應用小鼠及豬骨髓間葉幹細胞治療肝纖維化小鼠之研究

Abstract

根據行政院衛生署之報告顯示，因慢性肝炎與肝硬化而死亡之人口，長年於台灣十大死因中占據一席之地，而全球感染慢性肝炎者且高達3億人口之譜，由此可見，肝病確實對人類健康產生一定程度之威脅，因此治療肝臟疾病與幫助恢復肝臟功能，已成為目前研究之ㄧ重要課題。 肝纖維化 (liver fibrosis) 為各種慢性肝病進展為肝硬化之必經途徑；其成因為肝臟遭受慢性損傷後反覆癒合等反應導致細胞外間質 (extracellular matrix-ECM) 過量堆積形成纖維狀傷疤，逐步惡化將破壞肝組織之結構並影響其功能，最後演變為肝硬化與肝功能衰竭（liver failure）；因此探討如何阻止或延緩肝纖維化的發生，將對肝病之治療與後續之研究發展有重要之意義；然而肝臟移植為目前嚴重肝臟疾病患者常採用之治療方式，但其受限於捐贈肝之來源短缺、移植後可能具有免疫排斥反應及多發性併發症等問題，在臨床治療上有其限制；因此如何於惡化至肝硬化之不可逆階段前，予以治療並針對肝臟疾病發展新治療方式，為現下十分重要之課題。 近來已有研究報告指出，移植骨髓間葉幹細胞 (mesenchymal stem cells, MSCs) 於肝臟受損之動物模式，可有效減緩肝纖維化之病程，然而追蹤所施打之細胞遷移入肝臟之比例仍十分低，因此如若要提升治療之效果或許可藉由提升 MSCs 遷移入肝臟之比例著手。 為證實 MSCs 具有分化為肝細胞之潛能，本試驗首先會將分離純化後之小鼠與豬之骨髓間葉幹細胞於體外進行誘導分化，經由螢光免疫染色及即時定量 PCR (Q-RT-PCR) 證實分化後之小鼠與豬骨髓間葉幹細胞皆具有分化為肝細胞之特性；此外為追蹤施打入肝受損小鼠體內之間葉幹細胞，本試驗使用之骨髓間葉幹細胞皆分離自表現β-actin啟動子與綠色螢光蛋白質 (enhanced green fluorescent protein, EGFP) 之轉基因螢光小鼠與豬之骨髓。 由於本試驗假設所施打之骨髓間葉幹細胞可能因為其細胞團塊之大小不同，因而會卡於肝臟血管內進而提升骨髓幹細胞遷移入肝臟之比例，進而提升治療肝纖維化之效果；因此，首先將小鼠與豬骨髓間葉幹細胞經由純化及分離後培養於不貼附培養皿 (Ultra-low plate) 使其成為不同大小之球狀細胞團，隨後以過濾篩區分不同大小之細胞團包括: < 40 μm、 40~70 μm及 >70 μm，以脾內及肝門靜脈注射之方法分別將不同大小之細胞團塊注入肝纖維化小鼠體內，八週後綠色螢光小鼠及豬骨髓間葉幹細胞之遷移及參與改善肝纖維化進程則由血液生化值、肝臟切片之免疫組織染色及肝臟之羥脯胺酸 (hydroxyproline) 萃取等分析。 試驗結果顯示，無論是同種異體或異種之骨髓間葉幹細胞移植皆可顯著降低肝內羥脯胺酸之含量 (p < 0.05)，特別在小鼠骨髓間葉幹細胞團塊40~70 μm 移植組其不僅於細胞遷移比例之提升且於促使肝纖維化之改善皆顯著優於移植懸浮細胞而非細胞團塊組 (p < 0.05) ，而前人所測試之細胞皆為懸浮細胞而非細胞團塊。 綜合上述，骨髓間葉幹細胞具有分化為肝細胞之潛能，且同種異體或異種之骨髓間葉幹細胞不論以脾內注射或肝門靜脈注射移植入肝纖維化小鼠體內，皆發現會遷移至肝臟中且顯著改善肝纖維化現象及降低肝內羥脯胺酸含量，其中尤以小鼠骨髓間葉幹細胞團塊40 ~70 μm 移植組其細胞遷移比例及降低肝內羥脯胺酸含量皆顯著優於移植懸浮細胞而非細胞團塊組，鑑此，以骨髓間葉幹細胞為來源之細胞治療可採用細胞團塊移植法，其可有效改善實驗小鼠之肝纖維化，而或許此療法可提供一新觀點於臨床治療之研究。

According to the report of the Department of Health, Executive Yuan, R.O.C. (Taiwan), chronic liver disease and cirrhosis ranked the top ten in the major causes of death in Taiwan. In chronic liver diseases, liver fibrosis is one of the most common symptoms which is the excessive accumulation of extracellular matrix in the liver, and can subsequently lead to cirrhosis. One of the primary treatments for these various end-stage hepatic diseases is liver transplantation. However, this is limited by the lack of donor organs and the immuno-rejection from the recipient patients. Thus, it is necessary to devise better therapeutic methods for this disease. Recently, transplantation of bone marrow-derived mesenchymal stem cells (MSCs) has been shown to ameliorate liver fibrosis in animal models with only few cells migrated into damaged area. It is reasonable to suspect that liver fibrosis can be better mitigated by improving the migration rate of MSCs to the injured liver. In this study, MSCs were induced to differentiate into functional hepatocyte-like cells under defined conditions, which suggest the potential for therapeutic applications in healing liver diseases. To assess the potential of MSCs to ameliorate liver fibrosis in vivo, bone marrow-derived MSCs was isolated from the femur of transgenic porcine (pMSCs) and mice (mMSCs) harboring β-actin promoter constructed with EGFP (enhanced green fluorescent protein) cDNA serving as a tracing marker and EGFP-pMSCs or EGFP-mMSCs before/after Ultra-low plate culture were transplanted into the portal vein or spleen of carbon tetrachloride (CCl4)-treated mice. I hypothesized that spheres of MSCs in different sizes would show different potential into the injured liver due to three-dimensional bulkiness. To test this hypothesis, the transplantation was done in different sizes of cell spheres including suspended single cells, cell spheres of < 40μm, 40~70μm, and >70μm in diameter. After 4 weeks of transplantation, both the engraftments of EGFP-pMSCs and EGFP-mMSCs to the recipient livers were detected by immunohistochemistry with antibodies recognizing EGFP. The results showed that all of the cell transplanted mice could significantly ameliorate liver fibrosis compared to the non-transplanted control (p < 0.05). Moreover, the liver hydroxyproline content significantly decreased and the migration rate of MSCs increased significantly in the group with mMSCs spheres of 40~70μm than suspended single cells transplanted into portal vein (p < 0.05). Taken together, no matter allogenically or xenogenically, MSCs transplanted into mice with liver fibrosis were able to migrate into the liver and significantly decrease the level of liver hydroxyproline content. Moreover, the migration rate is significantly higher in the transplantation group with mMSC spheres of 40~70μm than the group with suspended single cells. These findings provide new insights that bone marrow MSCs-based cell therapy could be transplanted in spheres and is potentially useful for the treatment of liver fibrosis and can even contribute to liver regeneration.