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体外大鼠肝卵圆细胞的长期培养方法
引用本文:尹寿新,;周震,;马美雪,;杨景波,;羊东晔. 体外大鼠肝卵圆细胞的长期培养方法[J]. 中国临床康复, 2014, 0(29): 4663-4668
作者姓名:尹寿新,  周震,  马美雪,  杨景波,  羊东晔
作者单位:[1]中南大学湘雅二医院消化内科,湖南省长沙市410011; [2]长沙市第一医院消化内科,湖南省长沙市410005; [3]徐州市中心医院消化内科,江苏省徐州市221009
基金项目:2010年国家自然科学基金(81072037)
摘    要:背景:肝卵圆细胞是目前公认的成体肝干/祖细胞,但其体外长期培养时会不可避免地丢失干细胞的活性。目的:探索大鼠肝卵圆细胞体外长期培养的方法。方法:构建2-乙酰胺基芴/部分肝切除肝再生大鼠模型,通过酶消化和Percoll梯度离心分离纯化大鼠异质卵圆细胞并进行免疫染色鉴定,用含表皮生长因子、白血病抑制因子的培养基体外长期培养,后撤去表皮生长因子、白血病抑制因子,通过形态学的观察和分子标志物的检测判断其能否保持干/祖细胞活性。结果与结论:采用含表皮生长因子、白血病抑制因子的培养基体外培养卵圆细胞4个月后,大鼠异质卵圆细胞仍能表达肝细胞标志物ALB、胆管上皮细胞标志物CK-19,经不含表皮生长因子、白血病抑制因子的培养液继续培养后,卵圆细胞胎肝标志AFP表达量迅速下降。该研究结果表明大鼠肝卵圆细胞在表皮生长因子、白血病抑制因子等培养条件下可长期增殖并保持干细胞活性。

关 键 词:肝细胞  表皮生长因子  白血病抑制因子  细胞,培养的  组织构建  组织工程  卵圆细胞  长期培养  国家自然科学基金

Long-term culture of hepatic oval cells in rats in vitro
Affiliation:Yin Shou-xin, Zhou Zhen, Ma Mei-xue, Yang Jing-bo, Yang Dong-ye (1Department of Gastroenterology & Hepatology, the Second Xiangya Hospital of Central South University, Changsha 410011, Hunan Province, China; ZDepartment of Gastroenterology & Hepatology, the First Changsha Hospital, Changsha 410005, Hunan Province, China; 3Department of Gastroenterology & Hepatology, Xuzhou Central Hospital, Xuzhou 221009, Jiangsu Province, China)
Abstract:BACKGROUND: Hepatic oval cells are recognized as stem/progenitor cells currently, however, long-term culture of hepatic oval cells can inevitably result in the loss of cell activity. OBJECTIVE: To explore a long-term culture method of hepatic oval cells in vitro. METHODS: Partially hepatectomized rat model was established by using 2AAF/PH. The regenerated liver was digested by collagenase, and hepatic oval cells were isolated and purified by density gradient centrifugation and identified by immunocytochemistry. Hepatic oval cells were cultured in a medium containing epidermal growth factor and leukaemia inhibitory factor. Then epidermal growth factor and leukaemia inhibitory factor were removed after several months, and its ability of maintaining stem/progenitor cell activities was determined based on morphology and molecular markers. RESULTS AND CONCLUSION: Both hepatocyte marker ALB and biliary epithelial cell marker CK-19 were found after hepatic oval cells were cultured in a medium containing epidermal growth factor and leukaemia inhibitory factor for 4 months. While in the absence of epidermal growth factor and leukaemia inhibitory factor, the expression of fetal liver marker AFP was decreased quickly. These results indicated that hepatic oVal cells could expand in a medium containing epidermal growth factor and leukaemia inhibitory factor and maintain stem/progenitor cell activities for a long time.
Keywords:hepatocytes  epidermal growth factor  leukemia inhibitory factor  cells, cultured
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