Bojar H, Balzer K, Reiners K, Basler M, Reipen W, Staib W
Z Klin Chem Klin Biochem. 1975 Jan;13(1):25-30.
An enzymatic method is described for isolating intact parenchymal cells from rat livers. 3--4 g cells (wet weight) could be isolated from livers of rats weighing 180--230 g. After an in vitro preperfusion of 15 minutes with a Ca-free buffer, collagenase (200 mg/1) and calcium chloride (5.2 mmol/1) were added. Perfusion was continued for another 15 minutes at 37 degrees C. Micromorphological integrity of cell membranes was demonstrated by scanning electron microscopy. With regard to rates of gluconeogenesis and protein synthesis, parenchymal cells isolated according to our method were found to be superior to liver slices and cells isolated by other methods. Ratios of ATP/ADP (5.69) and of lactate/pyruvate (8.64) as parameters of the energetic situation and the redox state resp. were found within the physiological range. Integrity of cell surface receptors was proved by their sensitivity to epinephrine, glucagon and insulin. Glucagon (0.3 mumol/1) and epinephrine (1 mumol/1) and reduced glycogen deposition in hepatocytes of fasted rats by 84.9 % and 95.9 % resp. Both hormones stimulated glycogenolysis in parenchymal cells of fed rats to a similar extent. Urea synthesis was stimulated 29.5 % by glucagon (1 mumol/1), and inhibited 28.0 % by insulin (10 nmol/1). The stimulatory effect of glucagon (1 mumol/1) was abolished by insulin (10 nmol/1).
本文描述了一种从大鼠肝脏中分离完整实质细胞的酶法。从体重180 - 230克大鼠的肝脏中可分离出3 - 4克细胞（湿重）。先用无钙缓冲液进行15分钟的体外预灌注，然后加入胶原酶（200毫克/升）和氯化钙（5.2毫摩尔/升）。在37℃下继续灌注15分钟。通过扫描电子显微镜证实了细胞膜的微观形态完整性。就糖异生和蛋白质合成速率而言，发现按照我们的方法分离的实质细胞优于肝切片和用其他方法分离的细胞。作为能量状态和氧化还原状态参数的ATP/ADP比值（5.69）和乳酸/丙酮酸比值（8.64）均在生理范围内。细胞表面受体对肾上腺素、胰高血糖素和胰岛素的敏感性证明了其完整性。胰高血糖素（0.3微摩尔/升）和肾上腺素（1微摩尔/升）分别使禁食大鼠肝细胞中的糖原沉积减少84.9%和95.9%。两种激素对喂食大鼠实质细胞糖原分解的刺激程度相似。胰高血糖素（1微摩尔/升）使尿素合成增加29.5%，胰岛素（10纳摩尔/升）使其减少28.0%。胰岛素（10纳摩尔/升）消除了胰高血糖素（1微摩尔/升）的刺激作用。
