtain the recombinant fusion AIF genes inserted into the eukaryotic expression vectorpIRES2-EGFP, to observe the expression and location ofthe fusion AIF genes (3NE: PE(280-358)-AIF1-120, and4NE: PE(280...Objective: To obtain the recombinant fusion AIF genes inserted into the eukaryotic expression vectorpIRES2-EGFP, to observe the expression and location ofthe fusion AIF genes (3NE: PE(280-358)-AIF1-120, and4NE: PE(280-364)-AIF1-120), and to detect and compare their apoptosis inducing effects on the transfected HeLacells. Methods: Full-length human AIF gene was clonedby RT-PCR, and its N-terminal mitochondrial localization sequence (MLS) was replaced by part sequence ofPsuedomonas exotoxin A (PE) translocation domain(PEII(280-358/364)), then the recombinant fusion geneswere inserted into the pIRES2-EGFP eukaryotic expression vector. After these genes were transiently transfected into HeLa cells with LipofectAmine, the expression of therecombinant fusion AIF genes and their effects on HeLacells were detected by fluorescent microscopy, laser confocal microscopy and electron microscopy. Results: Theeukaryotic expression vectors containing the recombinant fusion AIF genes (pIRES2-EGFP-PEII(280-358/364)-AIF1- 120) were constructed successfully. It wasdemonstrated that the fusion AIF protein genes wereexpressed effectively in the transfected cells, with the GFP co-expressed in cells by indirect immunofluorescence staining analysis. After transfection, expression of the genes could induce HeLa cells to exhibit the typical apoptosis features: such as plasma membrane blebbing and peripheral chromatin condensation. As compared with control groups, the untreated cells and the void vector transfected cells, the living cell number of the AIF gene transfected cells reduced distinctly. Conclusion: Our data prove that the expression of the recombinant human AIF fusion genes could induce apoptosis in transfected HeLa cells, which provides new strategy for cancer killing.展开更多
Background Implanted medical catheter-related infections are increasing,hence a need for developing catheter polymers bonded to antimicrobials.We evaluated preventive effects of levofloxacin-impregnated catheters in c...Background Implanted medical catheter-related infections are increasing,hence a need for developing catheter polymers bonded to antimicrobials.We evaluated preventive effects of levofloxacin-impregnated catheters in catheterrelated Psuedomonas aeruginosa （strain PAO1） infection.Methods Drug release from levofloxacin-impregnated catheters was measured in vitro.Levofloxacin-impregnated catheters and polyvinyl chloride （PVC） catheters were immersed in 5 ml 50％ Luria Bertani medium containing 108 CFU/ml Pseudomonas aeruginosa then incubated for 6,12,24 or 48 hours at 37℃ when bacteria adhering to the catheters and bacteria in the growth culture medium were determined.Impregnated and PVC catheters were singly implanted subcutaneously in mice,50 μl （107CFU） of PAO1 was injected into catheters.After the first and fifth days challenge,bacterial counts on implanted catheters and in surrounding tissues were determined microbiologically.Bacterial colonization and biofilm formation on implanted catheters were assessed by scanning electron microscopy.Results Drug release from levofloxacin-impregnated catheters was rapid.Levofloxacin-impregnated catheters had significantly fewer bacteria compared to PVC in vitro.After first and fifth day of challenge,no or significantly fewer bacteria adhered to impregnated catheters or in surrounding tissues compared to PVC.Scanning electron microscopical images after first day displayed from none to significantly fewer bacteria adhering to impregnated implanted catheters,compared to bacteria and microcolonies adhering to PVC catheters.After the fifth day,no bacteria were found on impregnated catheters,compared to clusters surrounding mucus-like substance and coral-shaped biofilms with polymorphonuclear leukocyte on PVC catheters.After the first day of challenge,secretion occurred in all implanted catheters with surrounding tissues mildly hyperaemic and swollen.After the fifth day,minute secretions inside impregnated catheters and no inflammation in tissues,whereas purulent secretion inside PVC catheters and abscesses in surrounding tissues.Conclusion Levofloxacin-impregnated catheter is a promising new strategy for prevention of catheter-related Psuedomonas aeruginosa infection.展开更多
基金This work was supported by grants from The National Natural Sciences Foundation of China for Outstanding Youth Scholars (No.39925036) The Military Research Fundation of China for Outstanding Youth Scholars (No.98J009) and State 863 High Technology R
文摘Objective: To obtain the recombinant fusion AIF genes inserted into the eukaryotic expression vectorpIRES2-EGFP, to observe the expression and location ofthe fusion AIF genes (3NE: PE(280-358)-AIF1-120, and4NE: PE(280-364)-AIF1-120), and to detect and compare their apoptosis inducing effects on the transfected HeLacells. Methods: Full-length human AIF gene was clonedby RT-PCR, and its N-terminal mitochondrial localization sequence (MLS) was replaced by part sequence ofPsuedomonas exotoxin A (PE) translocation domain(PEII(280-358/364)), then the recombinant fusion geneswere inserted into the pIRES2-EGFP eukaryotic expression vector. After these genes were transiently transfected into HeLa cells with LipofectAmine, the expression of therecombinant fusion AIF genes and their effects on HeLacells were detected by fluorescent microscopy, laser confocal microscopy and electron microscopy. Results: Theeukaryotic expression vectors containing the recombinant fusion AIF genes (pIRES2-EGFP-PEII(280-358/364)-AIF1- 120) were constructed successfully. It wasdemonstrated that the fusion AIF protein genes wereexpressed effectively in the transfected cells, with the GFP co-expressed in cells by indirect immunofluorescence staining analysis. After transfection, expression of the genes could induce HeLa cells to exhibit the typical apoptosis features: such as plasma membrane blebbing and peripheral chromatin condensation. As compared with control groups, the untreated cells and the void vector transfected cells, the living cell number of the AIF gene transfected cells reduced distinctly. Conclusion: Our data prove that the expression of the recombinant human AIF fusion genes could induce apoptosis in transfected HeLa cells, which provides new strategy for cancer killing.
基金This study was supported in part by grants from the National Natural Science Foundation of China （No. 30760084） and by Guangxi Health Department Self Financing Scientific Research, China （No. Z2008154）.
文摘Background Implanted medical catheter-related infections are increasing,hence a need for developing catheter polymers bonded to antimicrobials.We evaluated preventive effects of levofloxacin-impregnated catheters in catheterrelated Psuedomonas aeruginosa （strain PAO1） infection.Methods Drug release from levofloxacin-impregnated catheters was measured in vitro.Levofloxacin-impregnated catheters and polyvinyl chloride （PVC） catheters were immersed in 5 ml 50％ Luria Bertani medium containing 108 CFU/ml Pseudomonas aeruginosa then incubated for 6,12,24 or 48 hours at 37℃ when bacteria adhering to the catheters and bacteria in the growth culture medium were determined.Impregnated and PVC catheters were singly implanted subcutaneously in mice,50 μl （107CFU） of PAO1 was injected into catheters.After the first and fifth days challenge,bacterial counts on implanted catheters and in surrounding tissues were determined microbiologically.Bacterial colonization and biofilm formation on implanted catheters were assessed by scanning electron microscopy.Results Drug release from levofloxacin-impregnated catheters was rapid.Levofloxacin-impregnated catheters had significantly fewer bacteria compared to PVC in vitro.After first and fifth day of challenge,no or significantly fewer bacteria adhered to impregnated catheters or in surrounding tissues compared to PVC.Scanning electron microscopical images after first day displayed from none to significantly fewer bacteria adhering to impregnated implanted catheters,compared to bacteria and microcolonies adhering to PVC catheters.After the fifth day,no bacteria were found on impregnated catheters,compared to clusters surrounding mucus-like substance and coral-shaped biofilms with polymorphonuclear leukocyte on PVC catheters.After the first day of challenge,secretion occurred in all implanted catheters with surrounding tissues mildly hyperaemic and swollen.After the fifth day,minute secretions inside impregnated catheters and no inflammation in tissues,whereas purulent secretion inside PVC catheters and abscesses in surrounding tissues.Conclusion Levofloxacin-impregnated catheter is a promising new strategy for prevention of catheter-related Psuedomonas aeruginosa infection.
