毛壳素对肝内胆管癌细胞的影响及其机制

The effect and mechanism of chaetocin on the intrahepatic cholangiocarcinoma cells

目的 观察毛壳素对人肝内胆管癌细胞CCLP-1活性的影响并探讨其相关作用机制。 方法 毛壳素以不同浓度分为1组（0 nmol/L）、2组（50 nmol/L）、3组（100 nmol/L）和4组（200 nmol/L）与CCLP-1细胞共培养，细胞增殖法检测CCLP-1细胞活性；吉姆赛染色观察CCLP-1细胞形态学改变；流式细胞技术检测细胞凋亡；荧光显微镜检测CCLP-1胞内活性氧（ROS）；蛋白质印迹法检测凋亡信号调节激酶1（ASK-1）、c-Jun氨基末端激酶（JNK）蛋白表达。结果 细胞增殖法提示毛壳素随浓度增加及作用细胞时间延长，细胞活性抑制率上升，呈剂量-效应关系（F＝102.369，P＝0.000）及时间-效应关系（F＝141.377，P＝0.000），并剂量与时间呈交互作用（F＝15.418，P＝0.000）；CCLP-1细胞经毛壳素作用后吉姆赛染色可见细胞核固缩，出现分页状、碎片形状及凋亡小体。流式细胞技术显示细胞总凋亡率：1组：（6.42±3.10）%；2组：（10.82±2.84）%；3组：（13.67±1.71）%和4组：（17.91±6.16）%，总凋亡率呈增多趋势并差异有统计学意义（F＝4.788，P＝0.034）；荧光显微镜检测实验组细胞内荧光值（24.126±9.587）高于空白组荧光值（5.114±1.937）并差异有统计学意义（P＝0.006）；Western blot法实验显示磷酸化ASK-1蛋白表达量：0.255±0.150 （1组）、0.680±0.039（2组）、1.356±0.125（3组）和1.916±0.367（4组）。磷酸化JNKs蛋白表达量：0.493±0.233（1组）、1.542±0.573（2组）、1.639±0.342（3组）、2.480±1.001（4组），提示随毛壳素浓度增加各蛋白表达量均上调（ASK-1：F＝36.946，P＝0.000，JNKs：F＝5.291，P＝0.027）。 结论 毛壳素对CCLP-1细胞有显著抑制作用并诱导其凋亡，并且ROS-ASK-1/JNKs信号参与CCLP-1细胞凋亡。

Objective To investigate the effect of chaetocin on the intrahepatic cholangiocarcinoma CCLP-1 and explore the potential mechanism.Methods The concentration of chaetocin were divided into 4 groups： Group 1： 0 nmol/L, Group 2： 50 nmol/L, Group 3： 100 nmol/L and Group 4： 200 nmol/L, and all were co-cultured with CCLP-1 cell, respectively. Cell viability was detected by Cell Counting Kit-8 assay; Giemsa staining was used to observe the cell morphological and flow cytometry technique were used to detect cell apoptosis; The intracellular reactive oxygen species （ROS） level was detected using fluorescent probe under fluorescence microscope; Western blotting were used to measure the expression of Apoptosis signal-regulating kinase 1 （ASK-1） and c-Jun N-terminal kinase （JNKs）.Results The proliferation of CCLP-1 cell was inhibited by chaetocin in dose dependent （F=102.369, P=0.000） or in time dependent （F=141.377, P=0.000）. Furthermore, both have interaction （F=15.418, P=0.000）. Giemsa staining showed chaetocin brought about CCLP-1 cell to present morphological characteristics of apoptosis, and flow cytometry indicated title rate of apoptosis of CCLP-1 cell were （6.42±3.10）%, （10.82±2.84）%, （13.67±1.71）% and （17.91±6.16）%, and apoptosis was significantly increased （F=4.788, P=0.034）. The intracellular ROS level of treatment group （24.126±9.587） was much higher than that of control group （5.114±1.937） and had significantly difference （P=0.006）. Western blotting showed the expressions of p-ASK-1 were 0.255±0.150, 0.680±0.039, 1.356±0.125 and 1.916±0.367. The expressions of JNKs were 0.493±0.233, 1.542±0.573, 1.639±0.342 and 2.480±1.001.Both proteins expressed increasingly as dose of chaetocin increased. （ASK-1： F=36.946, P=0.000, JNKs： F=5.291, P=0.027）. Conclusion Chaetocin could inhibit CCLP-1 cell and induce it apoptosis via promote ROS mediate ASK-1/JNK signal.