以羊水幹細胞治療膽汁淤積造成肝纖維化大鼠之療效

Abstract

根據行政院衛生福利部之報告顯示，慢性肝病及肝硬化 (liver cirrhosis) 已長年位居國人十大死因之一，而肝纖維化 (liver fibrosis) 是慢性肝病必經程，肝纖維化是由於肝臟慢性損傷後肝臟功能性細胞死亡及細胞外基質(extracellular matrix, ECM) 大量累積之結果。造成肝臟慢性損傷原因有許多，其中膽汁淤積所造成之肝纖維化更與許多疾病相關以及治療困難等特點，目前治療方式以手術為主，引此尋找替性療法為十分重要的課題。 羊水中含有源自發育中胎兒 (fetus) 的三胚層組織之羊水幹細胞 (amniotic-fluid-derived stem cells, AFSCs) (De Coppi et al., 2007)，AFSCs移植已證實不會造成畸胎瘤 (teratomas)，且無犧牲胚胎之倫理道德爭議，亦表現胚幹細胞之多分化潛能表面標誌 Oct-4 (Prusa et al., 2003)，目前在組織修復領域已有許多成果 (Berardis et al., 2015)。而幹細胞移植入肝纖維化動物後其細胞命運推測有三條，其一：幹細胞與肝臟細胞融合；其二：幹細胞分化為肝臟細胞；其三：幹細胞透過胞外分泌物質所調控，本實驗室先前已證明AFSCs 可透過前兩項方法減緩肝纖維化症狀，但尚未證明AFSCs是否能透過胞外分泌物質減緩肝纖維化症狀，有鑑於此，本研究目的為探討AFSCs是否能減緩因膽汁淤積所導致之肝纖維化，並透過注射AFSCs條件培養液探討AFSCs是否能透過胞外分泌物質以減緩肝纖維化之症狀。 本試驗由大鼠之羊水中分離出AFSCs，經由流式細胞儀檢測表面抗體發現純化出之大鼠AFSCs表現CD29, CD90等細胞標誌，且不表現CD11b, CD45及 MHC-II等細胞標誌；於體外誘導分化試驗結果證實AFSCs具三向分化為硬骨、軟骨與脂肪之潛能；此外，在RNA層次上表現Oct4，綜合上述結果可證實大鼠 III AFSCs已成功建立。本試驗係以膽管結紮 (bile duct ligation; BDL)建立膽汁淤積肝纖維化大鼠模型，在BDL後兩週，大鼠ALT與AST即有顯著上升(p＜0.05)，且肝臟ECM沉積較BDL一週更為明顯，因此以BDL後兩週做為移植用實驗動物。體內AFSCs移植肝纖維化大鼠接受異種豬羊水幹細胞 (pigAFSCs; pAFSC)與同種異體大鼠羊水幹細胞 (ratAFSCs; rAFSC)移植後二週，無論以pAFSC或rAFSC組別移植，皆可顯著降低AST及ALT之值 (p＜0.05)，且ECM相關基因如：TGFβ、αSMA、Col1與Col3表現量亦有顯著下降(p＜0.05)；此外，在幹細胞胞外分泌之檢測試驗，在注射pAFSC與rAFSC之條件培養液後，可顯著降低AST及ALT之值 (p＜0.05) 且ECM相關基因之Col3亦有顯著下降(p＜0.05)。 綜合上述試驗結果，移植異種與同種異體AFSC皆可有效減緩大鼠因膽汁淤積形成之肝纖維化症狀，並可能透過細胞胞外分泌物質所調控，但其移植後之治療機制尚未明確，仍需進一步研究釐清之。

According to the report of the Ministry of Health and Welfare, chronic liver disease and liver cirrhosis have been one of the top ten causes of death in Taiwan. Liver fibrosis is the result of extensive accumulation extracellular matrix (ECM) of after chronic liver injury. There are many causes of chronic liver injury, liver fibrosis which caused by cholestasis is related to many diseases and hard to heal. Thus, to find alternative therapy to treat liver fibrosis which caused by cholestasis is a very important issue. Amniotic fluid stem cells (AFSCs) can differentiate to all three germ layer cells (De Coppi et al., 2007) and express pluripotent stem cell marker, Oct4 (Prusa et al., 2003) and AFSCs have many achievements in the field of organizational repair (Berardis et al., 2015). These characteristics imply that AFSCs are more primitive than adult stem cells (ASCs). Furthermore, unlike embryonic stem cells, AFSCs do not induce teratoma in vivo and do not pose any ethical concerns. For these reasons, AFSCs is regarded as a new promising source of stem cells for tissue engineering and stem cell therapy. Thus, AFSCs are considered to be emerging cell therapy material. The cell fate that stem cells after transplanted into the liver fibrosis animals was estimated three ways: the stem cells fuses with the liver cells; stem cells differentiate into hepatocyte; stem cells secrete extracellular substances, we showed that AFSCs through the first two ways to ameliorate the symptom of liver fibrosis, but has not confirmed whether AFSCs ameliorate the symptom of liver fibrosis by extracellular substances In view of this, the purpose of this study is to investigate whether AFSCs can ameliorate the symptom of liver fibrosis caused by cholestasis. Later, investigating whether extracellular substance secret by AFSCs have the efficacy through injecting AFSCs conditioned medium to BDL rats. In the establishment of AFSCs, flow cytometry was used to assess surface markers expressed of rat AFSCs. It comes out that rat AFSCs express the markers of MHC-1, CD29 and CD90 but not express CD11b, CD45 and MHC-II. Besides, these AFSCs can expresses Oct-4 in RNA level and could differentiate into osteocytes, chondrocytes, adipocytes and hepatocytes in vitro, indicating that AFSCs have been established successfully. The model rats of liver fibrosis cause by cholestasis was established by bile duct ligation (BDL). After two weeks of BDL, serum ALT and AST were both significantly increased (P <0.05), and ECM deposition appeared to be much more obvious than the first week. Thus, we use the BDL rats after for two weeks as experimental animals. In vivo transplanted two kinds of AFSCs ( pigAFSCs ; pAFSC and ratAFSCs ; rAFSC) to BDL rats, after two weeks both of pAFSC and rAFSC liver fibrosis rat group after transplantation can reduced the AST and AST significantly (P <0.05), and the and ECM-related genes expression of TGF-β, αSMA, Col1 and Col3 were also significantly decreased (p <0.05). In addition, after injection of pAFSC and rAFSC conditioned medium. Serum AST and ALT was significantly decreased (p <0.05) and the Col3 of ECM-related genes was significantly decreased (p <0.05). Overall, our results provided the implication that AFSCs can ameliorate liver fibrosis in rat through extracellular secretion aiming to provide insight into future developmentof regenerative medicine. However, the therapeutic mechanisms and cell fates in transplanted AFSCs still need to be explored.