藉由豬來源羊水幹細胞治療肝纖維化小鼠之潛能

The Potential of Treating for Liver Fibrosis Mice by Using Porcine Amniotic Fluid Stem Cells

呂偉誠(Wei-Cheng Lu)

指導教授 ： 吳信志

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.10295

摘要

伴隨著國人飲食與生活型態改變，慢性肝病或肝硬化所造成的死亡率逐年攀升。在慢性肝病發生時，肝臟體內動態平和失調，肝臟受到損傷並自我修復的過程中，因具有功能性細胞死亡，細胞外基質 (extracellular matrix, ECM) 在肝間質組織中大量堆積，是為肝纖維化 (liver fibrosis) 的病徵。肝纖維化進而會逐漸變為肝硬化 (cirrhosis)，此時肝臟將喪失其大部分原有之功能，而必須藉由器官移植方式，始得能恢復肝臟正常生理機能。但在此同時，肝臟捐贈來源極為短缺、醫療費高昂，以及在接受肝臟捐贈後，尚有免疫排斥之問題，且在國內對於親屬之間器官捐贈的法律限制，造成多數罹患肝病患者，常無法適時獲得捐贈器官之移植而致死。對於上述諸多原因，近年來，許多研究乃積極投入肝病治療相關研究，在肝臟損傷的不可逆過程中，力謀尋求得能有效取代原有器官移植的方式，發展出一種新興療法，從而有效地減緩肝病發生的進程。
在胚胎動物懷孕期，母體中的羊水 (amniotic fluid)，包覆在胎兒周遭，主要能讓胚胎免除來自子宮壁壓力與摩擦，胚胎能自由地活動，並且在胚胎生長發育時，提供溫度恆定的生長環境。另外，絕大多數的懷孕婦女，為了掌握胎兒的健康情形，會選擇藉由羊膜穿刺 (amniocentesis) 的方式，抽取少量羊水，並試圖從中分離出源自胎兒器官發育過程，脫落於羊水中之細胞，經於體外進行培養後，再針對彼等細胞進行核型分析，於婦女妊娠初期，檢測胎兒是否罹患遺傳性疾病，並檢視胎兒發育情形。而近期有許多研究指出，自抽出的羊水液中，能分離出一群細胞群，而此細胞群表現了幹細胞所表現的細胞標誌，並且也能在體外分化為許多種類的細胞，是為羊水幹細胞 (amniotic fluid stem cells, AFSCs)。羊水幹細胞乃幹細胞種類中的新起來源，近年來，才逐漸受到學界的關注。羊水幹細胞在增殖與分化能力上，雖不如胚幹細胞佳，但相較與其他體幹細胞，因其取得時間點較早，具有較佳增殖與分化能力的特性，但在活體移植中，並不會產生畸胎瘤 (teratoma)，同時會表現胚胎時期的特定標誌SSEA-4，以及複能性幹細胞的特定標誌Oct4等特性。根據以上特性，羊水幹細胞被視為介於胚幹細胞與體幹細胞之間，表現出一種過渡型態的細胞群。此外，羊水實屬於醫療廢棄物，而在「垃圾變黃金」的轉變，也使羊水幹細胞大幅提升其價值。為因應肝臟疾病對現代人們的威脅，利用羊水幹細胞，治療於肝臟疾病的研究，為刻不容緩須積極探討的課題。
本試驗利用帶有Ds-red螢光轉基因的懷孕母豬，在妊娠期70天時，抽取其羊水後，利用幹細胞體外培養時，具有貼附型 (adhesion) 的特性，進行繼代純化所得之幹細胞，是為豬來源羊水幹細胞 (porcine AFSCs)。並經由流式細胞儀 (flow cytometry) 分析，證實此類細胞表現CD44與CD90等，幹細胞特有標誌，且在體外成功分化為脂肪細胞 (adipocyte)、軟骨細胞 (chondrocyte) 及硬骨細胞 (osteoblast) 等。此外，利用豬來源羊水幹細胞，在體外繼代至6至10代時，利用添加FGF與HGF等生長因子，證實此類細胞具有分化成為類肝臟細胞 (hepatocyte-like cells) 的潛能，而後再將試驗擴及活體動物實驗。經由四氯化碳 (carbon tetrachloride, CCl4) 處理的小鼠，在灌食第四週後，分析其血清中的天門冬胺酸轉胺酶 (GOT/AST)、麩醯胺酸丙酮酸轉胺酶 (GPT/ALT) 及白蛋白 (ALB) 皆有顯著性的上升，且在移植單一細胞的組別中，白蛋白 (ALB) 有顯著性的，而在肝臟外觀與切片結果中，都發現肝纖維化的病癥，而後將小鼠隨機分成注射單一細胞 (single cell)、與細胞團塊 (sphere) 小於40um、40-70um、大於70um等組別，分別將不同組別的豬來源羊水幹細胞，經由腸系膜注射移植入小鼠體內後，進行後續觀察。在細胞移植後第四週，由觀測血液生化值可發現，接受豬來源羊水幹細胞移植四週後，在各個細胞移植組別，與僅接受CCl4處理八週、並未給予細胞移植的PBS組別相比，其血液生化值天門冬胺酸轉胺酶 (GOT/AST) 及麩醯胺酸丙酮酸轉胺酶 (GPT/ALT) 皆有顯著下降的結果 (P < 0.05)。但在各細胞移植組之間，未能發現有任何差異。經梅森氏三色染色結果可發現，經由豬來源羊水幹細胞移殖的各組別，在移植手術與四週後，小鼠犧牲後所製作的肝臟病理切片，透過梅森氏三色染色，可發現肝小葉的結構漸趨完整，並且細胞浸潤的也較為和緩。此結果暗示，豬來源羊水幹細胞在移植入小鼠肝臟後，能減緩受損肝臟的惡化進程。此外，透過細胞移植後24, 48及72小時的切片，都偵測到表現紅色螢光的細胞，此外隨著移植時間的拉長，表現紅色螢光蛋白的細胞，比例隨之遞減。並且經過免疫染色法，發現在細胞移植後的72小時之後，小鼠肝臟中，可發現豬種類的白蛋白表現。以上結果顯示，移植豬來源羊水幹細胞，具有影響受損肝臟的功能。
綜觀上述實驗結果，豬來源羊水幹細胞在體外具有分化成為類肝臟細胞的能力，並且在活體移植的體內試驗中，也發現能幫助減緩小鼠肝纖維化，使肝臟恢復其功能。此外，在追蹤移植細胞的試驗中，直到移植第三天，尚能找尋到移植的豬羊水幹細胞，且開始表現類似肝臟細胞的功能性細胞，然而，其在動物體內，協助受損肝臟恢復的作用機制，還待再進一步的試驗，加以探討與釐清之。

關鍵字

豬來源羊水幹細胞 ； 肝纖維化 ； 異種細胞移植

並列摘要

According to official statistics, there are many people in Taiwan suffering from liver disease due to diet and lifestyle change in recent years. Most liver failure patients show the liver fibrosis symptoms in the beginning stage, because the liver is injured repeatedly and produce the number of hepatic extracellular matrix (ECM) through self-healing process. To treat the liver disease, the most effective way is to implement the liver transplantation. However, the orthotopic transplantation faces many difficulties, involving in the organ donor shortage, financial considerations, and also the requirement for lifelong immunosuppression. For above reasons, there is an urgent need to develop a new therapy for curing liver disease effectively.
Amniotic fluid is surrounding the embryo to protect the developing baby against with abdomen, moreover, is usually used to determine the fetus health by amniocentesis during pregnancy. In recent studies suggest amniotic fluid, where is full of shed fetal tissues and cells, is rich in stem cells (amniotic fluid stem cells, AFSCs). AFSCs perform not only high telomerase activity but also surprising proliferative ability than other stem cells from adult tissues. Most importantly, AFSCs, sharing the similar characteristics with ESC, can differentiate into three germ layer cells in vitro and express pluripotent stem cell marker, Oct4. Besides, in contrast to ESC, AFSCs are not teratogenic after transplantation and get rid of ethical issues. Therefore, it likely implies AFSCs are a new source to deal with the thorny problem of liver disease.
At of all, this study isolated the AFSCs from transgenic Ds-red pig in the tenth week pregnancy. The subculture purified Ds-red porcine AFSCs, determining surface antigen of by flow cytometry analysis, express CD44 and CD99 but not express CD31 and CD4a markers. Ds-red porcine AFSCs could be rise to osteoblasts, adipocytes and chondrocytes in differentiation potential test. In addition, porcine AFSCs could differentiate into hepatocyte-like cells in vitro by giving the appropriate growth factors (e.g. FGF and HGF) when cultured for thirteen days. The differentiated porcine AFSC cells transformed to a cubical shape and express liver-specific protein, albumin (ALB). These evidences indicate the potential to develop the therapy in liver disease by using porcine AFSCs.
The experiment in vivo utilized the male ICR mice treating by CCl4, a liver failure inducing drug, for 4 weeks and then the porcine AFSC cells are injected through portal vein. After treated CCl4 4weeks later, the serum detections perform a significantly high level of glutamic oxaloacetic transaminase (GOT/AST), glutamic pyruvic transaminase (GPT/ALT) and albumin (ALB) (P < 0.01). At the same time, the mice were randomly allocated to each trial group, including the single cells, sphere with diameter smaller than 40um (< 40um), between 40um and 70um (40-70um) and larger than 70um (> 70um), for cells injection surgery. After cells transplanted 4 weeks, liver enzyme level decrease significantly (P < 0.01) and ALB level show a tendency to a normal range (P <0.05) when compared with control group. Unfortunately, there is no difference between each group with cell treatment. The liver section were evaluated by Masson’s trichrome stain show the less liver scarring fibrosis in each cells transplantation group after cells transplanted 4 weeks. These results suggest the AFSCs could significantly improve the liver function of mice with fibrosis liver and prevent deterioration process. Furthermore, in order to figure out the migration of injected cells, the liver slide sections were observed at 24, 48, and 72 hours individually after cells transplanted. The transplanted Ds-red AFSCs distributed in interstitial space instead of located around liver vessels at 24 hours. Especially, by using immunostaining, the external AFSCs were found could form ALB expression cells in 72 hours group. Thus, above results implicate that transplanted cells may to affect the function of fibrosis liver.
In conclusion, the results indicate that transplanted AFSCs may to affect the fibrotic liver function. AFSCs derived from pig exhibit a positive potential in cell therapy of liver fibrosis, and it is considerable way to carry out cells injection via portal vein in patients.

並列關鍵字

Porcine amniotic fluid stem cells ； Liver fibrosis ； Stem cell transplantation

參考文獻

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