CAJ | 학술논문

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,既可在体外用丁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,体质量约150～200 g的健康雄性Wistar大鼠,南方医科大学实验动物中心提供.方法;将雄性Wistar大鼠行乙硫氨酸灌喂和2/3肝切除.采用两步法灌注和Percoll密度梯度离心法分离纯化肝卵圆细胞,行体外培养并添加肝细胞生长因子、肿瘤抑制因子M和成纤维细胞生长因子4诱导其分化.主要观察指标:肝卵圆细胞和诱导分化细胞的鉴定.结果:①体外分离每只模型大鼠肝脏中可获得约1.34×108L-1肝卵圆细胞,细胞为圆形、椭圆形或多角形,约为正常肝细胞1/6～1/3,核质比例大,2周后可呈克隆样增殖生长.②激光扫描共聚焦显微镜显示,肝卵圆细胞胞浆和胞膜表达干细胞标志Thy-1及C-kit.③免疫细胞化学检测其原始细胞标志甲胎蛋白呈阳性表达.肝卵圆细胞能稳定传代,肝卵圆细胞在肝细胞生长因子、肿瘤抑制因子M和成纤维细胞生长因子4刺激下细胞形态逐渐发生改变,细胞伸展且体积渐增大,贴壁能力减弱,诱导14d后分化细胞Alb明显阳性表达,且随着诱导时间的延长,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. 揹景:肝卵圓細胞具有彊大的自我更新複製及在一定的條件下剋隆增殖併分化為成熟肝細胞的能力,既可在體外用丁構建生物人工肝的生物材料部分,也可以進行體內移植,還可為組織工程提供種子細胞,在治療肝病方麵具有廣闊的前景.目的:建立成年Wistar大鼠肝卵圓細胞增殖模型,行體外分離和培養肝卵圓細胞,探索體外誘導其分化為肝細胞的可行性.設計:觀察性實驗.單位:南方醫科大學南方醫院消化病研究所.材料:實驗于2003-12/2006-02在南方醫院消化病研究所實驗室完成.36隻三四箇月齡,體質量約150～200 g的健康雄性Wistar大鼠,南方醫科大學實驗動物中心提供.方法;將雄性Wistar大鼠行乙硫氨痠灌餵和2/3肝切除.採用兩步法灌註和Percoll密度梯度離心法分離純化肝卵圓細胞,行體外培養併添加肝細胞生長因子、腫瘤抑製因子M和成纖維細胞生長因子4誘導其分化.主要觀察指標:肝卵圓細胞和誘導分化細胞的鑒定.結果:①體外分離每隻模型大鼠肝髒中可穫得約1.34×108L-1肝卵圓細胞,細胞為圓形、橢圓形或多角形,約為正常肝細胞1/6～1/3,覈質比例大,2週後可呈剋隆樣增殖生長.②激光掃描共聚焦顯微鏡顯示,肝卵圓細胞胞漿和胞膜錶達榦細胞標誌Thy-1及C-kit.③免疫細胞化學檢測其原始細胞標誌甲胎蛋白呈暘性錶達.肝卵圓細胞能穩定傳代,肝卵圓細胞在肝細胞生長因子、腫瘤抑製因子M和成纖維細胞生長因子4刺激下細胞形態逐漸髮生改變,細胞伸展且體積漸增大,貼壁能力減弱,誘導14d後分化細胞Alb明顯暘性錶達,且隨著誘導時間的延長,其暘性率逐漸升高.④細胞化學方法顯示誘導分化細胞胞漿G-6-P染色呈棕黑色沉澱,PAS染色呈紅色顆粒.結論:乙硫氨痠灌餵聯閤2/3肝切除可複製成年Wistar大鼠的肝卵圓細胞增殖模型.膠原酶灌註結閤Percoll密度梯度離心分離純化可穫得滿意的肝卵圓細胞.大鼠肝卵圓細胞能在體外傳代培養,在一定條件刺激下可誘導分化為肝細胞.
배경:간란원세포구유강대적자아경신복제급재일정적조건하극륭증식병분화위성숙간세포적능력,기가재체외용정구건생물인공간적생물재료부분,야가이진행체내이식,환가위조직공정제공충자세포,재치료간병방면구유엄활적전경.목적:건립성년Wistar대서간란원세포증식모형,행체외분리화배양간란원세포,탐색체외유도기분화위간세포적가행성.설계:관찰성실험.단위:남방의과대학남방의원소화병연구소.재료:실험우2003-12/2006-02재남방의원소화병연구소실험실완성.36지삼사개월령,체질량약150～200 g적건강웅성Wistar대서,남방의과대학실험동물중심제공.방법;장웅성Wistar대서행을류안산관위화2/3간절제.채용량보법관주화Percoll밀도제도리심법분리순화간란원세포,행체외배양병첨가간세포생장인자、종류억제인자M화성섬유세포생장인자4유도기분화.주요관찰지표:간란원세포화유도분화세포적감정.결과:①체외분리매지모형대서간장중가획득약1.34×108L-1간란원세포,세포위원형、타원형혹다각형,약위정상간세포1/6～1/3,핵질비례대,2주후가정극륭양증식생장.②격광소묘공취초현미경현시,간란원세포포장화포막표체간세포표지Thy-1급C-kit.③면역세포화학검측기원시세포표지갑태단백정양성표체.간란원세포능은정전대,간란원세포재간세포생장인자、종류억제인자M화성섬유세포생장인자4자격하세포형태축점발생개변,세포신전차체적점증대,첩벽능력감약,유도14d후분화세포Alb명현양성표체,차수착유도시간적연장,기양성솔축점승고.④세포화학방법현시유도분화세포포장G-6-P염색정종흑색침정,PAS염색정홍색과립.결론:을류안산관위연합2/3간절제가복제성년Wistar대서적간란원세포증식모형.효원매관주결합Percoll밀도제도리심분리순화가획득만의적간란원세포.대서간란원세포능재체외전대배양,재일정조건자격하가유도분화위간세포.
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/n vitro isolation and culture of HOCs, and to study the possibility of induction and differentiation of HOCs into hepatucytes. 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 1.34×108 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 confocal microscopy indicated positive expression of stem cells markers Thy-1 and C-kit in cytoplasm and membrane of HOCs. Immunocytochemistry 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.