分離與鑑定SSEA-1+肺部前驅幹細胞治療過敏性氣喘

Abstract

氣喘主要的症狀為慢性呼吸道發炎與呼吸道收縮阻力（airway hyperresponsiveness, AHR）增加。目前尚無探討肺部幹細胞/前驅幹細胞（pulmonary stem/progenitor cells, PSCs）與調控過敏性呼吸道發炎的相關研究。為了尋找並進一步研究PSCs，我們利用特殊的酵素分解法，由新生小鼠肺組織中分離出單一細胞懸浮液。利用流式細胞儀、即時聚合酶連鎖反應、免疫螢光染色、共軛焦顯微鏡及掃描式電子顯微鏡鑑定PSCs的特性。在體外細胞培養模式中，以流式細胞儀、酵素連結免疫吸附法、即時聚合酶連鎖反應及免疫轉漬法，分析PSCs的細胞調控機制；同時也探討PSCs對卵白蛋白引發過敏性呼吸道發炎動物模式的影響。結果發現，由新生鼠肺部組織所分離的單一細胞懸浮液，分別表現階段特異性胚胎抗原-1（stage-specific embryonic antigen-1, SSEA-1）或幹細胞抗原-1（stem cell antigen-1, Sca-1）。SSEA-1+ PSCs大量表現於新生鼠，但成鼠中則非常稀少。進一步純化後的SSEA-1+ PSCs可分化為肺細胞（penumocytes）與氣管上皮細胞（tracheal epithelial cells）。此外，與SSEA-1- 肺部細胞相較，SSEA-1+ PSCs表現高量的克拉拉細胞分泌蛋白（Clara cell secretory protein, CCSP）。移植SSEA-1+ PSCs至過敏性呼吸道發炎小鼠體內，可降低嗜酸性白血球（eosinophils）浸潤、抑制細胞趨化素/細胞激素、增加調節型T細胞（regulatory T cells）及維持肺部CCSP表現，進而達到減少呼吸道收縮阻力與肺部受損情形。本研究發現新生鼠之SSEA-1+ PSCs可降低肺部傷害與抑制發炎反應，進而對於氣喘進程具有免疫調節能力。深入了解SSEA-1+ PSCs免疫調控能力的分子機制將有助於開發未來治療呼吸道發炎的新策略。

Asthma is characterized by chronic airway inflammation and hyperresponsiveness (AHR). Little is known about the role of pulmonary stem cells (PSCs) in allergic airway inflammation. To identify the role of PSCs population in the bronchial epithelium of neonatal mice, we developed an enzyme-based digestion method to isolate PSCs from lung tissues. Characterization of PSCs was done using flow cytometry, real-time PCR, immunofluorescence staining, confocal microscopy, and scanning electron microscopy. The effects of SSEA-1+ PSCs was studied in an in vivo model of ovalbumin-induced allergic inflammation and an in vitro model of cell-based regulation using flow cytometry, enzyme-linked immunosorbent assay, real-time PCR, and immune-blotting. Cell suspensions derived from neonatal lung tissue contained cells expressing either SSEA-1+ (stage-specific embryonic antigen-1) or Sca-1+ (stem cell antigen-1) that represent PSCs phenotype. The SSEA-1+ PSCs were prevalent in neonatal mice, but rare in adult mice. Enriched SSEA-1+ PSCs had the ability to differentiate into pneumocytes and tracheal epithelial cells. The expression of CCSP (Clara cell secretory protein) were higher in SSEA-1+ PSCs as compared with that of SSEA-1- pulmonary cells. Transplantation of SSEA-1+ PSCs in asthmatic mice reduced AHR and airway damage by decreasing eosinophil infiltration, inhibiting chemokines/cytokines production, increasing regulatory T cells, and preserving the level of CCSP. Collectively, our results indicated that neonatal SSEA-1+ PSCs contribute to ameliorate the progression of asthma by reducing lung damage and inhibiting inflammatory responses. Study about the molecular mechanisms of neonatal SSEA-1+ PSCs might shed light on etiology of airway inflammation.