卵圆细胞体外扩增过程中分子标志物的变化及意义

In.vitro amplification of oval cells with preservation of stem cell phenotype

目的 探讨体外大量扩增卵圆细胞并保持其干细胞生物学特征的细胞培养方法.方法用RPMI 1640加上15%的胎牛血清和终浓度为20μg/L表皮生长因子（EGF）培养卵圆细胞株OC3,每隔5代收细胞并用逆转录聚合酶链反应、免疫细胞化学、酶细胞化学等方法检测OV-6、c-kit、γ-谷氨酰转肽酶、胎盘型谷胱甘肽硫转移酶、M2型丙酮酸激酶、L型丙酮酸激酶和白蛋白等分子标志物.结果卵圆细胞株OC3在体外大量扩增,不同程度表达OV-6、c-kit、γ-谷氨酰转肽酶、胎盘型谷胱甘肽硫转移酶、M2型丙酮酸激酶等幼稚肝细胞的生物标记,而不表达L型丙酮酸激酶和白蛋白这类成熟肝细胞的分子标记.结论用RPMI 1640加上15%的胎牛血清和终浓度为20μg/L的鼠源性EGF,在体外能大量扩增卵圆细胞株OC3,至第79代仍有很高的增殖活性,同时仍保留肝内干细胞的生物学特征.

Objective To explore cell culture techniques for amplification of oval cells with preservation simultaneously of the stem cell characteristics. Methods Oval cell line OC3 was cultured in RPMI 1640 supplemented with 15% fetal bovine serum and 20 μg/L EGF. Cells were harvested every 5passages and were examined with biomarkers including OV-6, c-kit, gamma-glutamyl transpeptidase,placental form of glutathione-S-transferase （ GST-P）, pyruvate kinase M2, pyruvate kinase L and albumin using techniques including RT-PCR, immunocytochemistry, and enzymo-cytochemistry. Results OC3 cell lines could be amplified abundantly in-vitro associating with expression of infant liver cell markers at various level, including OV-6, c-kit, gamma-glutamyl transpeptidase, GST-P, pyruvate kinase M2, but no expression of mature hepatocyte markers detected including pyruvate kinase L and albumin. Conclusions Amplification of OC3 cells with preservation of the stem cell phenotype and high proliferation index can be achieved up to the 79th passages by culturing in RPMI 1640 supplemented with 15% fetal bovine serum and 20 μg/L EGF.