不同氧氣處理下小鼠羊水幹細胞對急性肝損傷治療之潛力

Abstract

急性肝炎由病毒、藥物或毒物引起，將導致急性肝衰竭 (acute liver failure, ALF)。幹細胞具臨床治療潛力，而體外擴增是臨床應用幹細胞的必要步驟，然而在培養過程中幹細胞時常會喪失其幹性、增殖停滯與老化等。過去研究指出，低氧氣 (hypoxia, H) 處理細胞有助於維持幹細胞性能，但不同氧氣處理幹細胞的策略及其對小鼠ALF治療之效果仍須進一步釐清。 試驗一為探討不同氧氣處理策略之小鼠羊水幹細胞 (mouse amniotic fluid stem cells, mAFSCs ) 對其增殖和幹性 (stemness) 的影響，進一步探討細胞在體外培養過程中，前期所處氧氣濃度是否對其性狀造成長期影響，並判斷細胞性狀改善是否單純源自氧氣條件的轉換。mAFSCs 分為四組培養，分別為1.NN：全程常氧培養、2. NH：常氧培養至第四代移至低氧、3. HN：低氧培養至第四代移至常氧、4. HH：全程低氧培養。使用第十代mAFSCs進行試驗。常氧條件為21％ O2，低氧條件為5％ O2。流式細胞儀分析顯示，所分離之 mAFSCs 表面抗原Sca-1、CD90、CD29 與 MHCI 為高表現，CD34與MHCII為低表現， CD117 (c-kit) 則占比約1%。三系分化試驗中，mAFSCs可在體外誘導分化為硬骨、脂肪和軟骨細胞，以上結果證明成功建立 mAFSCs 細胞系。形態觀察顯示，NN與HN組細胞多呈扁平狀，而 NH 與 HH 組則呈紡錘狀。細胞增殖能力評估顯示，第六日時 HH 組顯著高於其餘組別；第八日則以 NH 表現最佳，其次為 HH 組別。幹性基因表現方面，NH組之Oct-4、Sox-2 表現量顯著高於其他組別。試驗二中，選用表現最佳之 NH 組與對照組 NN 進行體內試驗，使用9-10週齡 C57BL/6 小鼠，腹腔注射脂多醣 (lipopolysaccharide, L) 和D-半乳糖胺 (D-galactosamine, D) 誘發急性肝損傷，以腸繫膜注射2.5 ×105 cells/mL的方式來進行幹細胞治療，試驗分為1.對照組 2.急性肝損傷組 (L+D) 3.溶劑對照組 (Vehicle) 4. NN細胞治療組 5. NH細胞治療組。結果顯示，NN 與 NH 治療組肝臟外觀壞死與出血情形較 L+D 組明顯改善；H&E染色顯示病理發炎與壞死反應減輕，小葉結構相對清晰，NH組於門脈血管周圍可觀察到卵圓細胞 (oval cell) 輕度增生。肝功能指標方面，NN 組別與 NH 組皆能顯著緩解血清中的天門冬胺酸轉胺酶 (glutamic oxaloacetic transaminase, GOT/AST ) 及丙胺酸轉胺酶 (glutamic pyruvic transaminase, GPT/ALT ) ，兩組間沒有顯著差異。存活率分析顯示，L+D 與 Vehicle 組存活率顯著低於對照組，NN 與 NH 治療後則與對照組差異不顯著。 綜合上述結果，顯示低氧環境可有效促進 mAFSCs 之體外增殖能力，其中氧氣條件的轉換亦具維持幹性基因表現效果。雖在急性肝損傷模型中，低氧與常氧細胞皆可改善肝功能指標，但未觀察到差異顯著性，因此推論，低氧氣培養策略主要應用於提升細胞擴增效率及品質，為後續細胞製備與臨床應用提供潛在優勢。

Acute hepatitis, often caused by viral infection, drug toxicity, or chemical injury, may progress to acute liver failure (ALF), a life-threatening condition. Stem cell therapy holds promising clinical potential, with in vitro expansion being a critical prerequisite for therapeutic application. However, stem cells frequently experience a decline in stemness, proliferative arrest, and senescence during extended culture. Previous studies have suggested that hypoxic (H) conditions may help preserve stem cell properties, though the optimal oxygenation strategy and therapeutic efficacy for mouse ALF remains to be fully clarified. In the first part of this study, we investigated how different oxygenation strategies affect the proliferation and stemness of mouse amniotic fluid stem cells (mAFSCs), particularly whether prior oxygen conditions exert lasting effects and whether enhanced stem cell performance results from the hypoxic transition. mAFSCs were cultured under four conditions: (1) NN: continuous normoxic culture; (2) NH: normoxia switched to hypoxia at passage 4; (3) HN: hypoxia switched to normoxia at passage 4; and (4) HH: continuous hypoxic culture. Experiments were performed using passage 10 mAFSCs. Normoxia and hypoxia were defined as 21% and 5% O₂, respectively. Flow cytometry analysis revealed high expression levels of Sca-1, CD90, CD29, and MHC I, while CD34 and MHC II were expressed at low levels.; CD117 (c-kit) was expressed in approximately 1% of cells. Trilineage differentiation assays demonstrated the successful derivation of osteogenic, adipogenic, and chondrogenic lineages, indicating that the mAFSC line was successfully established. Morphologically, NN and HN groups displayed flattened shapes, while NH and HH groups exhibited spindle-shaped morphology. Proliferation assays revealed that HH had the highest cell counts on day 6, while NH showed superior proliferation by day 8. In terms of stemness genes, NH group expressed significantly higher levels of Oct-4 and Sox-2 compared to other groups. In the second part, the NH group, which demonstrated superior in vitro performance, along with the NN control group, was selected for in vivo evaluation. Acute liver injury was induced in 9- to 10-week-old C57BL/6 mice via intraperitoneal injection of lipopolysaccharide (LPS, L) and D-galactosamine (D-Gal, D). Stem cell therapy was administered by injecting 2.5 × 10⁵ cells/mL via the mesenteric vein. The experimental groups were as follows: (1) Control group, (2) Acute liver injury group (L+D), (3) Vehicle group, (4) NN-treated group, and (5) NH-treated group. The results show that the NN and NH treatment groups exhibited significant improvements in the morphology of the liver, necrosis, and hemorrhage compared to the L+D group. H&E staining revealed reduced inflammatory infiltration and necrosis, with clearer lobular architecture observed particularly in the NH and HH groups Notably, mild oval cell proliferation was observed near the portal area in the NH group. Serum analyses indicated that both NN and NH treatment groups significantly reduced levels of glutamic oxaloacetic transaminase (AST) and glutamic pyruvic transaminase (ALT), with no significant difference between them. Survival analysis showed that the survival rates of the L+D and Vehicle groups were significantly lower than that of the control group, whereas the NN and NH treatment groups showed no significant difference compared to the control. In summary, hypoxic conditions effectively enhanced the in vitro proliferative capacity of mAFSCs, with oxygen-switching strategies contributing to the maintenance of stemness gene expression. Although both hypoxic- and normoxia-cultured cells improved liver function in the acute liver injury model without significant differences. Therefore, hypoxic culture strategies are considered primarily beneficial for enhancing cell expansion efficiency and quality, offering potential advantages for future cell preparation and clinical applications.