大鼠肝细胞分离及体外损伤模型建立

Isolation of Rat Hepatocyte and Establishment of Necrotic Hepatocyte Injury Models in Vitro

目的:分离大鼠肝细胞,建立体外大鼠肝细胞损伤模型。方法:改进Seglen胶原酶(IV型)原位灌注分离大鼠肝细胞,培养液中加入不同浓度D-氨基半乳糖培养24 h,于培养1、3、6、12、24 h取上清测定ALT(丙氨酸氨基转移酶)、AST(天门冬氨酸转移酶)、LDH(乳酸盐脱氢酶),同步测定肝细胞的细胞增殖活性(MTT)反应。结果:平均肝细胞产量(8.92±0.47)×108,Trypan blue拒染实验细胞活性率96.23%±2.41%,培养体系中加入D-氨基半乳糖后,部分肝细胞胞膜破损,随剂量增加及时间延长而逐渐加重;各剂量组随时间延长,培养上清液中生化指标水平逐渐升高,而MTT反应水平逐渐下降,以3 h与6 h变化最为明显;各时间点生化指标及MTT反应变化随剂量增加呈现相同趋势。结论:该方法可获得高产量高活性大鼠肝细胞;D-氨基半乳糖可成功诱导大鼠肝细胞体外损伤模型。

OBJECTIVE To isolate the hepatocytes from rats and establish necrotic hepatocyte injury models in vitro. METHODS Hepatocytes were obtained from livers of rats by modified Seglen collagenase（type Ⅳ） perfusion method. The hepatocyte necrotic injuried models was established by incubating primary cultured hepatocyte with D - gaiactosamine [D-GaIN） at different dosage for 24 hours, and ALT, AST, LDH levels and MTr test were measured at various time for 1, 3, 6, 12 and 24 h points. Cell mor- phology was observed under inverted phase-contrast microscope. RESULTS Hepatocyte yield/rat：（8.92±0.47）×10^8 , Viability： 96.23%±2.41% by Trypan blue excluded test. The isolated hepawcytes kept single Free State with integrity membrane under microscope. After added D -GaIN into culture medium, portion of cell membrane became breakage and reunion, and level of ALT, AST and LDH increased as the time went by; but decreased in MTF test obviously at the 3 h and 6 h time point; Biochemical parameters bad the same trend with increasing of D-GaIN dosage at various time points （P〈0.01）. CONCLUSIONS Hepatocytes are isolated from rat liver in high viability and yield rate, The necrotic rat hepatocytes injury model are successfully induced by D-GaIN in vitro.