健脾消癌方对结肠癌外泌体诱导HFL1活化成CAFs的影响  

Effect of Jianpi Xiaoai Prescription on Activation of HFL1 into CAFs Induced by Exosomes in Colon Cancer

作　　者：刘佳琴 罗吉[2] 李勇敏[2] 杨晓 谭小宁[2] 罗燕[2] 吕元[2] 蒋益兰 LIU Jia-qin;LUO Ji;LI Yong-min;YANG Xiao;TAN Xiao-ning;LUO Yan;LYU Yuan;JIANG Yi-lan(Graduate School,Hunan University of Chinese Medicine,Changsha 410208,China;Hunan Key Laboratory of Chinese Medicine Oncology,Innovative Platform of Antitumor Chinese Drugs,Hunan Academy of Traditional Chinese Medicine Affiliated Hospital,Changsha 410006,China;The First Hospital of Hunan University of Chinese Medicine,Changsha 410007,China)

机构地区：[1]湖南中医药大学研究生院,长沙410208 [2]湖南省中医药研究院附属医院,中医肿瘤学湖南省重点实验室,抗肿瘤中药创新平台,长沙410006 [3]湖南中医药大学第一附属医院,长沙410007

出　　处：《中国实验方剂学杂志》2020年第22期40-46,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81774287);中央引导地方科技发展专项(2017CT5029);湖南省科技创新计划项目(2017SK50403);湖南中医药大学研究生培养质量工程项目(2019CX10)。

摘　　要：目的:探讨健脾消癌方对人结肠癌细胞(HCT116)来源外泌体诱导正常人胚肺成纤维细胞(HFL1)向肿瘤相关成纤维细胞(CAFs)活化的影响。方法:以13.1 g·kg-1的健脾消癌方灌胃SD大鼠以制备含药血清,采取超高速离心法分别提取含10%无外泌体血清培养的HCT116细胞外泌体及含20%健脾消癌方含药血清培养的HCT116细胞外泌体,纳米颗粒跟踪分析仪(Zetaview)检测外泌体的粒径分布,蛋白免疫印迹法(Western blot)鉴定外泌体的标志蛋白凋亡转接基因2互作蛋白X(Alix),热休克蛋白70(HSP70),肿瘤易感基因101蛋白(TSG101),荧光显微镜观察HFL1对细胞膜染色试剂盒(PKH67)标记的外泌体的摄取情况;将HFL1细胞分6组:空白组,转化生长因子-β1(TGF-β1)组,TGF-β1联合HCT116外泌体2 mg·L-1组,TGF-β1联合HCT116外泌体4 mg·L-1组,TGF-β1联合健脾消癌方外泌体2 mg·L-1组,TGF-β1联合健脾消癌方外泌体4 mg·L-1组,均干预48 h,Western blot和实时荧光定量聚合酶链式反应(Real-time PCR)分别测定α-平滑肌肌动蛋白(α-SMA)蛋白和mRNA的表达水平。结果:Zetaview检测粒径分布主要集中在50~100 nm,结合标志蛋白Alix,HSP70和TSG101的表达证实其为外泌体,HFL1细胞与外泌体共孵育后,荧光显微镜下可看到大量外泌体被HFL1细胞所摄取;与空白组比较,TGF-β1组和TGF-β1联合HCT116外泌体组的α-SMA蛋白及mRNA表达水平升高(P<0.05);与TGF-β1联合HCT116外泌体组比较,TGF-β1联合健脾消癌方外泌体组的α-SMA蛋白及mRNA水平下降(P<0.01)。结论:人结肠癌细胞外泌体联合TGF-β1可诱导HFL1活化成CAFs,健脾消癌方可通过影响α-SMA的表达水平而降低HFL1的活化能力从而达到拮抗结肠癌肺转移的作用。Objective: To observe the effect of Jianpi Xiaoai prescription on the activation of normal human embryonic lung fibroblasts(HFL1)into tumor-associated fibroblasts(CAFs)induced by human colon cancer cells(HCT116)derived exosomes. Method: SD rats were gavaged with 13.1 g·kg-1 of Jianpi Xiaoai prescription to prepare drug-containing serum,and HCT116 cell exosomes-containing 10% exosomes-free serum and 20% Jianpi Xiaoai prescription drug serum were isolated by ultra-high speed centrifugation. The particle size distribution of exosomes were detected by Nanoparticle tracking analyzer(Zetaview),and the exosomes’ marker proteins apoptotic transfer gene 2 interaction protein X(Alix),heat shock protein 70(HSP70),and tumorsusceptibility gene 101(TSG101)were identified by Western blot,and the uptake of exosomes labeled with cell membrane staining kit(PKH67)by HFL1 was observed by fluorescence microscope. HFL1 cells were divided into six groups:the blank group,the transforming growth factor-β1(TGF-β1)group,the TGF-β1 combined with HCT116 exosomes of 2 mg·L-1 group,the TGF-β1 combined with HCT116 exosomes of 4 mg·L-1 group,the TGF-β1 combined with Jianpi Xiaoai prescription exosomes of 2 mg·L-1 group,and the TGF-β1 combined with Jianpi Xiaoai prescription exosomes of 4 mg·L-1 group,and all groups were cultivated for 48 h. Western blot and Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)were used to determine the protein and mRNA expressions of α-smooth muscle actin(α-SMA). Result: The particle size distribution detected by Zetaview was mainly between 50-100 nm, and the exosomes were verified based on the expressions of marker proteins Alix,HSP70 and TSG101. After co-incubation of HFL1 cells with exosomes,a large number of exosomes were absorbed by HFL1 cells under fluorescence microscope. Compared with the blank control group,the protein and mRNA expressions of α-SMA in the TGF-β1 group and TGF-β1 combined with HCT116 exosome groups were increased(P<0.01). Compared with the TGF-β1

关 键 词：结肠癌 外泌体 正常人胚肺成纤维细胞 肿瘤相关成纤维细胞 Α-平滑肌肌动蛋白 健脾消癌方 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]