CAJ | 학술논문

目的观察甲基莲心碱对转化生长因子(TGF)-β1诱导的肝星状细胞(HSC)增殖及gremlin、TGF-β1、α-平滑肌动蛋白(α-SMA)表达的影响.方法体外培养HSC-T6细胞,分成正常对照组、TGF-β1干预组、TGF-β1+甲基莲心碱分别为2.0、4.0、8.0 mmol/L联合干预组,细胞计数试剂盒(CCK)-8检测细胞增殖抑制率,半定量RT-PCR、Western印迹法分别检测HSC-T6细胞gremlin、TGF β1、α-SMA mRNA及蛋白表达.组间差异采用单因素方差分析,方差齐性者两两比较采用LSD法,方差不齐者采用Dunnett T3检验,相关性采用Pearson直线相关分析.结果与正常对照组相比,TGF-β1对HSC-T6细胞增殖无影响(P＞0.05),但能上调gremlin、TGF-β1、α-SMAmRNA及蛋白的表达,差异有统计学意义(P＜0.05).与TGF-β1干预组相比,甲基莲心碱能抑制HSC-T6细胞增殖,呈时间、剂量依赖性,以TGF-β1+甲基莲心碱8 mmol/L联合作用72 h对HSC-T6细胞抑制最显著(F=2817.056,P＜0.01),抑制率为(69.08±1.05)％.甲基莲心碱可不同程度抑制gremlin mRNA及蛋白的表达,TGF-β1+8 mmol/L甲基莲心碱联合干预组表达量最低,为0.2483±0.0084、0.9307±0.0224.与TGF-β1干预组比较,gremlin、TGF-β1、α-SMA mRNA表达量随甲基莲心碱浓度的增加呈递减趋势(F=16.800、209.246、70.486,均P＜0.05),蛋白表达量也呈递减趋势(F=56.588、12.550、32.141,均P＜0.05).gremlin、TGF-β1、α-SMA的表达呈两两正相关(P＜0.05).结论甲基莲心碱能有效作用于HSC,gremlin可能参与肝纤维化的发生发展,有望成为肝纤维化治疗新靶点.
목적 관찰갑기연심감대전화생장인자(TGF)-β1유도적간성상세포(HSC)증식급gremlin、TGF-β1、α-평활기동단백(α-SMA)표체적영향.방법 체외배양HSC-T6세포,분성정상대조조、TGF-β1간예조、TGF-β1+갑기연심감분별위2.0、4.0、8.0 mmol/L연합간예조,세포계수시제합(CCK)-8검측세포증식억제솔,반정량RT-PCR、Western인적법분별검측HSC-T6세포gremlin、TGF β1、α-SMA mRNA급단백표체.조간차이채용단인소방차분석,방차제성자량량비교채용LSD법,방차불제자채용Dunnett T3검험,상관성채용Pearson직선상관분석.결과 여정상대조조상비,TGF-β1대HSC-T6세포증식무영향(P＞0.05),단능상조gremlin、TGF-β1、α-SMAmRNA급단백적표체,차이유통계학의의(P＜0.05).여TGF-β1간예조상비,갑기연심감능억제HSC-T6세포증식,정시간、제량의뢰성,이TGF-β1+갑기연심감8 mmol/L연합작용72 h대HSC-T6세포억제최현저(F=2817.056,P＜0.01),억제솔위(69.08±1.05)％.갑기연심감가불동정도억제gremlin mRNA급단백적표체,TGF-β1+8 mmol/L갑기연심감연합간예조표체량최저,위0.2483±0.0084、0.9307±0.0224.여TGF-β1간예조비교,gremlin、TGF-β1、α-SMA mRNA표체량수갑기연심감농도적증가정체감추세(F=16.800、209.246、70.486,균P＜0.05),단백표체량야정체감추세(F=56.588、12.550、32.141,균P＜0.05).gremlin、TGF-β1、α-SMA적표체정량량정상관(P＜0.05).결론 갑기연심감능유효작용우HSC,gremlin가능삼여간섬유화적발생발전,유망성위간섬유화치료신파점.
Objective To observe the impact of neferine (Nef) on the proliferation of transforming growth factor-β1 (TGF-β1)-induced hepatic stellate cells (HSC) and the expression of gremlin and TGF β1,a-smooth muscle actin (α-SMA).Methods HSC-T6 cells were cultured in vitro and divided into five groups:control group,TGF-β1 group,TGF-β1 + Nef 2.0 mmol/L group,TGF-β1 +Nef 4.0 mmol/L group and TGF-β1+Nef 8.0 mmol/L group.Inhibition of proliferation of HSC-T6 cells was measured by cell counting kit-8.Semi quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay were used to evaluate the mRNA and protein expressions of gremlin,TGF-β1 and α-SMA,respectively.The differences among groups were compared by oneway analysis of variance.Comparison between two groups were analyzed by LSD-t test and Dunnett T3 test.Correlation was tested by Pearson linear correlation analysis.Results TGF-β1 had no effect on the proliferation of HSC-T6 cells when compared with the control group (P＞0.05),but it could up-regulate the mRNA and protein expression of gremlin,TGF-β1 and α-SMA (P＜0.05).The proliferation of HSC-T6 cells were inhibited by Nef in a time and dose dependent way,when compared with the TGF-β1 group.The most significant inhibition was observed in the TGF-β1+Nef 8 mmol/L group at 72 hours (F=2817.056,P＜0.01),and the inhibition rate was (69.08±1.05)％.The expression of gremlin mRNA and protein could be inhibited by Nef in varying degrees,to be the lowest in the TGF-β1 + Nef 8mmol/L group (0.2483±0.0084 and 0.9307±0.0224,respectively).Compared with the TGF-β1 group,both the mRNA and protein expressions of gremlin,TGF-β1 and α-SMA were gradually decreased with the increasing concentrations of Nef (F=16.800,209.246 and 70.486,respectively; all P＜0.05; F=56.588,12.550 and 32.141,respectively; all P＜0.05),respectively.The expressions of gremlin,TGF-β1 and α-SMA were positively correlated pairwise (P＜ 0.05).Conclusion Nef can effectively act on HSC,and gremlin may play a role in the development of hepatic fibrogenesis,which might be a new target for the treatment of hepatic fibrosis.