乙型轉化生長因子對於調節小鼠肺幹/先驅細胞分化成第Ⅰ型肺泡上皮細胞的雙重特性

Abstract

嬰兒呼吸窘迫症候群（IRDS），也稱為新生兒呼吸窘迫症候群（NRDS），是一種由肺部表面張力素分泌不足和肺部結構不成熟引起的早產兒症候群。在妊娠年齡29週之前出生的嬰兒有大約60％的機會會發生嬰兒呼吸窘迫症候群，且此症候群為早產兒死亡的主要原因。在台灣，每年約有20萬新生兒，而其中約有8％至10％的嬰兒是早產兒。為了提高早產兒的存活率，地塞米松等類固醇在臨床上被廣泛用於孕婦。當孕婦在妊娠24~34週時有早產跡象，對孕婦使用地塞米松可加速胎兒肺部發育成熟並產生足夠的表面張力素。然而，地塞米松在肺部發育成熟和促進表面張力素生合成的機轉仍不清楚。因此，建立一個研究地塞米松在肺部成熟過程的機轉的細胞模型是必要的。藉由先前的無血清培養系統，我們已經發現了一種罕見的小鼠肺幹/先驅細胞（mPSCs）群體，這群細胞表達具有特異性的細胞標誌，柯薩奇病毒/腺病毒受體（CAR），並且將細胞命名為CAR + / mPSC。透過我們的研究，CAR + / mPSCs可以有效地大量培養並分離純化（> 1,000,000個細胞數）。這些細胞能夠在7-10天內幾乎同步地分化為第I型肺泡上皮細胞。我們假設藉由第I型肺泡上皮細胞分化過程，可以用於評估地塞米松在肺部發育成熟和促進表面張力素生合成中的治療效果。在我們的研究中指出，地塞米松治療可以抑制α-SMA的表現，並促進第I型肺泡上皮細胞的分化和表皮細胞蛋白EpCAM的基因表現。此外，我們的研究還發現TGF-β抑制劑具有增加緊密連接蛋白claudin-18的協同作用，這意味著在分化過程中能夠更像典型的表皮細胞。除此之外，若另外分化的過程中加入ROCK 抑制劑，能夠進一步的抑制α-SMA的表現和增加claudin-18和EpCAM的表現量。總之，藉由CAR+/mPSCs分化的細胞模型，可以幫助了解地塞米松的治療效果和機轉，並進一步評估更有效的肺部發育治療的療法。

Infant respiratory distress syndrome (IRDS), also called neonatal respiratory distress syndrome (NRDS), is a syndrome in premature infants caused by developmental insufficiency of pulmonary surfactant production and structural immaturity in the lungs. Babies born before 29 weeks of gestation have about 60 % chance of developing IRDS, which is the leading cause of death in premature infants. In Taiwan, there are about 200,000 newborns in each year, and about 8 to 10% of the babies are preterm. In order to improve the survival rate of premature infants, steroid, such as dexamethasone, is widely used clinically for the pregnant women, who have an unavoidable sign of preterm birth at 24 to 34 weeks of gestation, to accelerate fetal lung maturation and produce pulmonary surfactant, effectively. However, the pharmacological rationale for the therapeutic effects of dexamethasone in lung maturation and producing pulmonary surfactant was still unclear. Therefore, establishing a model to reveal the biological function of dexamethasone in lung maturation will be necessary. Recently, we have identified a rare population of mouse pulmonary stem/progenitor cells (mPSCs) expressed with a specific cellular surface marker, coxsackievirus/adenovirus receptor (CAR), and named the cells as CAR+/mPSCs precisely. By our studies, CAR+/mPSCs could be effectively enriched and isolated for a pure population( > 1,000,000 cell numbers), which were able to differentiate almost in synchronized pace into type I alveolar epithelial-like cells in 7-10 days. We hypothesize that the differentiation process could be used to evaluate the therapeutic effects of dexamethasone in lung maturation and surfactant production, indicated by type I alveolar epithelial-like cells formation. Our studies showed that dexamethasone treatment could down-regulate α-SMA expression, and promote the differentiation of type I alveolar epithelial-like cells by increasing T1α and EpCAM expression. Moreover, our studies also found that TGF-β inhibitor (ALK5) had the synergistic effects to up-regulate tight junction protein claudin-18, which implies a typical epithelial phenotype for type I alveolar epithelial-like cells sheet in the differentiation process, and epithelial marker, EpCAM, expression. In addition, combined with ROCK inhibitor, the expression of α-SMA was further decreased and the expression of claudin-18, EpCAM were increased. Taken all together, we could build a cell model of type I alveolar epithelial cells and CAR+/mPSCs differentiation cell model could help pharmacological studies to reveal the therapeutic effects of dexamethasone and to evaluate a more effective treatment combination for lung development.