CAJ | 학술논문

目的研究糖原合成酶激酶(GSK)3β过表达及GSK3β抑制剂SB-216763通过Wnt通路对大鼠肝卵圆细胞增殖的影响及其调控机制.方法肝卵圆细胞WBF-344分为空白对照组、GSK3β过表达慢病毒组(过表达组)、二甲基亚砜对照组和GSK3β抑制剂组,抑制剂组设1、5、10 u.mol/L 3个浓度梯度.以鉴定正确的GSK3β过表达慢病毒和不同浓度SB-216763处理WBF-344细胞,相差及荧光显微镜分别观察细胞形态及慢病毒组细胞荧光表达量；CCK8检测细胞增殖,Annexin V-碘化丙啶检测细胞凋亡;Western blot法检测GSK3β、β-catenin及cyclin DI蛋白表达.结果镜下见GSK3β过表达组细胞较少,老化明显,且出现大量绿色荧光；各抑制剂组细胞分裂旺盛,状态良好,且细胞数随SB-216763浓度升高而增多.CCK8及流式细胞术显示GSK3β过表达组细胞增殖减慢(t=7.178,P＜0.01),凋亡明显,抑制剂组增殖加快(F =45.030,P ＜0.01).Western blot 法显示过表达组GSK3β呈高表达趋势,3-catenin和cyclin D1表达减弱,抑制剂组GSK3β表达量无明显差别,但随抑制剂浓度增高,β-catenin和cyclin D1表达增强.结论 GSK3β过表达可下调Wnt通路使细胞增殖减慢,促进凋亡.GSK3β抑制剂能激活Wnt通路促进细胞增殖.
목적 연구당원합성매격매(GSK)3β과표체급GSK3β억제제SB-216763통과Wnt통로대대서간란원세포증식적영향급기조공궤제.방법 간란원세포WBF-344분위공백대조조、GSK3β과표체만병독조(과표체조)、이갑기아풍대조조화GSK3β억제제조,억제제조설1、5、10 u.mol/L 3개농도제도.이감정정학적GSK3β과표체만병독화불동농도SB-216763처리WBF-344세포,상차급형광현미경분별관찰세포형태급만병독조세포형광표체량；CCK8검측세포증식,Annexin V-전화병정검측세포조망;Western blot법검측GSK3β、β-catenin급cyclin DI단백표체.결과 경하견GSK3β과표체조세포교소,노화명현,차출현대량록색형광；각억제제조세포분렬왕성,상태량호,차세포수수SB-216763농도승고이증다.CCK8급류식세포술현시GSK3β과표체조세포증식감만(t=7.178,P＜0.01),조망명현,억제제조증식가쾌(F =45.030,P ＜0.01).Western blot 법현시과표체조GSK3β정고표체추세,3-catenin화cyclin D1표체감약,억제제조GSK3β표체량무명현차별,단수억제제농도증고,β-catenin화cyclin D1표체증강.결론 GSK3β과표체가하조Wnt통로사세포증식감만,촉진조망.GSK3β억제제능격활Wnt통로촉진세포증식.
Objective To research the effects of glycogen synthase kinase (GSK3β) overexpression and GSK3β inhibitor SB-216763 on the proliferation of hepatic oval cells in rats and its regulatory mechanisms by Wnt signaling pathway.Methods The hepatic oval cells WBF-344 were divided into the blank control group,GSK3 β over-expression group,DMSO control group and GSK3 β inhibitor groups,while the inhibitor groups set up three concentration gradients,that was 1,5,10 μmol/L.Using the GSK3β overexpression lentivirus,which had been identified correctly,and SB-216763 dealt with the cells WBF-344.The cells morphology of each group was observed under the phase contrast inverted microscope,and the expression of fluorescence in the lentivirus-transfected group was observed under the fluorescent microscope.The proliferation of each group cells was tested by CCK8 kits.The cells' apoptosis was detected by AnnexinV-FITC/PI kits.The expression of GSK3β,β-catenin and cyclin D(I) were detected by Western blot.Results The cells of GSK3β over-expression group were fewer and obvious aging.However,in each inhibitor added group,the cells' division and proliferation was vigorous,and the condition was good.Moreover,the cells' proliferation was getting stronger with the concentration of SB-216763 increasing.A large number of green fluorescence was expressed in the lentivirus-transfected cells.The cells' proliferation in GSK3β over-expression group restrained (t =7.178,P ＜ 0.01,as compared with control),while the cells' proliferation was vigorous in inhibitor groups (F =45.030.P ＜ 0.01,as compared with control).Flow Cytometry showed that the cells apoptosis was significant in GSK3β over-expression group.Western blot showed that the expression of GSK3β was increased,while the expression of β-catenin and cyclin D1 was decreased in the over-expression group.The expression of GSK3β had no significant difference among the control group and inhibitor groups.However,the expression of β-catenin and cyclin D1 was significantly increased with the concentration of SB-216763 increasing.Conclusions The overexpression of GSK3β can inhibit the Wnt signaling pathway,thus restrain the cells' proliferation and promotes apoptosis.SB-216763can activate the Wnt pathway,thus promotes cells' proliferation.