健脾化瘀方体外通过外泌体影响肝癌细胞的迁移、侵袭及上皮间质转化

Jianpi Huayu Decoction Inhibits Migration,Invasion and EMT of Hepatoma Carcinoma Cell through Regulation of Exosome

目的探讨健脾化瘀方(JHD)通过外泌体对肝癌细胞迁移、侵袭及上皮间质转化(EMT)的影响。方法利用转化生长因子β1(TGF-β1)刺激MHCC97H细胞,构建肝癌细胞EMT模型。根据处理条件,将细胞分为4组,(1)对照组:不进行干预;(2)模型组:TGF-β1(10 ng·mL^(-1));(3)JHD低浓度组:TGF-β1(10 ng·mL^(-1))+健脾化瘀方(2 mg·mL^(-1));(4)JHD高浓度组:TGF-β1(10 ng·mL^(-1))+健脾化瘀方(4 mg·mL^(-1))。干预48 h后,采用超速离心法提取各组细胞培养上清液的外泌体,通过透射电镜(TEM)、纳米颗粒追踪分析(NTA)、Western Blot法检测外泌体标志蛋白(CD63、CD81、HsP70、CD9),以及外泌体内吞实验对外泌体进行鉴定。将提取的4组外泌体分别与MHCC97H细胞共培养24 h/48 h后,分离MHCC97H细胞,进行划细胞划痕实验、Transwell细胞侵袭实验及Western Blot法检测EMT相关蛋白(E-cadherin、N-cadherin、Vimentin)。结果透射电镜下观察到外泌体的形态呈囊泡状,分布不一,有单个分布,也有聚集成组,背景清晰,无明显污染物,各组外泌体形态相近,无明显差异,直径在30~140 nm之间;纳米颗粒追踪分析仪检测到4组外泌体粒径的峰值在130~150 nm之间,与电镜观察结果基本一致;各组均可检测到外泌体标志蛋白CD63、CD81、HsP70、CD9表达。被PKH26标记的外泌体可被MHCC97H细胞重新吸收内化。与对照组比较,模型组的MHCC97H细胞迁移能力(24、48 h)及侵袭能力均显著增强(P<0.01),E-cadherin蛋白表达显著下调(P<0.01),Vimentin、N-cadherin蛋白表达显著上调(P<0.01)。与模型组比较,JHD高、低浓度组的MHCC97H细胞迁移能力(24、48 h)及侵袭能力显著减弱(P<0.01),E-cadherin蛋白表达显著上调(P<0.01),Vimentin、N-cadherin蛋白表达显著下调(P<0.01)。结论健脾化瘀方体外可通过外泌体抑制MHCC97H细胞的迁移、侵袭及上皮间质转化。

Objective To study the effects of Jianpi Huayu decoction(JHD)on migration,invasion and epithelialmesenchymal transition(EMT)of liver cancer cells by regulation of exosome.Methods The liver cancer cells of MHCC97 H strain were stimulated with TGF-β1 to establish the EMT model.The MHCC97 H cells were cultured in vitro,and divided into four groups according to the stimulations,including Control group cells( no intervention),Model group cells(TGF-β1 group,10 ng·mL^(-1)),JHD low dose group(TGF-β1 10 ng·mL^(-1)+ JHD 2 mg·mL^(-1)),and JHD high dose group(TGF-β1 10 ng · mL^(-1)+ JHD 4 mg · mL^(-1)).After treatment for 48 hours,cell culture supernatants were collected and exosomes were isolated by ultracentrifugation.The exosomes were then identified using transmission electron microscope(TEM),nanoparticle tracking analysis(NTA).Western Blot was used to detect the marker proteins of exosomes(CD63,CD81,HsP70,and CD9);and were identified by endocytosis experiment.The exosomes from four groups isolated as above were respectively cocultured with MHCC97 H cells for48 hours,then the MHCC97 H cells were collected for wound healing assay,transwell assay,and Western Blot was used to detect EMT related proteins(E-cadherin,N-cadherin,and Vimentin).Results TEM observation showed that,the exosomes extracted from different groups were vesicles of different types,similar in morphology,some were singly distributed,and some were gathered together,with a diameter of 30-140 nm.The background was clear without obvious pollutants.NTA revealed the peak values of exosomes from four groups were between 130-150 nm,almost the same with those observed under TEM.All extracted nanoparticles expressed CD63,CD81,HsP70 and CD9,which were further confirmed that the isolated nanoparticles were exosomes.Exosomes labeled with PKH26 can be absorbed and internalized by MHCC97 H cells.Compared with the Control group,the cell migration(24 h,48 h)and invasion abilities of Model group cells were significantly increased(P<0.01);expression of E-cadherin was significantly downregulated(P<0.01);the expression levels of Vimentin and N-cadherin were upregulated obviously(P<0.01).Compared with Model group,the vertically-migrating(24 h,48 h)and invasion abilities of JHD low dose group and JHD high dose group were significantly reduced(P<0.01).The expression levels of Ecadherin in JHD low dose group and JHD high dose group were higher(P<0.01);the expression levels of Vimentin and N-cadherin were lower than those in Model group(P<0.01).Conclusion Jianpi Huayu decoction inhibits migration,invasion and EMT of MHCC97 H cells possibly through regulation of exosomes.