S100A16 基因缺失导致新生小鼠因肺不张而死亡的机制研究

In the prvious studies, we generated S100A16 knoct-out (S100A16fl/-) mouse through homologous recombination and found that S100A16-/- mouse died shortly after birth. Further study rervealed that comparing with S100A16 wild-type (S100A16+/+), ① pulmonary surfactants SP-B and C protein expressions decreased in knock-out (S100A16-/-) mouse lung tissue; ② HE staining results showed that lung from newborn S100A16-/- mouse atelectasis, but the heterozygous (S100A16fl/-) did not have any difference; ③ electron microscopy detected that there were ultrastructural abnormalities in the lung of S100A16-/- newborn mouse, large and loose structure of lamellar bodies. Therefore, we postulate that S100A16-/- mouse had substantially reduced peripheral alveolar expansion before birth and failed to initiate breathing at birth. We will test this hypothesis by mouse model and separation of primary cells, as well as combination with co-IP, HE staining, Gene chip, etc. Results of this project will reveal the roles and molecular mechanisms of S100A16 in lung development, thus provide the theoretical and experimental basis for the treatment of respiratory distress syndrome of newborn (RDSN).

S100A16基因敲除杂合子小鼠是我们前期成功构建的一种模型小鼠，繁育过程中难以得到纯合子小鼠，发现此基因型新生小鼠出生后24小时内死亡。排除心脏发育问题后，检测纯合子新生小鼠肺组织，与野生型小鼠相比，缺乏S100A16基因导致 ①肺泡不张或不全张；②肺表面活性物质结合蛋白SP-B和SP-C表达下降；③II型肺泡上皮细胞中板层小体数目减少、结构松散。由此，我们推测：缺乏S100A16基因引起SP-B和SP-C表达减少，诱发新生小鼠肺不张，最终致其死亡。本项目拟采用体内和体外实验，结合HE染色、免疫共沉淀、及基因芯片等手段对此假设进行验证，并揭示缺乏S100A16基因下调SP-B和SP-C的分子机制。本项目的研究结果将揭示S100A16基因对小鼠肺发育的作用及分子机制，从而为临床新生儿呼吸窘迫综合征的防治提供理论依据和生物学靶标。

我们严格执行原研究计划，完成项目计划书中制定的大部分研究内容，而且增加了课题研究的广度和深度，为该项目的后续研究奠定了坚实的理论和实验基础。主要研究结果如下：.我们在饲养小鼠过程中发现，得不到敲除S100A16基因的纯合子小鼠（S100A16ko-/-）。我们重复观察记录5代小鼠生育情况，合计225只小鼠的出生情况。结果显示，没有得到S100A16ko-/-小鼠。首先，我们怀疑是胎鼠心脏发育问题，超声检测孕鼠胚胎，未发现异常。其次，我们怀疑是胎鼠肺发育问题。HE染色显示：S100A16fl/fl和S100A16fl/-的肺泡张开，肺泡形态正常，而S100A16ko-/-没有明显的肺囊泡及肺泡形成，肺间质增厚，并伴有充血。Werstern blot检测结果显示敲除S100A16 下调小鼠肺表面活性物质结合蛋白SP-B 和 SP-C的表达。S100A16 ko-/-小鼠II型肺泡上皮细胞内板层小体数目减少结构松散。我们采用基因芯片技术筛查受S100A16基因调控的基因，Werstern blot的方法检测了小鼠肺组织中信号分子FOXA-2和 CEBP/α的表达，很遗憾，没有找到变化明显的分子。.同时，我们发现S100A16是通过影响11β-HSD1降解而发挥其促进前体脂肪细胞分化脂质聚集的作用。高脂喂养，S100A16转基因小鼠更易发生肥胖和胰岛素抵抗，脂肪组织中11β-HSD1蛋白表达增加。高表达S100A16或11β-HSD1都能促进前体脂肪细胞分化，而干扰S100A16或11β-HSD1都起到反作用。S100A16和11β-HSD1蛋白表达呈正相关，而并未影响其mRNA表达水平，我们推测S100A16影响了11β-HSD1蛋白降解。抑制新蛋白合成的情况下，发现高表达S100A16抑制11β-HSD1降解。