Notch信号通路对先天性膈疝大鼠模型肺泡上皮分化的调控作用

Pulmonary hypoplasia is one of the most important reasons for the high morbidity and mortality of congenital diaphragmatic hernia. Despite surgical advances and advances in neonatal intensive care, mortality has still remained high. Then the research on how to improve prenatal fetal lung growth and then improve the survival rates of congenital diaphragmatic hernia has become a focus. Our previous researches showed that, in the late stage of the growth of the lungs, typeⅡalveolar epithelial cells were difficult in differentiating into typeⅠalveolar epithelial cells, and this maybe the main cause of pulmonary hypoplasia in congenital diaphragmatic hernia. Those researches found a new way to study the pulmonary hypoplasia in congenital diaphragmatic hernia. Notch signaling is closely related to the differentiation of alveolar epithelial, and it is the critical factor in the differentiation process. Our previous study showed that, besides the pulmonary hypoplasia, the expression of Notch1 and Nocth3 were abnormal in the CDH rat model. This indicated that the Nocth signaling disorder may be involved in the obstacle of differentiation in CDH. Based on those previous studies, our project team assumed that Notch signaling is involved in the differentiation process, and try to illustrate the mechanism of how Notch signaling control differentiation process of the alveolar epithelial in CDH. If we can certify this hypothesis, it would provide a new way to research and treat CDH and even other pulmonary hypoplasia diseases in newborn.

先天性膈疝所致的肺发育不良是产后患儿高死亡率主要原因，如何促进胎肺发育，提高产后患儿生存率仍然是全球性难题，迫切需要找到新突破点。项目组前期研究发现在肺发育晚期，肺泡II型上皮向I型上皮分化障碍是导致膈疝肺发育不良的重要原因，为治疗膈疝肺发育不良打开了新窗口。Notch信号通路与肺上皮分化密切相关，对调控肺泡上皮分化起关键性作用。项目组前期研究发现，先天性膈疝胎鼠肺内存在肺泡上皮分化障碍的同时，还存在Notch1和Notch3的表达异常，说明该分化障碍可能与Notch信号通路的异常调控作用有关。本项目在已有研究结果的基础上，应用RNAi干扰、重组腺病毒等技术和方法，拟证实Notch信号通路对先天性膈疝肺内II型上皮向I型上皮分化过程的影响；拟阐明Notch受体胞内段对膈疝胎肺I上皮分化的调控机制。以期为先天性膈疝及其他原因引起的新生儿肺发育障碍的研究和临床治疗提供新的方向和理论基础。

背景与目的 先天性膈疝（CDH）肺发育不良是CDH患儿高病死率的主要原因。本实验前期研究发现，肺泡Ⅱ型上皮细胞（AECⅡ）向肺泡Ⅰ型上皮细胞（AECⅠ）分化障碍，使AECⅠ数量减少是导致CDH肺发育不良的关键。Notch信号通路是调节多种组织和器官早期发育的信号通路。本研究前期预实验发现，Notch1的表达异常可能与CDH肺泡上皮细胞分化障碍调控过程相关。因此，本试验在前期研究的基础上，拟探讨CDH大鼠模型胎仔AECⅡ分化情况和Notch1信号通路的表达变化，以及二者关系。.方法成年雌性SD大鼠配种成功后，于孕9.5天随机分为3组：对照组（Control组），膈疝组，阻断组。膈疝组及阻断组均灌胃给予Nitrofen（除草醚）100mg/只(溶于花生油中)，Control组灌胃给予等量花生油。孕18.5天时，阻断组给予姜黄素（与1%CMC150mg/ml的悬浮液）灌胃（2ml/kg/d，连续3天），对照组和膈疝组给予等量1%CMC灌胃。孕21.5天经剖宫取出胎鼠，测胎鼠体重，观察记录胎鼠膈疝诱导情况，取出胎鼠双侧肺组织，记录肺重；光镜对各组肺发育情况进行组织学观察与测量；所取肺组织分别行免疫组化以及qRT-PCR检测，测定AECⅠ标志物T1α、AECⅡ标志物TTF1、Jagged1、NICD1、Hes1的表达情况，各组所得数据进行统计学比较分析。 .结果 膈疝组和阻断组共有52只胎仔膈疝形成，膈疝致畸率为63.4％。膈疝组与阻断组间致畸率差异无统计学意义（P>0.05）。膈疝组胎肺组织镜下表现存在肺明显发育不良，滞后于对照组。免疫组化与qRT-PCR结果显示：膈疝组与对照组相比，T1α的表达减弱（P<0.01），TTF1的表达增强（P<0.01）。Jagged1表达无统计学差异（P>0.05），NICD1与Hes1表达均高于对照组（P<0.01）。部分阻断Notch1表达后，阻断组较膈疝组肺发育明显改善，免疫组化与qRT-PCR结果显示：阻断组与膈疝组相比，T1α表达增强（P<0.01），TTF1表达降低（P<0.01）。 .结论 先天性膈疝大鼠模型肺发育不良存在AECⅡ向AECⅠ分化障碍， Notch1信号通路的表达异常可能是导致CDH大鼠模型肺发育不良及分化障碍的重要因素。以上结果为CDH的病因与治疗提供了新的研究方向。