活性维生素D3联合LY294002体外抑制大鼠肝星状细胞增殖作用研究*

目的探究维生素D的活性形式—1,25-(OH）2D3联合磷脂酰肌醇-3激酶/蛋白激酶B(PI3K/Akt）信号通路的特异抑制剂LY294002对体外培养大鼠肝星状细胞的抑制作用。方法将HSC-T6细胞分为正常对照、 DMSO组、1,25-(OH）2D3、LY294002组和1,25-(OH）2D3+ LY294002 联合组,采用MTT法检测细胞增殖;在倒置显微镜下观察细胞形态变化;使用流式细胞术检测HSC-T6细胞凋亡;采用Western blotting法检测Phospho-Akt（Ser473）、AKT（PAN）、半胱氨酸天冬氨酸蛋白酶（caspase）-3蛋白表达。结果体外干预肝星状细胞48 h后,两药联合干预组细胞抑制率为87.5%,显著高于1,25-（OH）2D3组的50.3%或LY294002组的40.3%（P<0.05）;联合组细胞凋亡率为92.12%,显著高于1,25-(OH）2D3组的71.0%或LY294002组的34.0%（P<0.05）;在显微镜下观察,两药处理组细胞形状由星形变为不规则形,凋亡数量增多,表现为正常活细胞数目明显减少,胞间隙增宽,细胞体积缩小,胞浆浓缩,胞核固缩,可见空泡及细胞碎片,有的细胞出现数量不等的凋亡小体;与单药干预组比,联合组细胞Bax/Bcl-2比值上升明显（P<0.05）,活化的凋亡蛋白caspase-3表达显著增加（P<0.05）。结论1,25-（OH）2D3可协同LY294002 共同诱导 HSC-T6 细胞凋亡,增强其抗肝纤维化的效果。

Inhibitory and apoptotic effect of LY 294002in combination with 1,25-dihydroxyvitamin D3 on HSC T6 cells in vitro

Objective To invastigate the anti-proliferating and apoptotic effect of LY294002 and 1,25-dihydroxyvitamin D3（1,25-（OH）2D3） on hepatic stellate （HSC-T6） cells in vitro. MethodsThe HSC-T6 cells were divided into five groups, e.g. control, DMSO, 1,25-（OH）2D3,LY294002 and 1,25-（OH）2D3 plus LY294002. The inhibitory effects were detected by MTT assay; The cell morphology was observed under microscopy; The apoptosis was detected by FCM, and the expression of Ser473,AKT and caspase-3 protein were assayed by Western blotting.Results The inhibitory rate in 1,25-（OH）2D3 plus LY294002 group was 87.5%,much higher than 50.3% in 1,25-（OH）2D3 or 40.3% in LY294002 group （P<0.05） after 48 h intervention;the apoptotic rate in 1,25-（OH）2D3 plus LY294002 group was 92.12%,much higher than 71.0% in 1,25-(OH）2D3 or 34.0% in LY294002-intervented group（P<0.05）;the ratio of Bax/Bcl-2 increased (P<0.05）,and the expression of caspase-3 also increased obviously in 1,25-（OH）2D3 plus LY294002 group(P<0.05）.ConclusionLY294002 has an anti-fibrotic effect with the assistance of 1,25-（OH）2D3 on T6 cells in vitro.