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目的:研究柯里拉京对转移性人肝癌细胞SMMC7721,MHCC97H增殖、迁移和侵袭的影响,并探讨其作用机制。方法:分别通过四甲基偶氮唑蓝(MTT)比色法,划痕实验,transwell实验检测不同浓度的柯里拉京对肝癌细胞SMMC7721,MHCC97H的增殖、迁移和侵袭的作用;通过细胞免疫荧光法观察不同浓度柯里拉京对肝癌细胞SMMC7721,MHCC97H肌动蛋白纤维的分布、丝状和片状伪足形成等的影响;通过蛋白免疫印迹法(Western blot)研究柯里拉京抑制肝癌细胞增殖、迁移、侵袭的作用机制。结果:MTT实验显示,柯里拉京抑制人肝癌细胞SMMC7721,MHCC97H增殖的半数抑制浓度(IC50)分别为388.67,314.42μmol·L~(-1)。划痕实验显示,200μmol·L~(-1)柯里拉京对SMMC7721细胞迁移的抑制作用最强,迁移率为12.16%;120μmol·L~(-1)柯里拉京对MHCC97H细胞迁移的抑制作用最强,迁移率为9.84%。Transwell实验显示,200μmol·L~(-1)柯里拉京对SMMC7721细胞和MHCC97H细胞侵袭的抑制作用最强,侵袭的细胞数分别为(40.44±3.01),(36.69±3.36)个。细胞免疫荧光显示,柯里拉京可使SMCC7771,MHCC97H中F-肌动蛋白(F-actin)骨架发生重塑,浓度增高,细胞边缘的丝状伪足和片状伪足形成逐渐减少、张力纤维数目逐渐增多,这种影响在SMMC7721为200μmol·L~(-1)时最显著,MHCC97H为120μmol·L~(-1)最明显。Western blot显示,柯里拉京能明显下调肝癌细胞SMMC7721和MHCC97H上皮-间充质转分化(EMT)的钙黏附蛋白-N(N-Cad)和波形蛋白(Vimentin)蛋白表达(P<0.05),下调有丝分裂原活化蛋白激酶(MAPK)通路的磷酸化-细胞外信号调节激酶(p-ERK)蛋白表达(P<0.05),上调磷酸化-p38(p-p38)蛋白表达(P<0.05)。结论:柯里拉京能抑制2种人肝癌细胞SMMC7721,MHCC97H的增殖、迁移及侵袭,其作用机制可能是通过抑制EMT通路中的N-Cad,Vimentin,MAPK信号通路p-ERK蛋白表达和上调p-p38蛋白表达,从而调控细胞骨架重塑来实现的。
Objective: To study the effect of corilagin on the proliferation, migration and invasion of human hepatocellular carcinoma cell line SMMC7721 and MHCC97H and to explore its mechanism. Methods: The effects of different concentrations of corilagin on the proliferation, migration and invasion of hepatoma cells SMMC7721 and MHCC97H were detected by MTT assay, scratch assay and transwell assay respectively. The effects of different concentrations of corilagin on the distribution of hepatocellular carcinoma cells SMMC7721 and MHCC97H actin fibers and the formation of filiform and lamellipodia were observed. Corilagin inhibited the proliferation of hepatoma cells by Western blot, Migration, invasion mechanism of action. Results: MTT assay showed that the half-inhibitory concentration (IC50) of corilagin inhibited the proliferation of human hepatocellular carcinoma cells SMMC7721 and MHCC97H respectively 388.67 and 314.42μmol·L -1. Scratch experiments showed that 200μmol·L -1 corilagin had the strongest inhibitory effect on the migration of SMMC7721 cells, with a migration rate of 12.16%. The inhibitory effect of 120μmol·L -1 corilagin on the migration of MHCC97H cells The strongest, the mobility was 9.84%. Transwell experiments showed that 200μmol·L -1 corilagin had the strongest inhibitory effect on invasion of SMMC7721 and MHCC97H cells, and the number of invasive cells was (40.44 ± 3.01) and (36.69 ± 3.36), respectively. Cell immunofluorescence showed that corilagin could remodel the F-actin skeleton in SMCC7771 and MHCC97H, and the concentration of F-actin was increased. The formation of filopodia and lamellipodia at the edge of the cells decreased gradually. The number increased gradually. The effect was the most significant when SMMC7721 was 200μmol·L -1, the most obvious was MHCC97H 120μmol·L -1. Corilagin could obviously down-regulate the expression of N-Cad and vimentin in SMMC7721 and MHCC97H epithelial-mesenchymal transdifferentiation (EMT) cells by Western blot (P <0.05) Down-regulated phosphorylation of p-ERK (P <0.05) and up-regulated phosphorylation of p38 (p-p38) in mitogen-activated protein kinase (MAPK) pathway. CONCLUSION: Corilagin can inhibit the proliferation, migration and invasion of two human hepatocellular carcinoma cell lines SMMC7721 and MHCC97H. The possible mechanism is that Corilagin can inhibit the expression of p-ERK protein and up-regulate the expression of N-Cad, Vimentin and MAPK signaling pathways in EMT pathway -p38 protein expression, thereby regulating the cytoskeletal remodeling to achieve.