经心脏逆行置管灌注法在小鼠原代肝细胞分离中的应用

Isolation of Mouse Primary Hepatocytes by Retrograde Liver Perfusion with Catheterization via Heart

目的观察采用经心脏逆行置管灌注肝脏的方法分离小鼠原代肝细胞的效果。方法对传统的分离原代肝细胞方法(Seglen两步胶原酶灌注法)进行改良,即从小鼠右心房的裂口处经肝上下腔静脉向下方置管,并以输液器滴注含体积分数10%胎牛血清的胶原酶灌注液进行肝脏灌注。评价此法与传统方法的置管成功率及所得肝原代细胞的数量及活力。结果改良组总的置管操作成功率高达95%,且在一次置管成功率上改良组高于传统组(94.7%vs.68.8%,P<0.05)。改良组小鼠肝脏灌注均匀,肝细胞产量高达1.07×106/g小鼠体质量,细胞活力平均为92.16%,两者均高于传统组(分别为0.99×106/g小鼠体质量及86.44%,P<0.05或P<0.01)。结论经心脏逆行置管灌注法分离小鼠原代肝细胞简便易学,重复性高,且能保证分离所得的肝细胞产量和质量。

Objective To explore the effects of retrograde liver perfusion with catheterization via heart on the isolation of primary mouse hepatocytes. Methods In order to more efficiently isolate primary mouse hepatocytes, we improved the traditional two-step collagenase perfusion method. The liver perfusion catheter was inserted through right atrium and suprahepatic vena cava, and the perfusion velocity was controlled by the drip infusion with collagenase perfusate containing 10% of fetal calf serum. Results Total success rate of catheterization in the improved group was as high as 95%, and the success rate of first attempt in the improved group was significantly higher than that in the traditional group （94.7% vs. 68.8% ）. Liver perfusion in the improved group was symmetrical with the high yield of hepatocytes up to 1.07×10^6 per gram of mouse weight and 92.16% of average cell vitality, which were higher than those in the traditional group. Conclusion The retrograde liver perfusion through the heart is a simple and easy-to-learn method to isolate mouse primary hepatocytes, which also could guarantee the satisfactory yield and vitality of primary hepatocytes.