AIM： To study the effects of adeno-associated virus （AAV） delivered short hairpin RNAs （shRNAs） on adult CD-1 mouse cochlea damaged by aminoglycoside anti-biotic kanamycin. METHODS： Three different shRNAs were d...AIM： To study the effects of adeno-associated virus （AAV） delivered short hairpin RNAs （shRNAs） on adult CD-1 mouse cochlea damaged by aminoglycoside anti-biotic kanamycin. METHODS： Three different shRNAs were designed （p27^Kip1, p53 and p27^Kip1＋p53） and tested in COS cells. A total of 20 adult CD-1 mice were used in the experiment. Mice were divided into fve different groups （four animals/group） depending on the AAV-shRNA construct they received and whether they received kanamycin or not. Saline and AAV-EGFP injected animals were used as controls. All constructs were injected through the round window membrane （RWM） into the cochlea. Cochleae were harvested after 1 mo. Apoptosis was detected with Tunel labeling from paraffin-embedded cochlear tissue sections.RESULTS： AAV2/2-p27^Kip1-shRNA and AAV2/2-p53-shRNA were tested in COS cells. Western blotting analysis confirmed that both constructs silenced their target genes effectively in the cell culture. AAV2/2-shRNA constructs were injected into the cochlea of CD-1 mice through the intact RWM. Cotransductionof Cotransduction of individual AAV2/2-shRNAs with AAV2/2-EGFP resulted in EGFP expression in the organ of Corti.Kanamycin Kanamycin treatment had no effect on the expression pattern of the EGFP. AAV2/2-shRNA treated mice （either with p53 or p27Kip1and p53 together） showed fewer apop-totic hair cells in the cochlea than the control group （P 〈 0.05; AAV2/2-p53-shRNA vs saline P = 0.00014; AAV2/2-p27＋p53-shRNA vs saline P = 0.0011）. AAV2/2-p27-shRNA injected cochleae showed no significant difference in the number of apoptotic cells when compared to the saline injected cochleae.CONCLUSION： Silencing of p53 protein in the kana-mycin treated ears may decrease cell death in the organ of Corti.展开更多
基金Supported by grants fromm a Helsinki University Central Hospital Research Fundsthe Sigrid Jusélius Foundation+1 种基金the Instrumentarium Research Foundationthe Finnish Medical Foundation
文摘AIM： To study the effects of adeno-associated virus （AAV） delivered short hairpin RNAs （shRNAs） on adult CD-1 mouse cochlea damaged by aminoglycoside anti-biotic kanamycin. METHODS： Three different shRNAs were designed （p27^Kip1, p53 and p27^Kip1＋p53） and tested in COS cells. A total of 20 adult CD-1 mice were used in the experiment. Mice were divided into fve different groups （four animals/group） depending on the AAV-shRNA construct they received and whether they received kanamycin or not. Saline and AAV-EGFP injected animals were used as controls. All constructs were injected through the round window membrane （RWM） into the cochlea. Cochleae were harvested after 1 mo. Apoptosis was detected with Tunel labeling from paraffin-embedded cochlear tissue sections.RESULTS： AAV2/2-p27^Kip1-shRNA and AAV2/2-p53-shRNA were tested in COS cells. Western blotting analysis confirmed that both constructs silenced their target genes effectively in the cell culture. AAV2/2-shRNA constructs were injected into the cochlea of CD-1 mice through the intact RWM. Cotransductionof Cotransduction of individual AAV2/2-shRNAs with AAV2/2-EGFP resulted in EGFP expression in the organ of Corti.Kanamycin Kanamycin treatment had no effect on the expression pattern of the EGFP. AAV2/2-shRNA treated mice （either with p53 or p27Kip1and p53 together） showed fewer apop-totic hair cells in the cochlea than the control group （P 〈 0.05; AAV2/2-p53-shRNA vs saline P = 0.00014; AAV2/2-p27＋p53-shRNA vs saline P = 0.0011）. AAV2/2-p27-shRNA injected cochleae showed no significant difference in the number of apoptotic cells when compared to the saline injected cochleae.CONCLUSION： Silencing of p53 protein in the kana-mycin treated ears may decrease cell death in the organ of Corti.
