半夏泻心汤含药血清对胃癌来源外泌体诱发腹膜间皮细胞上皮间质转化的影响  

Effect of Banxia Xiexintang-containing Serum on Epithelial-mesenchymal Transition of Peritoneal Mesothelial Cells Induced by Gastric Cancer-derived Exosomes

作　　者：董俊刚[1] 刘喜平[1] 李沛清[1] 王庆苗[1] 朱中博 何玲[1] 李永玉 王宇[1] DONG Jun-gang;LIU Xi-ping;LI Pei-qing;WANG Qing-miao;ZHU Zhong-bo;HE Ling;LI Yong-yu;WANG Yu(Gansu University of Chinese Medicine,Lanzhou 730000,China)

机构地区：[1]甘肃中医药大学,兰州730000

出　　处：《中国实验方剂学杂志》2021年第21期19-26,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81860813,81860782)。

摘　　要：目的:探讨半夏泻心汤对胃癌来源外泌体(Exo)诱发人腹膜间皮细胞(HMrSV5)上皮间质转化(EMT)的影响。方法:制备半夏泻心汤含药血清,提取人胃癌NCI-N87细胞外泌体(NCI-N87-Exo),采用透射电镜及蛋白免疫印迹法(Western blot)进行鉴定,并用细胞膜红色荧光探针(Dil)标记。实验分为空白组、模型组、半夏泻心汤低、中、高剂量组(13.5,27,54 g·kg^(-1))。空白组将HMrSV5细胞单独培养;模型组在HMrSV5中加入的NCI-N87-Exo(100 mg·L^(-1));半夏泻心汤低、中、高剂量组在模型组的基础上分别加入10%的半夏泻心汤低、中、高剂量含药血清。激光共聚焦显微镜观察24,48,72 h时HMrSV5细胞对NCI-N87-Exo的摄取情况,72 h后观察HMrSV5细胞形态学的变化,Western blot检测HMrSV5细胞中E-钙黏附蛋白(E-cadherin),细胞角蛋白19(CK19),α-平滑肌肌动蛋白(α-SMA)和弹性蛋白(Elastin)及转化生长因子-β_(1)( TGF-β_(1)),Smad2/3,磷酸化(p)-Smad2/3蛋白表达。结果:透射电镜观察到NCI-N87-Exo呈椭圆或碟状的囊泡结构,粒径介于40~80 nm,高表达Exo标志蛋白CD9,CD63,不表达钙网蛋白(Calreticulin),证实为NCI-N87-Exo。共培养24,48,72 h后,荧显微镜下均可观察到NCI-N87-Exo被HMrSV5细胞摄取,与时间呈正相关。与空白组比较,半夏泻心汤可显著抑制HMrSV5细胞对NCIN87-Exo的摄取,以半夏泻心汤中、高剂量组含药血清最为明显(P<0.05,P<0.01)。半夏泻心汤含药血清干预后HMrSV5细胞排列较为紧密,细胞间隙明显缩小,以半夏泻心汤含药血清大剂量组最为明显。Western blot显示,与模型组比较,半夏泻心汤中、高剂量组含药血清干预后HMrSV5细胞E-cadherin,CK19蛋白表达显著升高(P<0.01),α-SMA,Elastin蛋白表达显著降低(P<0.01),半夏泻心汤低、中、高剂量组TGF-β_(1),p-Smad2/3蛋白表达明显降低(P<0.05,P<0.01),Smad2/3无明显变化。结论:NCI-N87-Exo能够被HMrSV5细胞摄取并诱发HMrSV5细胞EMT,半夏泻心汤能够阻抑HObjective:To investigate the effect of Banxia Xiexintang on the epithelial-mesenchymal transition(EMT)of human peritoneal mesothelial cell line(HMrSV5)induced by gastric cancer-derived exosomes(Exo).Method:Banxia Xiexintang-containing serum was prepared and the human gastric cancer NCI-N87-derived exosomes(NCI-N87-Exo)were extracted,followed by their identification by transmission electron microscopy and Western blotting and labeling with 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate(Dil).The cells were divided into the blank group,model group,and low-,medium-,and high-dose(13.5,27,54 g·kg^(-1))Banxia Xiexintang groups.HMrSV5 cells in the blank group were cultured alone,the ones in the model group with 100 mg·L^(-1) NCI-N87-Exo,and those in the low-,medium-,and high-dose Banxia Xiexintang groups with 100 mg·L^(-1) NCI-N87-Exo plus low-,medium-,and high-dose 10%Banxia Xiexintang-containing serum,respectively.Confocal laser microscope was used to observe the uptake of NCI-N87-Exo by HMrSV5 cells at 24 h,48 h and 72 h.Seventy-two hours later,the morphological changes in HMrSV5 cells were observed.The protein expression levels of E-cadherin,cytokeratin 19(CK19),α-smooth muscle actin(α-SMA),elastin,and transforming growth factor-β_(1)(TGF-β_(1)),Smad2/3,and p-Smad2/3 were assayed by Western blot.Result:It was observed under the transmission electron microscope that NCI-N87-Exo showed an oval or dish-shaped vesicle structure with a particle size ranging from 40 to 80 nm.Exo marker proteins CD9 and CD63 were highly expressed while calreticulin was not expressed,implying that the NCI-N87-Exo was confirmed.After 24 h,48 h,72 h of co-culture,it was observed under the fluorescence microscope that NCI-N87-Exo were taken up by HMrSV5 cells,which was positively correlated with time.Compared with the blank group,Banxia Xiexintang significantly inhibited the uptake of NCI-N87-Exo by HMrSV5 cells,with better effect noticed in the middle-and high-dose Banxia Xiexintang groups(P<0.05,P<0.01).After intervent

关 键 词：胃癌 外泌体 人腹膜间皮细胞 上皮间质转化 半夏泻心汤 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]