摘要
本文建立了原位胶原酶循环灌注分离猪肝细胞方法并与离体两步胶原酶灌注法进行了比较。猪门静脉和下腔静脉分剐插管,先用D-Hanks液灌注,再采用自制的循环灌流装置进行胶原酶循环原位灌注分离猪肝细胞,分离后的肝细胞以5×105/ml培养,观察分离和培养7d的肝细胞产量、活率、蛋白质合成功能、葡萄糖合成功能和LDH含量。同时测定离体组的上述指标。研究结果表明采用原位胶原酶循环灌注法每克肝组织分离获得的肝细胞总量为5.1×107,肝细胞活率98.6％,培养7d肝细胞活率89.5％。肝细胞的蛋白质合成功能在培养7d中保持稳定;葡萄糖合成功能从1d时1.05±0.15nmol/cell下降到3d时0.74±0.09nmol/cell;LDH含量在3d较高。原位胶原酶循环灌注法分离的猪肝细胞总量、活率高于离体法;蛋白质合成功能和葡萄糖合成功能强于离体法。因此,原位胶原酶循环灌注分离猪肝细胞方法可获得大量高活率和良好功能的猪肝细胞。
In this study,we established in situ recirculating collagenase perfusion method for isolation of pig hepatocytes and compared it(Group A)with extracorporeal two-step collagenase perfusion method(Group B). Portal vein and inferior vena cava in pigs were cannulated. Pig hepatocytes were isolated by in situ D-Hanks solution and recirculating collagenase perfusion method using domestic circulating perfusion apparatus, and cultured at 5×105/ml in serum-contained medium. Yield, viability, protein and glucose syntheses,and LDH activity were evaluated during 7d of culture. These indices were all determined in the Group B. The results showed that yield of isolated hepatocytes was 5.1×107/g liver tissue in group A. Cell viability was 98. 6% on day 0 and 89.5 % on day 7. Protein synthesis of hepatocytes was stable over the course of 7 d. Glucose synthesis of hepatocytes declined from 1.05±0.15 nmol/cell on 1d to 0.74±0.09 nmol/cell on day 3. The yield and viability were higher and protein synthesis and glucose synthesis of the hepatocytes in Group A were much effective than in Group B. Therefore, large quantities of hepatocytes with high viability and better function could be acquired by in situ recirculating collagenase perfusion method for isolation of pig hepatocytes.
出处
《细胞生物学杂志》
CSCD
北大核心
2003年第2期124-127,F003,共5页
Chinese Journal of Cell Biology
基金
江苏省卫生厅重点项目基金(BQ200020)
南京市科委科技发展计划(20012095)资助
