摘要
AIM: To study the effects of adeno-associated virus(AAV) delivered short hairpin RNAs(shR NAs) on adult CD-1 mouse cochlea damaged by aminoglycoside antibiotic kanamycin. METHODS: Three different sh RNAs were designed(p27Kip1, p53 and p27Kip1+p53) and tested in COS cells. A total of 20 adult CD-1 mice were used in the experiment. Mice were divided into five 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-p27Kip1-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. Cotransductionn off individual AAV2/2-sh RNAs with AAV2/2-EGFP resulted in EGFP expression in the organ of Corti. Kaannaammyycciinn treatment had no effect on the expression pattern of the EGFP. AAV2/2-sh RNA treated mice(either with p53 or p27Kip1 and p53 together) showed fewer apoptotic 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 kanamycin treated ears may decrease cell death in the organ of Corti.
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.
基金
Supported by grants fromm a Helsinki University Central Hospital Research Funds
the Sigrid Jusélius Foundation
the Instrumentarium Research Foundation
the Finnish Medical Foundation
关键词
英文
文摘
医学
杂志
Inner ear
Adeno-associated virus
Short hairpin RNA
p27
p53
Kanamycin
Apoptosis