Influence of TGF-β1 siRNA mediated by lentivirus vector on hyperoxia-induced lung injury in neonatal mice
AIM: To explore the influence of knock-down TGF-β1 gene by lentivirus vector-mediated small interfering RNA (siRNA) on hyperoxia-induced lung injury in neonatal mice. METHODS: One hundred newborn KM mice were randomly divided into hyperoxia group,interference group,negative control group and air group,with 25 mice in each group. PRNAT-u6. 2 vectors containing short hairpin (shRNA)of TGF-β1 or negative control sequence were constructed and packaged with lentivirus plasmids. Two types of lentivirus vectors were administered intranasally to lungs of mice in interference group and negative control group respectively at postnatal day 3. Mice in hyperoxia group,interference group and negative control group were exposed to 600 mL/L oxygen from postnatal day 4 to day 14 before restoring to room air condition. Mice in air group were exposed to room air condition. Five mice per group were randomly sacrificed and lung tissues were excised at postnatal days 4,7,14,21 and 28 respectively. Left upper lobes were processed for paraffin embedding and sectioning. Mean linear intercept ( MLI) and radical alveolar counts ( RAC) were counted. Right lobes were excised and frozen for detecting expression of TGF-β1 mRNA and protein by RT-PCR and Western blot methods respectively. RESULTS: Lentivirus vector of TGF-β1 shRNA was successfully constructed,which was confirmed by double restriction enzyme digestion and sequencing. Compared with that in the other 3 groups,the expression of TGF-β1 mRNA in lungs decreased significantly in interfer- ence group at postnatal day 4 (P < 0. 05). At postnatal days 7,14,21 and 28,the expression of TGF-β1 mRNA and TGF-β1 protein in lungs of interference group were lower significantly than those in hyperoxia group and negative control group (P < 0. 05),but higher than those in air group( P < 0.05). MLI of alveoli decreased and RAC increased in interference group compared with those in hyperoxia group and negative control group (P < 0. 05),and MLI of alveoli increased and RAC decreased in interference group compared with those in air group at postnatal days 7,14,21 and 28 (P < 0. 05). The results showed no significant difference between hyperoxia group and negative control group at each time-point. CONCLUSION: Knock-down TGF-β1 gene by lentivirus vector-mediated small interfering RNA may protect hyperoxia-induced lung injury in neonatal mice.
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万方数据
维普
