抑瘤素表达产物诱导大鼠肝干细胞WB-F344体外分化

Expression products of oncostatin inducing rat hepatic stem cell WB-F344 to differentiate in vitro

目的:抑瘤素是一种肝细胞促分化因子,能够诱导胚胎肝细胞分化为具有各种代谢功能的成熟肝脏。构建以抑瘤素为主的肝干细胞体外诱导微环境,观察其诱导大鼠肝脏来源的干细胞WB-F344体外分化。方法:实验于2002-10/2004-07在解放军军事医学科学院放射与辐射医学研究所完成。①实验材料:表达抑瘤素的CHO-OSM-EGFP细胞株由李晓利等构建。WB-F344细胞由姚鹏博士惠赠。②实验方法:将终浓度为20%稳定表达抑瘤素的中国仓鼠卵巢细胞上清中加入浓度为1U/mL胰岛素、1×10-7mol/L地塞米松,与WB-F344细胞共培养。空白对照组中未加入诱导分化成分CHO-OSM-EGFP细胞上清、胰岛素、地塞米松。③实验评估:采用流式细胞术分析细胞周期和细胞凋亡,光学显微镜观察细胞形态,RT-PCR分析白蛋白及甲胎蛋白基因表达。结果:①诱导后的WB-F344细胞较空白对照组G2/M+S期细胞所占比例明显减低,增殖指数下降约2.8倍,G0/G1细胞所占比例明显增加。②实验组WB-F344细胞形态发生明显改变,由最初的多角形变为长梭性,细胞伸出小管状伪足,核质比例下调,形态趋于成熟大鼠肝脏实质细胞。③RT-PCR结果显示,诱导后WB-F344细胞表达白蛋白,不表达甲胎蛋白。诱导前WB-F344细胞表达甲胎蛋白,不表达白蛋白。结论:在以抑瘤素为主的诱导微环境中,WB-F344细胞表现出生长抑制。

AIM： The oncostatin as a kind of differentiation promoters in hepatic cells, can induce the fetal liver cells to differentiate the mature liver with mechanical performance. This study is designed to investigate the factors, which induce liver-derived stem cell WB-F344 differentiation in vitro, through establishing oncostatin M-depended microenvironment. METHODS： The experiment was carried out in the Institute of Radiation Medicine, Academy of Military Medical Sciences of Chinese PLA from October 2002 to July 2004. ①Materials： Chinese hamster ovary （CHO）-OSM-EGFP strain expressing oncostatin was established by Li Xiao-li, et al. WB-F344 cells were offered from Doctor Yao Peng.②Methods： The cultured supernatant collected from CHO cells was added to WB-F344 culture plates with 20% final concentration, 1 U/mL insulin amd 1 ×10^-7 mol/L dexamethasone. While these differentiation inducers were not found in blank control group. ③Evaluations： The cell cycle and apoptosis were analyzed by flow cytometry, cell morphology was detected by using light microscope, the mRNA expression of Albumin （AIb） and α-fetoprotein （AFP） were analyzed by RT-PCR. RESULTS： ①Compared with blank control group, WB-F344 stem cells were arrested significantly at G0/G1 phase of cell cycle, and cell proportion in the G2/M＋S phase decreased obviously. Moreover the proliferation index reduced by 2.8 times.②In the experimental group, cellular phenotypic changes were found, transforming from polygon to fusiform, which was identical with mature liver parenchymal cells in rats. Cells appeared tubiform pseudopodia and decreased ratio of nucleus-cytoplasm.③RT-PCR result showed that, the level of AFP mRNA expression was down-regulated while the level of AIb mRNA expression was up-regulated in WB-F344 cells after the induction, and it was contrast to that before induction. CONCLUSION： The WB-F344 differentiation can be inhibited in the oncostatin M-depended microenvironment, and the cells are identical with the mature hepatic cells in rats.