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目的:研究原代小型猪肝细胞的分离、培养及肝细胞的形态学变化及其功能特性。 方法:用体外灌流装置,EDTA和胶原酶两步灌流法消化分离小型猪肝细胞。在含100ml/L小牛血清及其附助因子的RPMI 1640培养基中培养。对不同培养时间的肝细胞进行形态学观察。同时测定不同培养时期肝细胞生化合成及生物转化功能。 结果:(1)肝细胞产量为6.0×10~9～8.2×10~9(7.0×10~9±0.79×10~9)/每只猪肝;(2)肝细胞活率为90.0～99％(94.4±3.07％);(3)肝细胞具有正常肝细胞的超微结构特征;(4)接种培养后肝细胞增生生长旺盛;(5)接种后20h内每10~7个肝细胞尿素及白蛋白的合成量分别为1.21～1.50mmol/L及0.58～0.80g/L;(6)肝细胞数在1.25×10~7个～1.60×10~7个时,利多卡因的转化率在24h时均在80％以上,随着时间的延长及肝细胞数的增多,利多卡因的转化率逐渐增加。 结论:本方法分离获取的肝细胞产率高、活性高、增生生长旺盛、具有良好的生物合成及生物转化功能,可作为生物人工肝较为理想的细胞来源。
Objective: To study the isolation and culture of primary miniature pig hepatocytes and the morphological changes of liver cells and their functional properties. Methods: Miniature pig hepatocytes were digested and separated by two-step perfusion with EDTA and collagenase in vitro. In RPMI 1640 medium containing 100 ml / L bovine serum and its accessory factors. The morphology of hepatocytes in different culture time was observed. Simultaneous determination of hepatocyte biochemical synthesis and biotransformation in different culture periods. Results: (1) The yield of hepatocytes was 6.0 × 10 ~ 9 ~ 8.2 × 10 ~ 9 (7.0 × 10 ~ 9 ± 0.79 × 10 ~ 9) / each pig liver; (2) The survival rate of hepatocytes was 90.0 ~ 99 (94.4 ± 3.07%); (3) The ultrastructure of hepatocytes in normal liver cells was normal; (4) The proliferation of hepatocytes was vigorous after inoculation; (5) And albumin were 1.21-1.50mmol / L and 0.58-0.80g / L, respectively. (6) When the number of hepatocytes was 1.25 × 10 ~ 7 ~ 1.60 × 10 ~ 7, the conversion rate of lidocaine In 24h are more than 80%, with the extension of time and an increase in the number of liver cells, lidocaine conversion rate gradually increased. Conclusion: The isolated hepatocytes obtained by this method have high yield, high activity, strong proliferative growth, good biosynthesis and biotransformation function, and can be used as an ideal cell source for bioartificial liver.