【摘要】 目的构建转化生长因子β1（TGF-β1）诱导的人非小细胞肺癌（Non-small cell lung cancer,NSCLC）H1299细胞上皮-间充质转化（EMT）模型,并探讨莲子心新碱（Neoliensine,NeoL）对其侵袭和迁移能力的抑制作用及潜在机制。方法用5、10、15 ng·mL^-1的TGF-β1诱导H1299细胞构建EMT模型;以15 ng·mL^-1的TGF-β1诱导H1299细胞作为EMT模型组,以法舒地尔（Fas）为阳性药,设置1、2、3μmol·L^-1的莲子心新碱实验组。采用倒置荧光显微镜观察诱导后的细胞形态;用CCK-8法检测莲子心新碱对H1299细胞的抑制率;Western Blot法检测细胞黏附因子E-钙黏蛋白（E-cadherin）以及间充质标记物N-钙黏蛋白（N-cadherin）、波形蛋白（Vimentin）以及ROCK 1与P-Smad 2/3蛋白的表达;Transwell法检测莲子心新碱对TGF-β1诱导的H1299细胞侵袭、迁移的抑制作用;免疫荧光法检测H1299细胞内ROCK 1蛋白的表达及分布。结果 TGF-β1诱导后H1299细胞形态呈现间充质特性。与对照组比较,随着TGF-β1浓度的升高,E-cadherin表达明显降低（P<0.05,P<0.01）,N-cadherin与Vimentin表达明显升高（P<0.01,P<0.001）,并且ROCK 1蛋白的表达也在这一过程中升高（P <0.05）。莲子心新碱可以抑制H1299细胞的活力,IC50为5.04μmol·L^-1;同时与模型组比较,莲子心新碱抑制了TGF-β1促进的H1299细胞EMT过程及侵袭迁移作用（P<0.001）,中、高剂量组明显增加E-cadherin蛋白的表达（P<0.05）,明显减少N-cadherin和Vimentin蛋白的表达（P<0.001）,还明显降低了H1299细胞内因TGF-β1诱导而升高的ROCK 1（P<0.001）和P-Smad2/3（P<0.001）蛋白的水平。免疫荧光结果显示莲子心新碱可以抑制TGF-β1诱导的H1299细胞胞质中ROCK 1蛋白的表达及分布。结论 TGF-β1可以诱导H1299细胞发生EMT,莲子心新碱可以有效抑制这一过程,其机制可能是抑制了TGF-β1/Smad/RhoA/ROCK通路。更多还原

【Abstract】 Objective To build a TGF-β1 induced epithelial-mesenchymal transition（EMT）model of non-small cell lung cancer（NSCLC）H1299 cells and explore the inhibitory effect and potential mechanism of neoliensine（NeoL）on the invasion and migration of the EMT model cells.Methods H1299 cells were induced by different concentrations(5, 10, 15 ng · mL^-1) of TGF-β1 to construct EMT model. In NeoL intervention experiment,15 ng·mL^-1 of TGF-β1-induced H1299 cells were used as the EMT model group,fasudil（Fas）was used as the positive control medicine,and experimental groups were treated with 1,2 and 3 μmol·L^-1 of NeoL. The morphology changes of TGF-β1-induced H1299 cells were observed by inverted fluorescence microscope. CCK-8 assay was used to determine the inhibitory rate of NeoL on H1299 cells. The expressions of adhesion factor E-cadherin,mesenchymal markers N-cadherin,vimentin,ROCK 1 and P-Smad 2/3 in NeoL treated cells were detected by Western Blot（WB）. Transwell assay was operated to figure out the inhibitory effect of NeoL on invasion and migration of TGF-β1-induced H1299 cells. And immunofluorescence assay was used to detect the expression and distribution of ROCK 1 in H1299 cells.Results After TGF-β1 induction, H1299 cells showed characteristic of mesenchymal morphology. Compared with the control group,the expression of reduced E-cadherin（P < 0.05,P <0.01）and high N-cadherin and increased vimentin（P < 0.01,P < 0.001）significantly correlated with the increase of TGF-β1. Meanwhile,the expression of ROCK 1 also increased（P < 0.05）. NeoL suppressed the vitality of H1299 cells,and the IC50 value was 5.04 μmol·L^-1. Compared with the model group,NeoL could inhibit TGF-β1 induced EMT in H1299 cells,invasion and migration（P < 0.001）. NeoL intervention significantly increased the expression of E-cadherin proteins（P < 0.05）. The TGF-β1-induced increase of ROCK 1 and P-Smad2/3 proteins in H1299 cells also significantly reduced（P < 0.001）. The results of immunofluorescence showed that NeoL inhibited the expression and distribution of ROCK 1 protein in the cytoplasm of the TGF-β1-induced H1299 cells.Conclusion TGF-β1 can induce EMT of H1299 cells. NeoL can effectively inhibit this process possibly by inhibiting the TGF-β1/Smad/RhoA/ROCK pathway.更多还原