同種羊水幹細胞應用於改善肝纖維化小鼠之治療潛能

Abstract

根據行政院衛生福利部之報告顯示，慢性肝病及肝硬化已長年位居國人十大死因之一，肝纖維化 (liver fibrosis) 是由於肝臟慢性損傷後反覆癒合造成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。嚴重之肝纖維化將演變為肝硬化 (cirrhosis) 及肝功能衰竭 (liver failure)，此階段之肝病只能依靠器官移植做為治療方式，但礙於肝臟捐贈來源短缺、治療費用高及台灣法律對於器官捐贈親等因素之諸多限制，多數嚴重肝病患者往往無法等到合適器官之捐贈。因此發展肝臟疾病之新治療方式，在惡化至肝硬化及肝功能衰竭等不可逆階段之前得能有效減緩肝纖維化之進程，確有其迫切之需要性。 就臨床醫學而言，取用第二孕期之胎兒羊水 (amniotic fluid) 提供進行胎兒健康檢測乃是例行工作，在羊水中已知內含大量之幹細胞，是謂羊水幹細胞 (amniotic fluid stem cells, AFSCs)，AFSCs已知具較他種成體幹細胞更佳的端粒酶活性 (telomerase) 及較長之端粒 (telomere) 長度，除其具高度增殖性外，且能表現胚幹細胞特有之多分化潛能表面標誌 Oct-4，其應用不僅可以無實際道德爭議，且不會有發展成為畸胎瘤之危險，因此AFSCs乃被視為新興之細胞治療材料。 本試驗由小鼠之羊水中成功分離出羊水幹細胞，經由流式細胞儀完成檢測其表面抗體，結果發現純化出之小鼠羊水幹細胞，得能表現包括：CD29, CD44, Sca-1, 及 MHC-I 等細胞標誌，且不表現諸如：CD11b, CD166, CD34, CD45, CD31, CD105, CD86, CD133, CD117, 及 MHC-II等細胞標誌；此外，進一步在體外誘導分化之試驗結果，證實羊水幹細胞確實具有分化成為類肝臟細胞之潛能，以此可作為後續肝纖維化小鼠之幹細胞治療試驗之依據。進一步透過體內細胞移植試驗結果證實，彼等肝纖維化小鼠接受同種異體羊水幹細胞移植後四週，無論於小鼠羊水幹細胞之懸浮細胞與不同大小之球狀細胞團包括：＜40 μm, 40~70 μm, 及 ＞70 μm 等直徑大小不同之處理組別者，分別均呈現有顯著降低其肝臟細胞大量死亡導致其血液生化指標包括GOT/AST及 GPT/ALT等測定值之顯著改善效果外 (p＜0.05) ，且有降低其肝臟體重比及其肝臟內原蓄積纖維之含量，惟血清中白蛋白 (albumin) 之含量在細胞移植組及未移植之對照組中二者並未有顯著差異產生。此外，在細胞移植後之遷移命運，檢測結果發現，其中部分經植入源自綠色螢光小鼠之羊水幹細胞者於24小時後，呈現有與紅色螢光小鼠之肝臟細胞發生細胞融合之現象，而尚有部分植入之羊水幹細胞仍維持原單一綠色螢光蛋白之表現狀態，進一步試驗在48小時後取其組織切片中驗證結果，則發現有共同表現包括綠色及紅色螢光蛋白之細胞，惟於細胞移植四週後再次取樣測試結果顯示，無論於肝臟組織切片或流式細胞儀之檢測者，分別皆未能發現彼等表現有綠色螢光蛋白之細胞者。 綜合上述試驗結果，移植同種異體之小鼠羊水幹細胞，確能有效減緩彼等受試小鼠肝臟纖維化之症狀，惟其移植後呈現療效之確實作用機制，則有待進一步研究釐清之。

According to the statistic results from the Ministry of Health and Welfare, Executive Yuan, Taiwan, chronic liver disease and cirrhosis have been ranking on the top 10 major causes of death for decades in Taiwan. Liver fibrosis results from the wound-healing response of the liver to the chronic damage. Repeating injury raises the loss of functional hepatocytes and the accumulation of extracellular matrix (ECM) proteins on liver. Advanced liver fibrosis will result in cirrhosis and liver failure, and liver transplantation is often the only option for effective therapy. However, the shortage of available donor livers limits this treatment. Thus, alternatively investigate a new therapy for such kind of liver disease is essential. Amniotic fluid, which is routinely sampled for prenatal diagnosis in the second trimester of pregnant contains abundant of stem cells. Amniotic fluid stem cells (AFSCs) exhibit long telomere length, also have high levels of telomerase activity. In spite of the outstanding proliferative rate, AFSCs can also differentiate to all three germ layer cells and express pluripotent stem cell marker, Oct4. These characteristics imply that AFSCs are more primitive than other stem cells isolated form adult tissues. 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. This study is carried out to investigate whether mouse AFSCs are able to ameliorate the symptom of liver fibrosis through allogenic transplantation. In the establishment of amniotic fluid stem cells, flow cytometry was used to assess markers expressed by mouse AFSCs. It comes out that mouse AFSCs express the markers of CD29, CD44, Sca-1, MHC-I, and do not express CD11b, CD166, CD34, CD45, CD31, CD105, CD86, CD133, CD117, MHC-II. Besides, mouse AFSCs could differentiate into hepatocytes in vitro, which implies that AFSCs has the potential to be applied to the therapeutic strategy of liver disease. Allogenic AFSCs single cells and spheres with three groups of diameters including 40μm, 40~70μm and over 70μm were injected via portal vein of liver CCl4 treated mice respectively. The treated results of liver fibrosis mice show that all groups of AFSCs transplantation have a significant antifibrotic effect as evidenced by the significant decrease in the blood biochemical analysis of GOT, GPT and liver-body weight ratio compared to the control group (p<0.05). However, there is no significant difference in the albumin expression of the serum analysis between each group. In addition, the fibrotic tissues were evaluated less by Masson's trichrome staining 4 weeks after cell transplantation compared to the control group. To investigate the cell fate of transplanted AFSCs in liver injured mice, we transplant EGFP-mouse AFSCs into CCl4 treated Ds-red mice. Tissue section results show that most EGFP AFSCs are found around the blood vessel of liver in 24 to 48 hours after transplantation. Some of the transplanted cells are already fused with the cells of recipient and express EGFP and Ds-red protein in the same time. The same results can also be found in the fluorescent analysis of flow cytometry. However, no EGFP cells is found after four week of transplantation. In conclusion, AFSCs have a potential therapeutic effect on ameliorate the damage of liver fibrosis, and there might be somewhat antifibrotic processes would be clarified in addition to their capacity to differentiate into hepatocytes.