先天性膈疝模型胎鼠肺发育不良的超微结构动态观察

Sequential Ultramicrostructural Observation on the Hypoplastic Lung of Fetal Rat Model of Congenital Diaphragmatic Hernia

目的探讨先天性膈疝(CDH)肺发育不良的机制,研究CDH模型胎鼠肺组织不同发育阶段的超微结构特点。方法将7只Sprague-Dawley(SD)孕鼠用随机数字表法分为两组,膈疝组(n=4):于孕9.5 d用Nitrofen(125 mg/只,溶于2 ml橄榄油中)灌胃1次,分别于SD鼠孕16 d1、8 d和21 d取胎鼠3只、10只、17只;对照组(n=3):于妊娠9.5 d时用橄榄油2 ml灌胃1次,分别于SD鼠孕16 d、18 d和21 d各取胎鼠10只。将16 d胎鼠肺组织切片进行透射电子显微镜观察;18 d胎鼠肺组织进行HE染色,透射电子显微镜观察并统计膈疝的发生率;21 d胎鼠肺组织除进行HE染色、透射电子显微镜观察、统计膈疝的发生率外,计算胎鼠肺重与体重比(Lw/Bw)。结果 (1)膈疝组胎鼠肺重/体重比显著低于对照组(0.0238 vs.0.0430,P=0.04);膈疝组18 d和21 d时胎鼠膈疝发生率分别为90.00%和82.35%,表明膈疝组胎鼠肺发育不良,而对照组18 d和21 d时胎鼠均无膈疝发生;(2)与对照组相比,膈疝组16 d时胎鼠肺超微结构也呈现出肺发育不良的改变:基板较宽,内容物很少;肺上皮细胞常染色质为主,核糖体丰富;支气管腔无微绒毛;18 d和21 d的样品观察表明,随着孕期的延长,上述特征更明显;(3)对照组与膈疝组孕21 d时胎鼠肺泡Ⅱ型上皮细胞均有典型的板层小体,反映肺组织某些晚期出现的细胞亚结构尚正常。结论膈疝早期胎鼠肺既已存在发育不良,而非晚期发育时所形成。提示膈疝肺发育不良的处理应在肺发育早期即行干预。

Objective To explore the mechanism of pulmonary hypoplasia in case of congenital diaphragmatic hernia（CDH）,and study the ultramicrostructural features of lung tissue of CDH fetal rat models at different developmental stages.Methods Seven Sprague-Dawley（SD） pregnant rats were randomly divided into CDH group（n=4） and control group（n=3）.For the rats in the CDH group,Nitrofen was used to fill in the stomach once at day 9.5 of pregnancy（125 mg of Nitrofen dissolved in 2 ml of olive oil each）,and 3,10,17 fetal rats were collected at day 16,18 and 21 of pregnancy respectively.For the rats in the control group,2 ml of olive oil was used to fill in the stomach,and 10 fetal rats were collected at day 16,18,and 21 of pregnancy respectively.The lung tissue sections of the fetal rats collected on day 16 were observed under transmission electron microscope（TEM）.For the lung tissue of the fetal rats collected on day 18,hematoxylin-eosin（HE） staining and TEM observation were performed and the incident of CDH was detected.Besides the procedures carried out for the rats collected on day 18,the ratio of fetal lung to body weight was observed for the lung tissue of the fetal rats collected on day 21.Results（1） The ratio of fetal lung to body weight of fetal rats in the CDH group was significantly lower than that of fetal rats in the control group（0.0238 vs. 0.0430,P0.01）.The incidences of CDH in the 18th-day and 21st-day fetal rats in the CDH group were 90.00% and 82.35% respectively,while no CDH was observed in the corresponding fetal rats in the control group,suggesting pulmonary hypoplasia in the CDH group.（2） The ultramicrostructural observation showed that compared with the control group,pulmonary hypoplasia appeared in 16th-day fetal lungs in the CDH group,i.e.,broad breathing barrier substrate,little contents,predominant euchromatin and rich ribosomes in the alveolar epithelial cells,and no microvilli in the bronchial lumen.The observation on the 18th-day and 21st-day samples suggested that,with the progressing of pregnancy,the above-mentioned features became more obvious.（3） Typical lamellated body was observed in fetal lung type Ⅱ alveolar epithelial cells from the 21st-day fetuses in both the CDH group and the control group,suggesting that some late subcellular structures were normal.Conclusion Lung hypoplasia develops in the early period of fetal rats with CDH rather than in the late period,implying that the treatment of pulmonary hypoplasia of diaphragmatic hernia should be performed in the early stage of lung development.