半夏泻心汤对正常骨髓间充质干细胞摄取胃癌细胞来源外泌体的干预作用  

Intervention effect of Banxia Xiexin Decoction on the uptake of gastric cancer exosomes by BMSCs in gastric cancer microenvironment

作　　者：岳娟 刘喜平 崔国宁 朱中博 王庆苗 李沛清 王宇 董俊刚 YUE Juan;LIU Xi-ping;CUI Guo-ning;ZHU Zhong-bo;WANG Qing-miao;LI Pei-qing;WANG Yu;DONG Jun-gang(Basic Medical College,Gansu University of Chinese Medicine,Lanzhou 730000,China;Clinical College of Traditional Chinese Medicine,Gansu University of Chinese Medicine,Lanzhou 730000,China)

机构地区：[1]甘肃中医药大学基础医学院,兰州730000 [2]甘肃中医药大学中医临床学院,兰州730000

出　　处：《中华中医药杂志》2022年第4期1908-1913,共6页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：国家自然科学基金项目(No.81860813,No.81860782)。

摘　　要：目的:观察胃癌细胞来源外泌体能否靶向骨髓间充质干细胞(BMSCs)并被摄取及半夏泻心汤的干预效应。方法:分离大鼠BMSCs,倒置显微镜及免疫荧光染色法对其进行鉴定;制备半夏泻心汤含药血清,四甲基偶氮唑盐(MTT)法检测半夏泻心汤含药血清对BMSCs存活率的影响,筛选最佳实验浓度;提取人胃癌BGC-823细胞来源的外泌体,采用透射电镜观察及Western Blot法对其进行鉴定,并用荧光染料Dil标记,分为空白组,模型组,中药高、中、低剂量组及阳性药组,各组上室均加入BMSCs悬液,下室均加入无外泌体血清的培养基。模型组,中药高、中、低剂量组及阳性药组下室加入胃癌外泌体。模型组及空白组上室加入含10%正常对照血清,中药高、中、低剂量组上室内相应加入含10%中药高、中、低剂量含药血清;阳性药组上室加入含10μmoL/L氯丙嗪的10%正常对照血清。24、48、72 h时用激光共聚焦显微镜观察各组BMSCs对胃癌细胞来源外泌体的摄取情况。结果:MTT法筛选半夏泻心汤含药血清的最佳实验浓度为10%。免疫荧光结果显示:共培养24 h内各组未发现BMSCs内红色荧光标记,48 h后发现BMSCs内红色荧光标记,72 h时BMSCs内含大量红色荧光标记,红色荧光标记的MOD值显示:48、72 h与空白组同期比较,模型组MOD值显著升高(P<0.05);与模型组比较,中药中、高剂量组及阳性药组显著降低(P<0.05);与本组48 h比较,72 h时各组BMSCs红色荧光标记MOD值显著升高(P<0.05)。结论:胃癌细胞来源外泌体可靶向BMSCs并被摄取,提示BMSCs可能是胃癌细胞来源外泌体的靶细胞之一,半夏泻心汤能够抑制BMSCs对胃癌细胞来源外泌体的摄取。Objective:To observe whether gastric cancer exosomes can target and be ingested by bone marrow mesenchymal stem cells(BMSCs)in gastric cancer microenvironment and the intervention effect of Banxia Xiexin Decoction.Methods:The serum containing Banxia Xiexin Decoction was prepared;rat BMSCs were isolated and identified by inverted microscope and immunofluorescence staining;the effects of Banxia Xiexin Decoction containing serum on the survival rate of BMSCs was detected by MTT method,the exosomes derived from BGC-823 human gastric cancer cells were extracted and identified by transmission electron microscopy and Western Blot,and labeled with fluorescent dye Dil,they were divided into blank group,model group,high,medium and low dose groups and positive group,BMSCs suspension was added to the upper chamber of each group,and the culture medium containing serum without exosomes was added into the lower chamber.In the model group,high,medium and low dose groups of Chinese medicine and positive group,gastric cancer exosomes were added into the lower chamber.The model group and blank group were added with 10%normal control serum.High,medium and low dose serum containing 10%Chinese medicine was added in the upper room of high,medium and low dose groups.The positive group was added with 10%control serum containing 10μmol/L chlorpromazine.The uptake of gastric cancer exosomes by BMSCs in each group was observed by laser confocal microscope at 24 h,48 h and 72 h.Results:The optimal concentration of serum containing Banxia Xiexin Decoction was 10%by MTT method.The results of immunofluorescence showed that:no red fluorescent markers were found in BMSCs within 24 h,but red fluorescent markers were found in BMSCs after 48 h.At 72 h,BMSCs contained a large number of red fluorescent markers.At 48 h and 72 h,The MOD value of red fluorescent labeling showed that compared with the blank group,the MOD value of model group was significantly increased(P<0.05).Compared with the model group,the MOD value of midium and high dose groups and

关 键 词：胃癌 半夏泻心汤 骨髓间充质干细胞 外泌体 

分 类 号：R285[医药卫生—中药学]