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目的探讨鳖甲煎改良方(modified turtle shell decoction,MTSD)对肝星状细胞T6(hepatic stellate cell T6,HSC-T6)TGFβ1、Smad3及Smad7表达的影响。方法用含生药量为0.71 g/ml的MTSD处理HSC-T6作为实验组,与MTSD剂量相同的鳖甲煎丸(turtle shell pills,TSP)处理的HSC-T6作为阳性对照组,常规培养的HSC-T6作为正常对照组,倒置显微镜下观察比较各组HSC-T6的形态变化。CCK-8检测各组HSC-T6细胞的生长情况,并绘制生长曲线;采用Real time-PCR、免疫荧光细胞化学及Western blotting技术分别检测各组HSC-T6的TGFβ1、Smad3及Smad7基因及其编码蛋白的表达变化。结果倒置显微镜下观察显示,与阳性对照细胞比较,MTSD处理48 h的HSC-T6,胞体变小,部分细胞死亡碎裂。CCK-8检测证实,MTSD对HSC-T6生长具有抑制作用,与2个对照组比较,MTSD处理的HSC-T6的生长减慢,从48 h开始差异具有统计意义(P<0.01)。Real time-PCR、免疫荧光细胞化学及Western blotting检测显示,MTSD能在m RNA及蛋白水平明显下调HSC-T6的TGFβ1、Smad3的表达,上调其Smad7的表达,与2个对照组相比,差异均具统计学意义(P<0.01)。结论 MTSD能有效抑制HSC-T6的生长,下调其TGFβ1、Smad3的表达,同时使Smad7的表达上调。MTSD抑制HSC-T6的生长可能与干扰TGFβ1/Smad3信号通路密切有关。
Objective To investigate the effect of modified turtle shell decoction (MTSD) on the expression of TGFβ1, Smad3 and Smad7 in hepatic stellate cell T6 (HSC-T6). Methods HSC-T6 was treated with MTSD containing 0.71 g / ml crude drug and HSC-T6 treated with turtle shell pills (TSP) with the same dose of MTSD as the positive control. HSC -T6 as a normal control group, observed under inverted microscope to compare the morphological changes of HSC-T6 in each group. The growth of HSC-T6 cells in each group was detected by CCK-8, and the growth curve was drawn. The expression of TGFβ1, Smad3 and Smad7 in HSC-T6 and the coding of Smad7 in HSC-T6 were detected by Real-time PCR, immunofluorescence cytochemistry and Western blotting Changes in protein expression. Results Under inverted microscope, compared with the positive control cells, the HSC-T6 cells treated with MTSD for 48 h had smaller cell body and some cell death fragmentation. CCK-8 test confirmed that MTSD could inhibit the growth of HSC-T6. Compared with the two control groups, the growth of MTSD-treated HSC-T6 slowed down, and the difference was statistically significant at 48h (P <0.01). Real time-PCR, immunofluorescence cytochemistry and Western blotting showed that MTSD significantly down-regulated the expression of TGFβ1 and Smad3 in HSC-T6 and up-regulated the expression of Smad7 at m RNA and protein levels, compared with the two control groups All statistically significant (P <0.01). Conclusion MTSD can effectively inhibit the growth of HSC-T6, down-regulate the expression of TGFβ1 and Smad3, and up-regulate the expression of Smad7. MTSD inhibits the growth of HSC-T6 may be closely related to interference with TGFβ1 / Smad3 signaling.