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目的研究分离 Sprague-Dawley(SD)大鼠肝细胞的方法及短期培养后对细胞活力的影响,并研究新鲜分离的同种异体肝细胞移植对实验性急性肝功能衰竭的治疗作用。方法采用胶原酶原位循环灌注法分离肝细胞;游离肝细胞在合成 RPMI 1640培养基内培养6天并测定细胞内生化指标;用 D-氨基半乳糖将60只雄性 SD 大鼠制成急性肝功能衰竭模型并随机均分为移植组和对照组,将新鲜离体肝细胞悬液注入移植组大鼠腹腔内,对照组则注入等量生理盐水,观察两组长期存活率的差异并进行x~2检验。结果游离肝实质细胞(hepatocyte)的纯度可达95%,细胞活率约85%～95%,平均每只大鼠移植肝细胞2.5×10~8～2.8×10~8个;经过短期培养,肝细胞内的丙氨酸转氨酶、碱性磷酸酶仍具有较高活力,且肝细胞仍具有合成蛋白质及胆红素的能力;接受肝细胞移植的大鼠存活率(40.0%)显著高于对照组(13.3%)(P<0.01)。结论用上述方法分离的肝细胞纯度、细胞活率均较高,行同种异体肝细胞移植对大鼠急性肝功能衰竭有明显的治疗作用。
Objective To study the method of isolation of hepatocytes in Sprague-Dawley (SD) rats and the effect on cell viability after short-term culture, and to study the therapeutic effect of freshly isolated allogeneic hepatocytes transplantation on experimental acute liver failure. Methods The hepatocytes were isolated by collagenase in situ perfusion. The free hepatocytes were cultured in RPMI 1640 medium for 6 days and the intracellular biochemical markers were determined. Sixty male Sprague Dawley rats were made into acute liver by D-galactosamine The model of failure was randomly divided into transplantation group and control group, freshly isolated hepatocyte suspension was injected into the peritoneal cavity of the transplanted group, while the control group was injected with the same amount of normal saline. The difference of long-term survival rate ~ 2 test. Results The purity of free hepatocytes was up to 95%, cell viability was about 85% ~ 95%, and the average number of transplanted hepatocytes was 2.5 × 10 ~ 8 ~ 2.8 × 10 ~ 8. After short-term culture, ALT and ALP in hepatocytes still have high viability, and liver cells still have the ability to synthesize protein and bilirubin. The survival rate of rats receiving hepatocyte transplantation (40.0%) was significantly higher than that of control Group (13.3%) (P <0.01). Conclusions The purity and cell viability of hepatocytes isolated by the above method are high, and allogeneic hepatocyte transplantation has a significant therapeutic effect on acute liver failure in rats.