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BACKGROUND:Glial fibrillary acidic protein(GFAP) expression highly correlates with spinal glial scar formation,and is regarded as an important target for scar therapy.Efficient inhibition of expression could benefit recovery from spinal cord injury.OBJECTIVE:To investigate the inhibitory effects of synthetic small interfering RNAs(siRNAs) on astro-cytic GFAP expression in rats.DESIGN,TIME AND SETTING:A randomized,controlled,animal experiment at the cellular and mo-lecular level was performed at the First Hospital of Dalian Medical University between June 2005 and Feb-ruary 2006.MATERIALS:A total of 100 seven-day-old,Sprague Dawley rats were selected.GAPDH siRNA was pur-chased from Ambion,USA,And TransMessenger? Transfection Reagent from DAKO,Carpinteria,CA.METHODS:Rat astrocytes were isolated and cultured.Three pairs of 21-nucleotide(nt) siRNAs specific to rats GFAP mRNA,401,404 and 854,were synthesized and transfected in primary astrocytes at 1,2,3,and 4 g/L using TransMessenger? Transfection Reagent.Non-transfected astrocytes served as the blank group.Cells transfected with siRNA were regarded as the negative control group,with GAPDH siRNA as the posi-tive control group,and 401 siRNA,404 siRNA,and 854 siRNA as experimental groups.MAIN OUTCOME MEASURES:GFAP mRNA and protein expression were assessed by RT-PCR and Western blot,respectively,at 24,48,and 72 hours of culture.RESULTS:GFAP mRNA expression in the positive control group was significantly less than the negative control group(P < 0.01).GFAP mRNA expression in astrocytes from three pairs of siRNA was significantly less than the blank group after 48 hours(P < 0.01),while no differences were detected between the negative control and blank groups(P > 0.05).GFAP protein expression was remarkably less in siRNA-transfected as-trocytes compared to the blank control(P < 0.01).CONCLUSION:Transfected siRNAs could significantly inhibit GFAP gene expression in astrocytes after 72 hours in culture.
BACKGROUND: Glial fibrillary acidic protein (GFAP) expression highly correlates with spinal glial scar formation, and is considered as an important target for scar therapy. Efficient inhibition of expression could benefit recovery from spinal cord injury. OBJECTIVE: To investigate the inhibitory effects of synthetic Small interfering RNAs (siRNAs) on astro-cytic GFAP expression in rats. TIME AND SETTING: A randomized, controlled, animal experiment at the cellular and mo-lecular level was performed at the First Hospital of Dalian Medical University between June 2005 and Feb-ruary 2006.MATERIALS: A total of 100 seven-day-old, Sprague Dawley rats were selected. GAPDH siRNA was pur-chased from Ambion, USA, And TransMessenger® Transfection Reagent from DAKO, Carpinteria, CA. METHODS: Rat astrocytes were isolated and cultured. Thh pairs of 21-nucleotide (nt) siRNAs specific to rats GFAP mRNA, 401,404 and 854, were synthesized and transfected in primary astrocytes at 1,2,3, and 4 g / L using TransMessenger ™ Tran sfection Reagent. Non-transfected astrocytes served as the blank group. Cells transfected with siRNA were considered as the negative control group, with GAPDH siRNA as the posi-tive control group, and 401 siRNA, 404 siRNA, and 854 siRNA as experimental groups. MAIN OUTCOME MEASURES: GFAP mRNA and protein expression were assessed by RT-PCR and Western blot, respectively, at 24, 48, and 72 hours of culture .RESULTS: GFAP mRNA expression in the positive control group was significantly less than the negative control group (P <0.01) .GFAP mRNA expression in astrocytes from three pairs of siRNA was significantly less than the blank group after 48 hours (P <0.01), while no differences were detected between the negative control and blank groups (P> 0.05). GFAP protein expression was remarkably less in siRNA-transfected as-trocytes compared to the blank control (P <0.01) .CONCLUSION: Transfected siRNAs could significantly inhibit GFAP gene expression in astrocytes after 72 hours in culture.