| 张佳玲 张峰 边康麒 王晖晖 诸葛宇征.不同二步酶灌注法分离大鼠肝星状细胞的比较研究[J].,2014,14(25):4806-4810 |
| 不同二步酶灌注法分离大鼠肝星状细胞的比较研究 |
| Comparative Study on Isolation of Rat Primary Hepatic Stellate Cells byDifferent Two-step Enzyme Perfusion |
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| DOI： |
| 中文关键词: 肝星状细胞 链酶蛋白酶 原代培养 |
| 英文关键词: Hepatic stellate cells Pronase Primary culture |
| 基金项目:江苏省自然科学基金项目（BK2011094） |
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| 中文摘要: |
| 目的：在现有二步酶灌注法分离大鼠肝星状细胞（hepatic stellate cells，HSC）的基础上，探索更加高效的分离HSC 方法。方
法：分别采用链酶蛋白酶+胶原酶循环灌注、链酶蛋白酶非循环灌注+ 胶原酶循环灌注以及胶原酶单独循环灌注法分离大鼠
HSC，比较三种方法的细胞获得率、活性和纯度差异。应用0.4 %台盼蓝染色判断活性，结蛋白(desmin)、波形蛋白（vimentin）细胞
免疫荧光方法鉴定纯度。结果：链酶蛋白酶非循环灌注+胶原酶循环灌注法细胞获得率高于另两种方法，细胞活力高于链酶蛋白
酶循环灌注+胶原酶循环灌注法，三组得到的细胞纯度均高于90 %且无显著差异。结论：在三种二步酶灌注方法中，链酶蛋白酶
非循环灌注+胶原酶循环灌注法能显著提高HSC 获得率，且对细胞活力影响小，不降低细胞纯度，是一种高效的分离方法，有利
于HSC 相关肝脏疾病的生物学研究。 |
| 英文摘要: |
| Objective:To explore a more efficient method for HSC isolation based on the method of the two-step enzymatic
infusion.Methods:Three methods including circulatory pronase+circulatory collagenase perfusion, non-circulatory pronase + circulatory
collagenase perfusion and single collagenase perfusion were performed, and cell yield rate, viability and purity were compared. Cells
viability was examined by 0.4% trypan blue. Desmin and vimentin immunofluorescence was used to identify cell purity.Results:The
yield of HSC by non-circulatory pronase + circulatory collagenase perfusion was higher than that of the other two methods, and cell
viablity was higher than that by circulatory pronase + circulatory collagenase perfusion. Cell purity of all groups was over 90%, and there
was no significant difference.Conclusion:Among the three two-step enzymatic perfusion methods, non-circulatory pronase + circulatory
collagenase perfusion can significantly improve the obtaining rate of HSC, and have little effect on cell viability without reducing cell
purity, which is a highly efficient isolation method and benefits the biological research for HSC related liver diseases. |
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