健脾消癌方干预结肠癌细胞HCT116来源外泌体中miR-21调控CAFs的活化与糖酵解的研究  

Effect of Jianpi Xiaoai Formula(健脾消癌方) on the Activation and Glycolysis of CAFs Regulated by MiR-21 in HCT116-Derived Exosomes of Colon Cancer Cells

作　　者：罗燕 曾普华[1,2] 蒋益兰 李勇敏[1,2] 曾千 周融融[1,2] 吕元 罗吉[1,2] LUO Yan;ZENG Puhua;JIANG Yilan;LI Yongmin;ZENG Qian;ZHOU Rongrong;LV Yuan;LUO Ji(Hunan Provincial Hospital of Integrated Traditional Chinese and Western Medicine,Changsha 410006;The Affiliated Hospital of Hunan Academy of Traditional Chinese Medicine,Changsha 410006;Graduate School of Hunan University of Chinese Medicine,Changsha 410208)

机构地区：[1]湖南省中西医结合医院,长沙410006 [2]湖南省中医药研究院附属医院,长沙410006 [3]湖南中医药大学研究生院,长沙410208

出　　处：《中药药理与临床》2023年第6期2-7,共6页Pharmacology and Clinics of Chinese Materia Medica

基　　金：国家自然科学基金青年项目(编号:82004312);湖南省自然科学基金面上项目(编号:2021JJ30421);湖南省自然科学基金青年项目(编号:2021JJ40312);湖南省教育厅优秀青年项目(编号:20B443);湖南省卫健委一般项目(编号:20201248、20201066);湖南省中医药管理局青年项目(编号:2021248);长沙市自然科学基金(kq2014204);湖南省科技创新计划临床医疗技术创新引导项目(编号:2021SK51006);湖南省中医药研究院青年项目(编号:201919、202124)。

摘　　要：目的:研究健脾消癌方干预结肠癌细胞HCT116来源外泌体中小分子含氧核糖核酸-21(miR-21)调控肿瘤相关成纤维细胞(Cancer associated fibroblasts,CAFs)的活化与糖酵解的作用机制。方法:采用超高速离心法收集分泌HCT116细胞来源外泌体(Exosomes,EXO);透射电镜鉴定HCT116细胞来源的外泌体形态;纳米颗粒跟踪分析仪检测外泌体的粒径分布范围;Western blot检测外泌体标志性蛋白TSG101、Alix、Calnexin的表达。制备健脾消癌方无外泌体含药鼠血清,采用含药血清干预HCT116细胞,RT-PCR检测miR-21的表达;收集无外泌体胎牛血清、无外泌体鼠血清、健脾消癌方无外泌体含药鼠血清干预后及miR-21 inhibitor转染后HCT116细胞来源的外泌体(HCT116外泌体1μg/mL组、空白鼠血清处理HCT116外泌体1μg/mL组、含药鼠血清处理HCT116外泌体1μg/mL组、miR-21敲除HCT116外泌体1μg/mL组),分别干预人胚肺成纤维细胞HFL-1,Western blot、RT-PCR法分别检测α-平滑肌肌动蛋白(Alpha-smooth muscle actin,α-SMA)、血小板源生长因子受体-A(Platelet-derived growth factor A,PDGFRA)、丙酮酸激酶M2(Pyruvate kinase M2,PKM2)蛋白和mRNA的表达。结果:经透射电镜观察到HCT116细胞上清中提取到的外膜囊泡呈杯托样结构,粒径分析显示其直径主要分布在80 nm~200 nm之间,Western blot法检测到外泌体中标志蛋白TSG101、Alix均有表达;PKH67荧光标记的外泌体可被HFL-1细胞摄取;健脾消癌方可降低HCT116细胞来源miR-21的表达,且呈时间、浓度依赖;与空白对照组比较,HCT116外泌体1μg/mL组、空白鼠血清处理HCT116外泌体1μg/mL组α-SMA、PDGFRA、PKM2蛋白和mRNA相对表达上调(P<0.05或P<0.01);与空白鼠血清处理HCT116外泌体1μg/mL组比较,健脾消癌方含药鼠血清处理HCT116外泌体1μg/mL组、miR-21敲除HCT116外泌体1μg/mL组α-SMA、PDGFRA、PKM2蛋白和mRNA相对表达下调(P<0.05或P<0.01)。结论:HCT116细胞来源外泌体中miR-21可诱�Objective:To investigate the mechanism of Jianpi Xiaoai Formula(健脾消癌方) in intervening in the activation and glycolysis of cancer-associated fibroblasts(CAFs) regulated by small molecule microRNA-21(miR-21) in exosomes derived from HCT116 colon cancer cells.Methods:Exosomes secreted by HCT116 cells were collected using ultracentrifugation.Transmission electron microscopy was used to identify the morphology of HCT116-derived exosomes.Nanoparticle tracking analysis was performed to determine the size distribution of the exosomes.Western blot was conducted to detect the expression of exosomal marker proteins TSG101,Alix,and calnexin.Drug-containing serum was prepared with Jianpi Xiaoai Formula without exosomes,and HCT116 cells were treated with the drug-containing serum.The expression of miR-21 was measured by RT-PCR.Exosomes derived from HCT116 cells after treatment with exosome-free fetal bovine serum,exosome-free mouse serum,drug-containing exosome-free mouse serum,and transfection with miR-21 inhibitor(HCT116 exosomes 1.0 μg/mL group,blank mouse serum-treated HCT116 exosomes 1.0 μg/mL group,drug-containing mouse serum-treated HCT116 exosomes 1.0 μg/mL group,and miR-21 knockdown HCT116 exosomes 1.0 μg/mL group) were collected and used to intervene in human embryonic lung fibroblasts(HFL-1).Western blot and RT-PCR were performed to detect the protein and mRNA expression of alpha-smooth muscle actin(α-SMA),platelet-derived growth factor receptor-A(PDGFRA),and pyruvate kinase M2(PKM2).Results:Transmission electron microscopy revealed cup-shaped structures of exosome-like vesicles extracted from the supernatant of HCT116 cells.Particle size analysis showed that their diameter was mainly distributed between 80 nm and 200 nm.Western blot detected the expression of the exosomal marker proteins TSG101 and Alix in the exosomes.PKH67-labeled exosomes could be taken up by HFL-1 cells.Jianpi Xiaoai Formula reduced the expression of miR-21 derived from HCT116 cells in a time-and concentration-dependent manner.Co

关 键 词：健脾消癌方 结肠癌 外泌体 小分子含氧核糖核酸-21 肿瘤相关成纤维细胞 糖酵解 

分 类 号：R285[医药卫生—中药学]