【摘要】 目的：探讨盐酸石蒜碱（LH）对甲状腺乳头状癌TPC-1细胞增殖和周期的影响及其机制。方法：体外培养TPC-1细胞，用不同浓度（1.25、2.5、5、10、20、40、60、80和100μmol/L）的LH处理TPC-1细胞48 h,CCK-8法检测LH作用TPC-1细胞的IC₅₀。参照IC₅₀，将对数生长期TPC-1细胞随机分为7组：空白对照（control）组、溶剂对照（DMSO）组、1μmol/L LH组、2μmol/L LH组、4μmol/L LH组、NAC（3 mmol/L）组和LH（4μmol/L）+NAC（3 mmol/L）组。用CCK-8法、平板集落、EdU实验检测LH对细胞增殖的影响；透射电子显微镜观察细胞线粒体超微结构；流式细胞术检测细胞周期、活性氧（ROS）水平及线粒体膜电位变化；Western blot法检测MAPK家族蛋白（p38 MAPK、ERK1/2、JNK、p-p38 MAPK、p-ERK1/2和p-JNK）的水平；免疫荧光检测p-p38 MAPK、p-JNK和p-ERK1/2在细胞内的定位。结果：（1）LH抑制TPC-1细胞增殖，抑制率与LH浓度及作用时间呈正比，LH作用48 h的IC₅₀为2.314μmol/L;（2）4μmol/L LH处理后TPC-1细胞线粒体超微结构发生变化，LH呈浓度依赖性地使线粒体膜电位去极化（P<0.05）;（3）LH处理后TPC-1细胞S期比例显著升高，G₀/G₁期比例显著降低（P<0.05）;（4）LH处理后的TPC-1细胞ROS水平相较对照组显著升高（P<0.01）;（5）LH处理后TPC-1细胞p38 MAPK、ERK1/2、JNK、p-p38 MAPK、p-ERK1/2和p-JNK蛋白水平均显著升高，尤以磷酸化蛋白升高显著，具有一定的药物浓度依赖性，且p-p38 MAPK和pERK1/2在TPC-1细胞中都出现不同程度的入核现象；（6）NAC能抑制部分MAPK通路的激活。结论：LH在体外能抑制TPC-1细胞的增殖，诱导细胞线粒体超微结构受损、线粒体膜电位去极化、细胞S期阻滞和细胞ROS水平升高，其作用机制可能是通过ROS累积诱导激活部分MAPK通路而发挥作用。更多还原

【Abstract】 AIM:To investigate the effects of lycorine hydrochloride（LH）on the proliferation and cell cycle of thyroid papillary carcinoma TPC-1 cells and its mechanism.METHODS:The TPC-1 cells were treated with LH at different concentrations（1.25,2.5,5,10,20,40,60,80 and 100μmol/L）for 48 h,and IC₅₀of LH for TPC-1 cells was detected by CCK-8 method.According to the IC₅₀,TPC-1 cells at logarithmic growth stage were randomly divided into 7groups:blank control（control）group,solvent control（DMSO）group,1μmol/L LH group,2μmol/L LH group,4μmol/L LH group,NAC（3 mmol/L）group and LH（4μmol/L）+NAC（3 mmol/L）group.The effects of LH on cell proliferation were detected by CCK-8,plate colony and EdU assays.Mitochondrial ultrastructure was observed by transmission electron microscopy,and the changes of cell cycle,reactive oxygen species（ROS）and mitochondrial membrane potential were detected by flow cytometry.The levels of MAPK family proteins（p38 MAPK,ERK1/2,JNK,p-p38 MAPK,p-ERK1/2 and p-JNK）were measured by Western blot.The intracellular localization of p-p38 MAPK,p-JNK and p-ERK1/2 was observed by immunofluorescence.RESULTS:（1）The proliferation of TPC-1 cells was inhibited by LH,and the inhibitory rate was proportional to the concentration and action time of LH.The IC₅₀of LH at 48 h was 2.314μmol/L.（2）Mitochondrial ultrastructure of TPC-1 cells changed after 4μmol/L LH treatment,and LH depolarized mitochondrial membrane potential in a concentration-dependent manner（P<0.05）.（3）The proportion of S phase in TPC-1 cells after LH treatment were significantly increased,and the proportion of G₀/G₁phase was significantly decreased（P<0.05）.（4）The level of ROS in TPC-1 cells treated with LH was significantly higher than that in control group（P<0.01）.（5）After LH treatment,the protein levels of p38 MAPK,ERK1/2,JNK,p-p38 MAPK,p-ERK1/2 and p-JNK were increased,especially the phosphorylated proteins,with a certain drug concentration dependence.Moreover,p-p38 MAPK and p-ERK showed different degrees of nuclear translocation in TPC-1 cells.（6）Partial MAPK pathway activation was inhibited by NAC pretreatment.CONCLUSION:In vitro,LH inhibits the proliferation of TPC-1 cells,damages the ultrastructure of mitochondria,depolarizes mitochondrial membrane potential,and induces S-phase arrest and ROS increase.The mechanism may be related to ROS accumulation induction and partial MAPK pathway activation.更多还原