大鼠肝星形细胞、枯否细胞的同步分离培养与鉴定

Seperation and culture of hepatic stellate cells and Kupffer cells simultaneously

目的:建立经济、简便的同步分离培养肝星形细胞及枯否细胞的方法。方法:应用胶原酶和蛋白酶对大鼠肝脏进行原位灌流、消化,获得大鼠肝脏非实质细胞,经18%(W/V)的Nycodenz密度梯度离心,一步分离肝星形细胞及枯否细胞;肝星形细胞处在界面,枯否细胞则处在界面下;台盼蓝染色鉴定细胞活力;Desmin免疫细胞化学染色鉴定肝星形细胞;溶菌酶免疫细胞化学染色鉴定枯否细胞。结果:该方法分离的肝星形细胞和枯否细胞的纯度达95%左右。肝星形细胞得率(3.0±0.5)×107/肝,枯否细胞得率(4.0±0.5)×106/肝。结论:应用Nycodenz一步密度梯度离心法可以很好的同时分离肝星形细胞和枯否细胞。

Objective： To establish a method for separation and culture of hepatic stellate cells （HSCs） and Kupffer cells （KCs） simultaneously. Method： Non-parenchymal cells were fractionated by sequential collagenase and pronase digestion of liver. HSCs and KCs were separated by 18%（W/V）Nycodenz. HSCs presented themselves at the boundary, while KCs presented themselves below the boundary.Viability of HSCs and KCs was measured by the Trypan blue exclusion test. HSCs were established by the visualization of desmin with anti-desmin antibody by immunocytochemistry staining. KCs were established by the visualization of lysozyme by immunocytochemistry staining. Results：Purity of these cultures was about 95%. This method for cell isolation resulted in a yield of （3.0±0.5）×10^7 HSCs and （4.0±0.5）×10^6 KCs per liver. Conclusion： HSCs and KCs can be separated by one-step density gradient centrifugation with nycodenz.