乳兔和成年兔肝细胞体外分离及培养的比较研究

Comparison of isolation and culture in vitro of heparatocytes from suckling rabbits and adult rabbits

目的研究比较体外分离培养乳兔和成年兔肝细胞增殖活性及功能。方法采用非灌注胶原酶消化法分离乳兔和成年兔肝细胞,比较细胞产率和细胞活率,均采用DMEM培养液(含10%胎牛血清)培养,计数法观察原代细胞增殖变化,MTT法观察传代细胞增殖情况,检测培养液中白蛋白含量变化。培养过程采用相差显微镜进行形态学观察,培养细胞采用PAS染色法鉴定。结果分离收获乳兔肝细胞较成年兔肝细胞数目多,活率高。培养过程中,乳兔肝细胞较成年兔肝细胞折光性好,生长快,增殖能力强,培养液上清蛋白含量高并且增长速度较快,具有统计学意义。PAS染色观察,乳兔和成年兔肝细胞胞质中充满大量糖原颗粒,两者差异无显著性。结论乳兔肝细胞比成年兔肝细胞,更加适合体外培养研究。

[Objective] To compare the isolation and culture of liver cells from suckling rabbits and adult rabbits in vitro. [Methods] The suckling rabbit and adult rabbit hepatocyte were isolated by type I collagenase digestion , The viability of cells was measured by typan blue staining. The liver cells we,re incubated in DMEM medium conraining 10% fetal calf serum. The growth of primary liver cells was measured by cell counting and MTT assay was used to assess the cell growth in the passage. The synthetic function of albumin from suckling rabbit and adult rabbit hepatocyte were determined and compared. The cell morphology was observed by the phase contrast microscope. The cultured liver cells were identified by PAS （periodic acid-Schiff） staining. [Results] The yield and viability of the hepatocytes isolated from suckling rabbits were higher than those from adult rabbits. The cultured cells from suckling rabbits was of the better proliferation ability and albumin synthetic function than cells from adult rabbits. Numerous glycogen was detected in the cytoplasm of the cultured cells from beth suckling and adult rabbits by PAS staining. [Conelusions] The hepatocytes from suckling rabbits are more suitable for cell culture in vitro than hepatocytes from adult rabbits.