背景:诱导多功能干细胞具有与胚胎干细胞相似的自我更新、增殖及分化能力, 无来源限制, 又不存在伦理问题, 有望成为治疗肝病的细胞来源.目的:观察诱导多功能干细胞来源肝样细胞体内移植对肝纤维化的改善作用.方法:采用三步法将诱导多功能干细胞诱导分化为肝样细胞, 采用糖原染色、LDL摄取实验、免疫组织化学法检测诱导分化细胞合成糖原能力、摄取LDL能力和甲胎蛋白、白蛋白、CK18蛋白表达.取成年雄性SD大鼠45只 (由中南大学湘雅医学院附属海口医院医学实验动物中心提供), 随机分为3组, 分别为正常对照组、模型组、细胞移植组, 每组15只.模型组、细胞移植组以腹腔注射四氯化碳方法建立肝纤维化模型, 造模后第3天, 细胞移植组尾静脉注射0.5 mL诱导分化21 d的肝样细胞, 细胞浓度为2×109 L-1.细胞移植后4周, 取静脉血与肝组织标本, 分析肝功能及肝纤维化指标变化与肝病理改变.结果与结论: (1) 诱导分化21 d后, 人诱导多能干细胞克隆团已经松散, 以类圆形或多角形为主, 呈铺路石样分布并密集排列, 糖原染色可见细胞胞浆内有大量聚集的粉红色糖原, 具备摄取LDL的能力, 免疫组织化学法观察甲胎蛋白、白蛋白与CK18呈阳性表达; (2) 细胞移植后4周, 与正常组比较, 模型组白蛋白水平显著降低 (P <0.05), 直接胆红素、间接胆红素、谷草转氨酶、谷丙转氨酶及Ⅳ型胶原、血清透明质酸酶、血清Ⅲ型前胶原水平均显著升高 (P <0.05) .与模型组比较, 细胞移植组白蛋白水平显著升高 (P <0.05), 直接胆红素、间接胆红素、谷草转氨酶、谷丙转氨酶及Ⅳ型胶原、血清透明质酸酶、血清Ⅲ型前胶原水平均显著降低 (P <0.05); (3) 细胞移植后4周, 细胞移植组炎性细胞浸润、肝细胞变性与坏死程度较模型组均有不同程度的改善; (4) 结果表明, 诱导多功能干细胞来源的肝样细胞对大鼠肝纤维化具有明显改善作用.%BACKGROUND: Induced pluripotent stem cells have similar self-renewal, proliferation and differentiation abilities to embryonic stem cells. They have no source limitations, no ethical problems, and no current problems of cell xenogenesis, which are expected to be the source of cells for the treatment of liver diseases. OBJECTIVE: To observe the effect of induced pluripotent stem cell-derived hepatocyte-like cells on liver fibrosis. METHODS: The three-step method in vitro was used to induce the differentiation of induced pluripotent stem cells into hepatocyte-like cells. Glycogen staining, immunohistochemistry and low-density lipoprotein uptake assay were used to detect the ability of induced cells to synthesize glycogen, alpha-fetoprotein, albumin, CK18 protein and low-density lipoprotein uptake. Forty-five Sprague-Dawley rats (provided by the Experimental Animal Center, the Affiliated Haikou Hospital, Xiangya School of Medicine, Central South University) were randomized into three groups: normal control group, model group and cell transplantation group (n=15 per group). The rats in the latter two groups were intraperitoneally injected with carbon tetrachloride to establish liver fibrosis models. Cell transplantation group was given intravenous injectin of hepatocyte-like cells (induced for 21 days), 0.5 mL, 2×109/L, at 3 days after modeling. Four weeks after cell transplantation, venous blood and liver tissue samples were taken to analyze the changes of liver function, liver fibrosis index and liver pathology. RESULTS AND CONCLUSION: (1) After 21 days of induction, human induced pluripotent stem cell clonal clusters became loose, mainly round or polygonal in shape, and presented with a dense paving stone-like arrangement. A large amount of pink glycogens could be seen in the cytoplasm, indicating that induced pluripotent stem cells have the ability to synthesize glycogen. Low-density lipoprotein uptake test showed that induced pluripotent stem cells had the ability to uptake low-density lipoprotein. Immunohistochemistry detection showed that the cells were positive for alpha fetoprotein, albumin and CK18. (2) At 4 weeks after cell transplantation, the level of albumin in the model group was significantly lower than that in the normal control group (P < 0.05), while the levels of direct bilirubin, indirect bilirubin, aspartate aminotransferase, alanine aminotransferase, type IV collagen, serum hyaluronidase, serum type III procollagen in the model group were significantly higher than those in the normal control group (P < 0.05). Compared with the model group, the level of albumin in the cell transplantation group was significantly increased (P < 0.05), while the levels of direct bilirubin, indirect bilirubin, aspartate aminotransferase, alanine aminotransferase, type IV collagen, serum hyaluronidase, serum type III procollagen in the cell transplantation group were significantly decreased (P < 0.05). (3) Four weeks after cell transplantation, inflammatory cell infiltration, hepatocyte degeneration and necrosis in the cell transplantation group were improved to different extents. Therefore, hepatocyte-like cells derived from induced pluripotent stem cells could significantly improve liver fibrosis in rats.
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