大鼠肝卵圆细胞体外分离培养和向肝细胞的诱导分化(英文)

Isolation,culture and differentiation of rat hepatic oval cells into hepatocytes in vitro

背景:肝卵圆细胞具有强大的自我更新复制及在一定的条件下克隆增殖并分化为成熟肝细胞的能力,既可在体外用于构建生物人工肝的生物材料部分,也可以进行体内移植,还可为组织工程提供种子细胞,在治疗肝病方面具有广阔的前景。目的:建立成年Wistar大鼠肝卵圆细胞增殖模型,行体外分离和培养肝卵圆细胞,探索体外诱导其分化为肝细胞的可行性。设计:观察性实验。单位:南方医科大学南方医院消化病研究所。材料:实验于2003-12/2006-02在南方医院消化病研究所实验室完成。36只三四个月龄,其阳性率逐渐升高。④细胞化学方法显示诱导分化细胞胞浆G-6-P染色呈棕黑色沉淀,PAS染色呈红色颗粒。结论:乙硫氨酸灌喂联合2/3肝切除可复制成年Wistar大鼠的肝卵圆细胞增殖模型。胶原酶灌注结合Percoll密度梯度离心分离纯化可获得满意的肝卵圆细胞。大鼠肝卵圆细胞能在体外传代培养,在一定条件刺激下可诱导分化为肝细胞。

BACKGROUND： Hepatic oval cells （HOCs） possess the potential of self-renewal, replication, and clone, proliferation and differentiation into mature hepatocytes under a certain condition. HOCs can be used as biomaterial for constructing biological artificial liver in vitro, employed for in vivo transplantation, as well as for tissue engineering as seed cells. HOCs can be widely used for improving clinical treatment of liver diseases. OBJECTIVE： To establish adult Wistar rat models of HOC proliferation, to perform in vitro isolation and culture of HOCs, and to study the possibility of induction and differentiation of HOCs into hepatocytes. DESIGN： Observational study. SETTING： Institute of Gastroenterology, Nanfang Hospital, Southern Medical University. MATERIALS： Experiments were performed at the Laboratory of Institute of Gastroenterology of Nanfang Hospital from December 2003 to February 2006. Thirty-six healthy male Wistar rats aged 3-4 months （150-200 g） were provided by Experimental Animal Center of Southern Medical University. METHODS： Male Wistar rats were orally fed with ethionine received two-thirds partial hepatectomy （2/3 PH）. HOCs were harvested and purified by two-steps perfusion and Percoll density gradient centrifugation, and then cultured in vitro and induced with hepatocyte growth factor （HGF）, oncostatin M （OSM） and fibroblast growth factor-4 （FGF4）. MAIN OUTCOME MEASURES： Identification and differentiation of HOCs. RESULTS： The concentration of HOCs was about 1.34×10^8 L^- 1 in each rat model after in vitro isolation. These cells were round, oval or polygon, about 1/6-1/3 the size of normal hepatocytes. The nucleus-cytoplasm ratio was relatively large. After 2 weeks, clone-like proliferation of HOCs could be observed. Laser scanning con focal microscopy indicated positive expression of stem cells markers Thy-1 and C-kit in cytoplasm and membrane of HOCs. lmmunocytochemistry demonstrated positive stem cells marker alpha fetoprotein （AFP） in cytoplasm of HOCs. HOCs can stably passage and its shape gradually changed after inducing with HGF, OSM and FGF4. HOC volume became larger and HOCs lost their ability of sticking to the wall of culture flask. Apparent positive stain of cytoplasm albumin （Alb） was detected 14 days after induction, and the positive ratio increased along with the extension of inducing duration. Results of cytochemistry indicated a brown or black deposit after glucose-6-phosphotase （G-6-P） staining and red particles after periodic acid-Schiff （PAS） staining. CONCLUSION： Adult Wistar rat models of HOC proliferation are replicated by ethionine feeding combined with 2/3 PH. HOCs can be obtained through collagenase perfusion and Percoll density gradient centrifugation. Rat HOCs can be passaged and cultured in vitro. Under a certain condition, HOCs can be induced and differentiated into hepatocytes.