羊水幹細胞及其條件培養液治療肺纖維化大鼠之潛能

Abstract

肺纖維化之疾病歷程緩慢、漸進且不可逆，其特徵為肺泡結構被破壞和過度之細胞外基質沉積，肺功能將持續下降，最終呼吸衰竭甚至死亡。肺纖維化之死亡率很高，且肺纖維化的發生率與盛行率正逐年增加，惟目前仍未有可行之治療方法可以阻止或逆轉肺纖維化之進展。已有許多研究指出羊水幹細胞 (amniotic fluid stem cells, AFSCs) 對於多種疾病治療模式具修復損傷組織之特點，且尚未發現其有不利影響，故對於治療肺纖維化具有極大優勢。然而目前尚不清楚 AFSCs 對於不可逆之肺纖維化是否具有療效，且尚未證明 AFSCs 是否可透過旁分泌釋放之分子改善肺纖維化。此外，以傳統方式誘導之肺纖維化動物模式可能有纖維化自發性消退之侷限性，並不符合人類疾病進展，故此建立方法仍有待改善。有鑑於此，本研究擬探討 AFSCs 及其條件培養液 (AFSCs derived-conditioned medium, AFSCs-CdM) 治療博來黴素 (bleomycin, BLM) 誘發之大鼠單側左肺不可逆纖維化之功效。 本研究主要分為三個試驗。試驗一為建立綠色螢光大鼠之 AFSCs：自胚胎 (E12.5~E13.5) 之羊水中分離出 AFSCs，其外型呈現近似於纖維母細胞之紡錘狀，且經由螢光顯微鏡觀察與流式細胞儀分析後確認其可表現綠色螢光蛋白質 (green fluorescent proteins, GFP)；以流式細胞儀分析細胞表面抗原之結果顯示 AFSCs 會表現 CD90、CD29 及 MHC class I，但不表現 CD45、CD11b 及 MHC class II；AFSCs 於體外誘導分化試驗中顯示可分化為脂肪、硬骨和軟骨細胞；以上結果顯示本試驗成功建立可表現 GFP 之大鼠 AFSCs。試驗二為建立大鼠單側左肺纖維化模式：將大鼠分為正常組 (Normal group)、假手術組 (Sham group)、BLM 損傷第14天組 (BLM D14 group)、BLM 損傷第21天組 (BLM D21 group)、BLM 損傷第28天組 (BLM D28 group) 及 BLM 損傷第49天組 (BLM D49 group)，共6組；以自製軟管插入 8~9 週齡雄性 SD 大鼠之左支氣管，以適當角度注入 BLM 或 DPBS；Hematoxylin and eosin (H&E) staining 結果顯示 BLM 損傷組有細胞浸潤情形、肺泡間隔明顯增厚及肺泡結構被破壞；Masson’s trichrome staining 結果顯示以 BLM 損傷之大鼠左肺自第 14 天開始即有極顯著纖維化現象 (p < 0.001)，且損傷後第 21 天開始即維持更為嚴重但穩定之纖維化至損傷後第 49 天，沒有顯著差異 (p > 0.05)；以上結果顯示本試驗成功建立不可逆轉之大鼠單側左肺纖維化模式。試驗三為利用 AFSCs 及 AFSCs-CdM 治療左肺纖維化大鼠：將大鼠分成正常組 (Normal group)、DPBS 注射組 (BLM × DPBS group)、非條件培養液注射組 (BLM × non-CdM group)、AFSCs 移植組 (BLM × AFSCs group) 和 AFSCs-CdM 移植組 (BLM × AFSCs-CdM group)，共 5 組；於 BLM 損傷後第21天，經尾靜脈注射 DPBS、non-CdM、AFSCs 或 AFSCs-CdM 以進行治療；H&E staining 結果顯示 AFSCs 和 AFSCs-CdM 移植組減輕肺泡間隔增厚情形，此外發現其具有較完整之肺泡結構；Masson’s trichrome staining 結果顯示 AFSCs 和 AFSCs-CdM 移植組雖較正常組有極顯著纖維化情形 (p < 0.001)，但對於 DPBS 和 non-CdM 注射組仍極顯著降低膠原蛋白沉積量 (p < 0.001)；以上結果顯示 AFSCs 和 AFSCs-CdM 可改善不可逆轉之大鼠肺纖維化，減輕膠原蛋白沉積，甚至觀察到肺泡結構。 綜觀所述，本研究之結果有助於了解 AFSCs 和 AFSCs-CdM 治療不可逆肺纖維化之潛能，未來可再進一步探討 AFSCs 和 AFSCs-CdM 之治療劑量、路徑和修復機制，期望能對臨床試驗及相關研究提供參考依據。

Pulmonary fibrosis (PF) is a chronic, progressive and irreversible lung disease, which leads to damage of alveolar structures and excessive deposition of extracellular matrix. These consequences result in a gradual declining in lung function, respiratory failure and even death; moreover, the mortality rate of PF is very high, and worldwide incidence and prevalence of PF has increased over time. Worse still, there is no effective therapy to inhibit or reverse the progression of PF. Many studies have demonstrated that amniotic fluid stem cells (AFSCs) can repair damaged tissues, and they have not shown deleterious effects in a variety of disease models, thus AFSCs may have the great therapeutic potential for PF. However, it is unclear whether AFSCs are effective in irreversible PF, and it has not been proven whether AFSCs can improve PF by paracrine released molecules. In addition, the animal model of PF induced by traditional methods may have the limitation of spontaneous resolution of fibrosis, which is not consistent with the progression in human PF. Thus, the establishment method still needs to be improved. In view of this, this study intends to investigate the efficacy of AFSCs and its conditioned medium (AFSCs-CdM) in the bleomycin (BLM)-induced irreversible fibrosis of the left lung in rats. This research is mainly divided into three trials. The trial one is to establish AFSCs derived from the amniotic fluid of green fluorescent proteins (GFP) bearing rat embryos (E12.5~E13.5). Morphology of AFSCs were similar to fibroblasts, and these cells could show GFP by fluorescence microscopy and flow cytometry analysis. The cell surface antigen analysis showed that AFSCs expressed CD90, CD29, and MHC class I, but did not express MHC class II, CD45, and CD11b. Furthermore, AFSCs had the capacity to differentiate into adipocytes, osteoblasts and chondrocytes in vitro. The results above show the successful establishment of rat AFSCs which can express GFP. The second trial is to establish a one-sided left lung fibrosis model in rats. Rats were randomly divided into Normal group, Sham group, BLM D14 group, BLM D21 group, BLM D28 group, and BLM D49 group, a total of 6 groups. A handmade tube was inserted into the left bronchi of 8~9-week-old male SD rats, and then BLM or DPBS was injected at an appropriate angle. The Hematoxylin and eosin (H&E) staining results showed that the BLM injury groups had cell infiltration, thickened alveolar septa and alveolar structures destruction. Masson's trichrome staining results showed that the left lung of rats injured with BLM had significant fibrosis from day 14 after injury (p < 0.001), and fibrosis was more severe from day 21 after injury which was no significant difference between day 21 to day 49 after injury (p > 0.05). The results above show that the irreversible and one-sided left PF rat model is successfully established. The last trial is to treat PF rats with AFSCs and AFSCs-CdM. Rats were randomly divided into Normal group, DPBS injection group (BLM × DPBS group), non-CdM injection group (BLM × non-CdM group), AFSCs transplantation group (BLM × AFSCs group), AFSCs-CdM transplantation group (BLM × AFSCs-CdM group), a total of 5 groups. On day 21 of BLM injury, DPBS, non-CdM, AFSCs, or AFSCs-CdM were injected through tail vein for treatment; H&E staining results showed that AFSCs and AFSCs-CdM transplantation groups had less thickened alveolar septa, and more intact alveolar structures were found in both groups. Masson's trichrome staining results showed that although AFSCs and AFSCs-CdM transplantation groups had a very significantly fibrosis (p < 0.001) compared with Normal group, the collagen deposition was still significantly reduced compared with the DPBS and non-CdM injection groups (p < 0.001). The results above show that AFSCs and AFSCs-CdM can improve irreversible PF in rats by reducing collagen deposition; in addition, alveolar structures are even observed. In summary, this study helps us to understand the potential of AFSCs and AFSCs-CdM in the treatment of irreversible PF rats. In the future, we can further investigate the therapeutic dose, route and mechanisms of AFSCs and AFSCs-CdM, which can be provided as a reference for clinical trials and related studies.