豬脂肪幹細胞應用於異種移植改善小鼠肝纖維化之研究

Abstract

慢性肝病為國人十大死因之一，其可能原因為病毒性肝炎、酒精或藥物濫用、以及飲食習慣。肝纖維化是慢性肝病惡化必經之過程，過去研究指出肝纖維化可逆轉，如何找出有效的治療方式也成為熱門研究。許多文獻證實骨髓間葉幹細胞（BM-MSCs）在肝纖維化動物模型上之治療潛力。脂肪間葉幹細胞（AD-MSCs）為另一類型之成體幹細胞，其擁有與骨髓間葉幹細胞相似之表面抗原表現以及轉分化潛能，由於其分離過程之侵入性小於其他細胞來源，AD- MSCs在近期的再生醫學研究中極具吸引力。 本試驗自紅螢光蛋白轉基因豬脂肪組織中分離並純化出具有多分化潛能之脂肪幹細胞AD-MSCs，經過流式細胞儀分析，證實其表現與間葉幹細胞相似之表面抗原CD29、CD44與CD90，並可在體外經誘導分化為成骨細胞、軟骨細胞、脂肪細胞以及肝臟細胞，此外，AD-MSCs穩定表現紅色螢光蛋白DsRed，有利於體內試驗之後續追蹤，此結果使其在再生醫學以及細胞治療上極具應用價值。為在動物試驗中測試AD-MSCs是否具治療肝纖維化之潛能，本試驗採用八週齡之ICR公鼠，給予灌食四氯化碳溶液四週後，小鼠血清中GOT、GPT以及ALB顯著高於控制組 (P<0.05)。此外，為增加AD-MSCs遷移入肝臟組織間之效率，以立體培養之方式將細胞培養成不同大小之立體團塊 (sphere)，並分為四十微米以下、介於四十至七十微米、七十微米以上以及未經立體培養之懸浮細胞等四組，經由肝門靜脈注射技術，以最直接之方式將脂肪幹細胞及其團塊移植入小鼠體內 (每隻小鼠1x106細胞)，使其隨血流立即流經肝臟，以期能在治療效果上有所突破。研究結果顯示，經過細胞移植手術兩週後，小鼠血清中GOT、GPT及ALB比起控制組顯著下降 (P<0.05)，細胞移植手術四週後，取試驗小鼠肝臟組織，以梅森氏三色染色法標定其肝臟組織病理切片中之纖維化組織，發現接受細胞移植之組別其肝臟組織中纖維化傷疤含量明顯減少，而羥脯胺酸含量檢測結果也支持前述試驗之發現，但在各項數值上，並未因細胞團塊大小不同而有顯著差異，其結果與前人使用骨髓間葉幹細胞團塊所得之結果不甚相同。 綜上所述，本試驗結果證明豬脂肪幹細胞確實有治療小鼠肝纖維化之潛能，並提供未來再生醫學研究發展之新方向，然而AD-MSCs之治療機制與細胞後續之命運尚待釐清，若能有更進一步之研究成果，相信在不久之將來，此一研究能造福許許多多為肝臟疾病所苦之患者

Chronic liver disease, which has reached the top 10 death causes for the past decades in Taiwan, might be caused by viral hepatitis, alcohol or drug abuse and diet preference. Liver fibrosis is the ineludible process of chronic liver disease deterioration. However, previous reports mentioned that liver fibrosis might be attenuated by stem cell therapy. Bone marrow-derived mesenchymal stem cells (BM-MSCs) were used in the previous study, which their therapeutic potential in a chemical induced liver fibrosis animal model had been demonstrated. The adipose-derived mesenchymal stem cell (AD-MSC) is another type of somatic stem cells which shares several characteristics with BM-MSC. Due to its smaller invasion in collection process compare with acquiring cells from the bone marrow, the AD-MSCs became attractive in regenerative medicine. In this study, we isolated AD-MSCs with multipotency from adipose tissue and performed mesenchymal stem cells surface antigen CD29, CD44 and CD90 analysis by flowcytometry. Besides, AD-MSCs could differentiate into osteocytes, chondrocytes, adipocytes and hepatocytes in vitro, suggesting their advantages to be applied to clinical plastic and reconstructed surgery. To assess the potential of AD-MSCs to ameliorate liver fibrosis in vivo, AD-MSCs were isolated from dorsal adipose tissue of transgenic pigs which globally express fluorescent protein DsRed, for further examination. To generate liver fibrosis models, male ICR mice were treated with CCl4 via oral gavage for four weeks, and the levels of glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and albumin (ALB) in their serum were higher than the control group (P<0.05) following chemical induction. To provide the transplanted AD-MSCs a massive affinity that could easily located in the liver after portal vein injection, the AD-MSCs were cultured into advanced three dimensional spheres and were separated into three groups according to the diameter of the cell spheres. AD-MSCs spheres with diameter below 40μm, 40~70μm, over 70μm and single cells were injected via portal vein (1x106 cells/mouse) of transplanted mice with liver fibrosis, respectively. Two weeks after AD-MSCs transplantation, the serum GOT, GPT and ALB of recipient mice in AD-MSCs-injected groups were significantly decreased when compared with mice in the saline-injected group (P<0.05). Additionally, the fibrotic tissues were evaluated by Masson's trichrome staining at 4 weeks after cell transplantation. The shrinkage of fibrotic area was observed in AD-MSCs-injected groups. The tissue repairing effects were also confirmed by hydroxyproline content analysis. However, there was no significant difference between single cells and cell spheres transplantation, the exhibited result indicate the divergence with previous study that BM-MSCs displayed better outcomes in sphered structure. Overall, our results provided the intriguing implication that porcine AD-MSCs can ameliorate liver fibrosis in mice, aiming to provide insight into future development of regenerative medicine. However, the therapeutic mechanisms and cell fates in transplanted AD-MSCs still need to be explored.