新生大鼠在高氧环境下肺成纤维细胞P16-RB-E_2F_1通路的改变

Variation of P16-RB-E_2F_1 signal pathway in lung fibroblasts of neonatal rats treated with hyperoxia

目的探讨新生大鼠在高氧环境下肺成纤维细胞(lungfibroblasts,LF)P16-RB-E2F1蛋白信号传导通路的改变。方法足月新生SD大鼠于生后12h内分别持续吸入85%高氧和空气,于3、7、14和21d随机处死动物,无菌条件下采集肺组织,进行LF细胞的原代培养,应用免疫组织化学、Westernblotting法检测P16、RB、E2F1蛋白表达;Real-timePCR法检测其mRNA含量。免疫组织化学法检测磷酸化RB蛋白(p-RBser-795)表达。结果高氧组与空气组相比,生后7d时高氧组P16蛋白及mRNA表达开始减少,E2F1表达增加(P<0.05),14d和21d时P16蛋白及mRNA表达明显减少,E2F1表达则明显增加(P<0.01);各实验组LF的总RB蛋白表达无明显差异(P>0.05),生后7d时高氧组p-RB蛋白表达增加(P<0.05),14d和21d时明显增加(P<0.01)。结论高氧可能通过肺成纤维细胞P16基因表达失活,导致RB蛋白磷酸化(Rb灭活),E2F1基因表达水平增加,LF过度增殖最终导致肺间质纤维化。

【Objective】 To explore the variation of P16-RB-E2F1（retinoblastoma,RB） signal pathway in lung fibroblasts of neonatal rats treated with hyperoxia.【Methods】 In 12 hours after birth,full-term newborn rats were continuously exposed to oxygen（0.85） or room air.Pups were randomly selected to be sacrificed on postnatal 3 d,7 d,14 d and 21 d,lungs were immediately resected under sterile condition and primary cell culture was done.The protein expressions of p16,RB,E2F1 in LF were detected by immunohistochemistry and Western-blot;the mRNA expressions of them in LF were detected by Real-Time PCR.RB proteins phosphorylated（p-RB） at serine 795 in LF were detected by immunohistochemistry.【Results】 Exposure neonatal rats in hyperoxia for 3 days did not change the expression of p16 and E2F1 in lung fibroblast.（P〈0.05）.However,the expression of P16 in hyperoxia groups was significant down-regulated but the expression of p-RB and E2F1 was significant up-regulated on 7 day,14 day and 21 day compared with that in air-exposed groups（P〈0.05 on 7 day,P〈0.01 on 14 day and 21 day）.No notable difference was observed in the protein and mRNA expression of RB between hyperoxia groups and air-exposed groups.【Conclusion】 Exposure of neonatal rats in hyperoxia can down-regulate expression of p16 and up-regulate E2F1 expression,change the phosphorylation of RB protein,ultimately lead the pulmonary interstitial fibrosis.