探討間葉幹細胞在以N-nitrosodimethylamine誘發成肝炎、肝纖維化及肝癌之大鼠體內的生理、病理角色

Abstract

在台灣、亞洲及南非地區肝細胞癌是最嚴重的癌症。由於肝癌無論是手術治療或非手術治療，其治療效果均與腫瘤期別有密切關係，因此早期診斷、早期治療是非常重要的。然而高達80％的原發性肝細胞癌病人於五年內會再併發復發性肝細胞癌，其再復發的機制至今仍不十分清楚。因此對於肝癌之形成機轉、復發原因與預防或治療方式之探究是至為重要的。 隨著不同時期和程度的肝損傷，過度的活性氧(reactive oxygen species, ROS) 如O2˙-、H2O2 以及NO會造成脂質過氧化(lipid peroxidation)、蛋白質氧化(protein oxidation)、DNA損傷(DNA damage) 與突變產生(mutagenesis)。這些活性氧(reactive oxygen species, ROS)d可能主要來自肝細胞之粒線體、活化的巨噬細胞(activated macrophages, 或稱Kupffer cells)與入侵之中性球(infiltrating neutrophils)。活性氧(reactive oxygen species, ROS)會啟動細胞核因子nuclear factor-kappa B (NF-kB) 與activator protein-1 (AP-1) 移位至細胞核而造成許多發炎細胞激素的釋放引起發炎。過量活性氧(reactive oxygen species, ROS)透過Kupffer cell內的 NADPH oxidase 或是肝細胞之cytochrome P4502E1 (CYP2E1) 會促進肝臟內之stellate cells 活化而造成肝纖維化與肝癌。 肝損傷時肝臟內許多細胞受原位細胞或發炎細胞氧化壓力增加引起Bax/Bcl-2 family啟動細胞凋亡 (apoptosis)、caspase 1/IL-1調節之發炎性細胞死亡(pyroptosis)或Beclin-1/LC-3β調節之細胞自噬 (autophagy)造成肝功能損傷進而形成肝腫瘤。間葉幹細胞(MSC)具有極佳自我更新（self-renewal）及增生（proliferation）能力，MSC同時也具有旁分泌（paracrine）的功能，其培養基(CM)中可能含有高量的growth factors、cytokines及prostaglandins。這些物質會增加細胞增生、存活及血管生成而提升組織修復。 為探究MSC之治療效益，我們利用以N-nitrosodiethylamine (DEN)刺激肝發炎、硬化與腫瘤之大鼠動物模式，評估MSC給予對DEN誘發肝發炎、硬化與腫瘤之病理生理效應。研究發現靜脈給予MSC後，其主要匯集於受傷的肝臟組織。另外，MSC的給予改善DEN引起之肝發炎、硬化與腫瘤等病理特徵，包括降低Masson染色之損傷指標、細胞凋亡、細胞自噬，發炎性細胞死亡表現，並降低肝功能AST與ALT之數值。綜合研究結果，MSC有相當之潛力發展成為對肝損傷患者之細胞治療製劑。Hepatocellular carcinoma (HCC) is the most severe cancer in Taiwan, Asia, and South Africa. The therapeutic strategy to treat HCC, either operative or non-operative methods, is related to the tumor stages. Therefore, an earlier diagnosis and treatment of HCC is important. However, 80% of the patients with primary HCC will be accompanied by relapsing HCC in five years. The mechanism of HCC recurrence is not clearly understood yet. Based on the information, it is necessary to investigate the pathophysiologic mechanisms of HCC formation and recurrence and to discover novel techniques and methods to prevent and cure HCC. Hepatogenesis begins in the setting of chronic liver inflammation and hepatic fibrosis, which are possibly caused by hepatotoxins. Increased oxidative stress, inflammatory cytokines and growth factors subsequently lead to hepatocyte proliferation. Prolonged cell damage by chronic inflammation is critical in cancer development. Overproduction of reactive oxygen species (ROS) including O2˙-, H2O2 and NO, can cause lipid peroxidation, protein oxidation, DNA damage and mutagenesis associated with various stages of liver injury. ROS can trigger translocation of nuclear factor-kappa B (NF-B) and activator protein-1 (AP-1) to nucleus and activation of several inflammatory cytokines and adhesion molecules that contribute to further production of ROS and consecutive cell death. The increased ROS can enhance Bax/Bcl-2 ratio, caspase 3 (CPP32) expression, and poly-(ADP-ribose)-polymerase (PARP) fragments subsequently resulting in apoptotic cell death, enhance the expression of the autophagy-promoting protein Beclin-1 expression, leading to autophagy and caspase 1/IL-1 mediated pyroptosis. Mesenchymal stem cells (MSC) provide excellent self-renewal and proliferation ability. In addition, MSC may release high levels of growth factors, cytokines and prostaglandins in the microenvironment or condition medium (CM) by the paracrine mechanism to enhance cell proliferation, survival and angiogenesis to repair injured tissue. To investigate the therapeutic efficiency of MSC treatment, we used N-nitrosodiethylamine (DEN) as an inducer to develop hepatocellular inflammation, fibrosis and carcinoma on rats, and evaluated the pathophysiological effects of MSC treatment on these rat models. MSC application improves DEN-induced hepatocellular inflammation, fibrosis and carcinoma. The degree of fibrosis by Masson staining was also decreased. Furthermore, the marker of apoptosis, pyroptosis, and autophagy was enhanced by DEN injury and was decreased by MSC treatment. The increased level of AST and ALT activity by DEN injury was also reduced by MSC treatment. In conclusion, MSC has a considerable potential to be a cell therapy for patients with liver injury and HCC.