宫内炎性预敏和生后高氧暴露对早产大鼠肺Bax和Bcl-2基因表达和肺细胞增殖及凋亡的影响

Increased apoptosis after intra-amniotic endotoxin priming plus hyperoxic exposure in lungs of preterm newborn rats

目的观察宫内炎性预敏及生后60％氧暴露对肺增殖性细胞核抗原（PCNA）及肺细胞凋亡影响的动态变化规律及Bcl-2家族中Bax、Bcl-2基因的表达对肺细胞凋亡的调控作用；探讨其与新型支气管肺发育不良（BPD）发病机制之间的关系。方法早产大鼠随机分为生理盐水＋高氧组、脂多糖（LPS）＋高氧组、LPS组和正常对照组，于生后第1、7和14天利用简单随机抽样方法取8只，采用免疫组织化学染色方法检测各组肺组织PCNA表达水平及脱氧核糖核酸转移酶介导的细胞凋亡标记技术（TUNEL）和逆转录聚合酶链反应技术（RT—PCR）检测各组肺组织凋亡和Bax、Bcl-2基因表达水平。结果①PCNA的表达：LPS组和生理盐水＋高氧组在生后第1天表达明显低于对照组（LPS组：0．18±0．01；生理盐水＋高氧组：0．53±0．11；对照组：1．16±0．31；P=0．005，0．021）；LPS＋高氧组在生后第14天明显低于其他3组（对照组：0．89±0．22；LPS组：1．03±0．07；生理盐水＋高氧组：0．96±0．16；LPS＋高氧组：0．47±0．08；P=0．048，0．019，0．030）。②凋亡指数（AI）：LPS组在生后第1天明显高于对照组（17．73±2．21VS7．16±0．31，P=0．021）；生理盐水＋高氧组在生后第7天最高；LPS＋高氧组在生后第14天表达明显高于其他3组（对照组：20．53±4．51；LPS组：13．99±1．69；生理盐水＋高氧组：35．08±4．96；LPS＋高氧组：49．92±7．93；P=0．005，0．002，0．048）。③BaxmRNA的表达：LPS组在生后1d明显高于其他3组（LPS组：0．73±0．06；对照组：0．16±0．03；生理盐水＋高氧组：0．23±0．03；LPS＋高氧组：0．24±0．13；P=0．001，0．002，0．002）；生理盐水＋高氧组在生后第7天表达明显高于对照组（0．58±0．06 vs 0．19±0．05，P=0．002）；LPS＋高氧组在出生后第14天明显高于对照组（0．58±0．01 vs 0．29±0．09，P=0．009）。④Bel-2mRNA的表达：LPS组在生后在第1天明显低于其他3组（LPS组：0．18±0．02；对照组：0．41±0．09；生理盐水＋高氧组：0．48±0．03；LPS＋高氧组：0．59±0．05；P=0．019，0．007，0．012）；生理盐水＋高氧组及LPS＋高氧组在生后第7天明显低于对照组（0．28±0．05／0．21±0．02 vs 0．49±0．09，P=0．02，0．008）。结论宫内炎性预敏及生后高氧暴露可抑制肺细胞增殖，可能通过提高Bax／Bcl-2的表达途径使肺组织细胞过度凋亡，进而导致BPD的发生。

Objective Apoptosis has been shown to be involved in lung remodeling in both rat lung and human fetal lung explants. Mounting evidence suggests the pro- and anti-apoptotic members of the Bcl-2 family exert most of their function at the mitochondrial level and play pivotal roles in the process of apoptosis.Molecules such as Bax （death agonist） and Bcl-2 （death antagonist） act in competition, and their relative abundance and dimerization can determine cell death or cell survival. It is not known whether apoptosis is in part responsible for the impaired lung growth found in preterm developing lung exposed to intra-amniotic endotoxin priming plus hyperoxia, and imbalance of Bax/Bcl-2 contributes to it. The purpose of this study was to investigate the expression apoptosis index （AI）, Bax and Bcl-2 in premature lungs of rats with intraamniotic endotoxin priming and/or exposed to 60% O2 and to elucidate the relationship between intrauterine inflammatory/chronic O2 exposure and the pathogenesis of bronchopulmonary dysplasia （BPD）. Methods Timed pregnant Spragne Dawley （SD） rats were randomly divided into two groups： lipopolysaccharide （LPS） group and saline group. LPS or saline were intra-amniotically injected into the sacs on gestational age day 15 （70% of term）. Six days after intra-amniotic injection, the preterm rats were delivered and randomized to put in 60% O2 exposure or in room air. On days 1, 7 and ld after birth, the lungs were removed and dissected from the main bronchi for analysis. Total RNA were extracted from the right frozen lung tissues. Lung AI was measured by terminal transferase nick end labeling （TUNEL） and Bax and Bcl-2 mRNA levels were measured by semi-quantitative reverse transcription polymerase chain reaction （RT- PCR）. Results （1）To quantify the proliferation of preterm lungs after intra-amniotic endotoxin priming and/ or exposed to 60% O2, proliferating cell nuclear antigen （PCNA） in the four experimental groups were measured： PCNA increased along with the lung development from dl to d14 in both control and endotoxin alone groups. However, in endotoxin alone group, PCNA was significantly lower than that of controls from dl to d14. In hyeroxia alone group, PCNA begun to decreased on day 7, and became significantly lower than that of the control group on day 14 （P 〈 0. 05 ）. In endotoxin plus hyperoxia group, PCNA was significantly lower than that of controls on days 1 and 7. （2）To quantify the apoptosis of immature lungs after intra-amniotic endotoxin priming and/or exposed to 60% O2, AI increased along with the lung development from d 1 to d 7 in both control group and endotoxin alone group. However, in endotoxin alone group, AI was significantly higher than that of controls from dl to d7. In hyperoxia alone group, AI begun to increased on day 7, and became significantly lower than that of the control group on day 14 （ P 〈 0. 05 ）. In endotoxin plus hyperoxia groups, AI was higher than controls on days 1 and 7, and became significantly higher than those of the other 3 groups on day 14. （3）RT-PCR showed that mRNA of Bax decreased while Bcl-2 mRNA increased from day 1 to day 14 in endotoxin alone group, and they were significantly higher than those of controls on day 1 （P 〈 0. 05 ）. Conversely, Bax mRNA increased while Bcl-2 mRNA decreased from day 1 to day 7 in hyperoxia treated groups, and was significantly different from that of the control group on days 7 and 14 in both hyperoxia treated groups. Conclusions These results suggest that intra-amniotic endotoxin presensitization and exposure to hyperoxia may inhibit proliferation of lung cells. Increased expression of Bax and Bel-2 may be related to the evolution of lung injury and may contribute to the pathogenesis of BPD.