大鼠脐带间充质干细胞对肝细胞及卵圆细胞体外增殖与功能的影响

摘要

目的：探讨大鼠脐带间充质干细胞（UMSC）对原代大鼠肝细胞及肝卵圆细胞增殖与功能的影响。方法采用2-乙酰氨基芴加肝脏三分之二切除术建立肝卵圆细胞增殖模型，通过原位二步胶原酶灌流法分离到单个肝脏细胞，再经过 Percoll 密度梯度离心分离到肝卵圆细胞。原代大鼠肝细胞/肝卵圆细胞各分为3组：UMSC 组、原代大鼠肝细胞组/肝卵圆细胞组及 UMSC 与原代大鼠肝细胞共培养组/UMSC 与肝卵圆细胞共培养组，分别于第1、3、6、8天通过MTT 法检测各组细胞增殖能力，于第3天通过 ELISA 法检测各组细胞培养上清液中白蛋白含量。结果UMSC 与原代大鼠肝细胞共培养组在第3、6、8天的 A 值均比相应时间点的 UMSC 组 A 值与原代大鼠肝细胞组 A 值之和大（P ＜0．05）；UMSC 与肝卵圆细胞共培养组在第3、6、8、10天的 A 值均比相应时间点的 UMSC 组 A 值与肝卵圆细胞组 A 值之和大（P ＜0．05）。UMSC 无白蛋白分泌能力，UMSC 与原代大鼠肝细胞共培养组培养上清液中白蛋白水平为（266．21±50．44）ng/mL，较原代大鼠肝细胞组（130．79±22．10）ng/mL 高（P ＝0．013）；UMSC 与肝卵圆细胞共培养组培养上清液中白蛋白水平（（49．64±3．56）ng/mL）较肝卵圆细胞组（13．54±1．53）ng/mL 高（P ＝0．000）。结论UMSC 在体外可以促进原代大鼠肝细胞及肝卵圆细胞的存活和增殖，并可增强其分泌白蛋白的作用。%Objective To explore the effects of UMSC on proliferation and function of primary rat hepatocytes and hepatic oval cells in vitro . Methods Rat model for hepatic oval cell proliferation was established by 2-acetylaminofluorenne and two third partial hepatectomy (2-AAF/PH).Liver cells were isolated by two steps collagenase perfusion via portal vein in site,and then separated to hepatic oval cells by density gradient centrifugation of Percoll. Primary rat hepatocytes/hepatic oval cells were respectively divided into three groups:UMSC group,primary rat hepatocytes/hepatic oval cells group and UMSC co-culture with primary rat hepatocytes/hepatic oval cells group.Cells proliferation rate of each group was measured by MTT on days 1 ,3,6,8,and concentration of albumin in culture medium was measured by ELISA on day 3.Results OD value of UMSC co-culture with primary rat hepatocytes group was significantly higher than the sum of OD values of UMSC group and primary rat hepatocytes group on days 3,6 and 8 (P <0.05);OD value of UMSC co-culture with hepatic oval cells group was higher than the sum of OD values of UMSC group and hepatic oval cells group on 3,6 and 8(P <0.05).UMSC didn’t secrete albumin,but concentration of albumin in culture medium of UMSC co-culture with primary rat hepatocytes group [(266.21 ±50.44)ng/mL]was higher than that in primary rat hepatocytes group [(130.79 ± 22.10)ng/mL,P =0.0013];Concentration of albumin in culture medium of UMSC co-culture with hepatic oval cells group [(49.64±3.56)ng/mL]was higher than that in hepatic oval cells group [(13.54 ± 1 .53 )ng/mL,P = 0.000 ].Conclusion UMSC can promote proliferation of primary rat hepatocytes and hepatic oval cells in vitro ,and increase secretion of albumin.