大鼠肝细胞的分离、短期培养及异体移植治疗实验性急性肝功能衰竭

The isolation,short-term culture of hepatocytes and allograft transplantation of fresh prepared rat hepatocytes in the treatment of murine acute experimental hepatic failure

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 evaluate the efficacy of isolation,short-period culture on the viability of rat hepatocytes and the effect of transplantation of fresh-prapared hepatocytes on acute experimental hepatic failure of the rat.Meth- otis Hepatocytes were isolated by an in-situ collagenase cycle perfusion technique modified after Seglen; Some of the isolated hepatocytes were cultured in RPMI-1640 culture medium supplemented with 20% newborn calf serum for six days,during the period,the activity of glutamie-pyruvie transaminase (AST) and alkaline phosphatase (ALP) and the content of total protein (TP) and total bilirubin (T-Bil) in the hepatocytes were de- termined;sixty male SD rats with acute hepatic failure induced by D-galactosamine were randomly divided into transplantation group (n=30) and control group (n=30),the fresh hepatocytes were transplanted intraperi- toneally to the former and the normal saline to the later.Results The purity of the isolated hepatocytes was 95% and of them,85-95% were viable,hepatocytes yielded amount to 2.5～2.8×10~8 per rat;After short- term culture,the activities of AST and ALP were still within normal range and the hepatoeytes still showed the ability of protein synthesis and bilirubin secretion;The long-term survival rate of the transplantation group (40%,12/30) was significantly higher than that of the controls (13.3%,4/30)(P<0.01,χ~2 test).Conclu- sion The purity and viability of hepatoeytes isolated by the aforementioned method were high and allograft transplantation of rat hepatoeytes effeeted a significant decrease in the mortality of murine acute experimental hepatic failure.