CD44阳性人脐带间充质干细胞扰乱大鼠血小板稳态研究  

CD44+ human umbilical cord mesenchymal stem cells disrupt platelet homeostasis in rats

作　　者：杜伟[1] 杨雪[1,2] 安康 梅小利 何白英[5,6] 龙成燕 于冰 黄崇刚 DU Wei;YANG Xue;AN Kang;MEI Xiaoli;HE Baiying;LONG Chengyan;YU Bing;HUANG Chonggang(Chongqing Academy of Chinese Materia Medica,Chongqing Drug Safety Evaluation Center,Chongqing 400065,China;Chongqing College of Traditional Chinese Medicine,Chongqing 402760,China;Shandong Institute of Pharmaceutical Research,Jinan 250062,China;Shandong First Medical University&Shandong Academy of Medical Sciences,Jinan 250062,China;Nan Jiao Hospital of Chongqing City,Chongqing 400055,China;Chongqing Attractiveness&Distinctiveness Health Technology Co.,Ltd.,Chongqing 400054,China)

机构地区：[1]重庆市中药研究院/重庆市药物安全评价中心,重庆400065 [2]重庆中医药学院,重庆402760 [3]山东省药物研究院,山东济南250062 [4]山东第一医科大学(山东省医学科学院),山东济南250062 [5]重庆南郊医院,重庆400055 [6]重庆悦目可爱生命健康科技有限公司,重庆400054

出　　处：《药物评价研究》2023年第1期92-99,共8页Drug Evaluation Research

基　　金：重庆市技术创新与应用发展专项(cstc2020jscx-lyggX0004)。

摘　　要：目的 基于生物信息分析与动物实验探讨人脐带间充质干细胞(hUCMSCs)与机体血小板稳态的关系。方法 运用DisGeNET、GeneCards、STRING 11.5等数据库,分析hUCMSCs诱发血小板减少的靶点及通路。将SD大鼠随机区组分为5组:阴性对照组(0.9%氯化钠注射液)、溶媒对照(80 mg·kg^(-1)白蛋白)组和hUCMSCs高、中、低剂量(1.0×10^(7)、5×10^(6)、2.5×10^(6)个·kg^(-1),分别为临床等效剂量的10.0、5.0、2.5倍)组,每组6只,雌雄各半。每周按10 mL·kg^(-1)尾iv 1次,共4次,与临床拟用方法一致。全自动血液分析仪分析血小板相关的指标:血小板计数(PLT)、血小板压积(PCT)、血小板分布宽度(PDW)、平均血小板体积(MPV);剖取大鼠胸骨,取骨髓,制备骨髓涂片,瑞特-吉姆萨复合染液染色,光学显微镜下计数全片巨核细胞总数、产血小板巨核细胞数;测量脾脏总长度;苏木精-伊红(HE)和过碘酸-雪夫(PAS)染色脾脏作组织病理学检查;实时荧光定量PCR(qRT-PCR)分析肝脏促血小板生成素(TPO)、血小板生成素受体(Mpl)、Krüppel样因子1(Klf1)、Friend白血病整合素1(FLI1)、GATA结合蛋白1(GATA1)mRNA变化。结果 hUCMSCs与血小板减少相关的靶点有209个,作用于158条信号通路,与血小板生成最密切的通路为造血细胞谱通路,包含了20个靶点。以平均连接度筛选出14个核心靶点:IL6、TNFRSF11A、CD34、KIT、IL4、CSF2、CSF3、IL3、IL2RA、TPO、EPO、TFRC、CD44、IL11。CD44既是核心靶点又是hUCMSCs阳性表面标志物。与阴性对照、溶媒对照组比较,hUCMSCs高、中、低剂量组PLT、PCT显著降低(P<0.05),PDW、MPV差异无统计学意义;hUCMSCs组全片巨核细胞总数、产血小板巨核细胞数差异无统计学意义;hUCMSCs高、中剂量组脾脏长度显著增加(P<0.05);PAS染色见hUCMSCs高、中剂量组脾脏血小板储存增加;hUCMSCs高、中、低剂量组肝脏组织TPO、Mpl、KLF1 mRNA表达显著增加(P<0.05),FLI1、GATA1 mRNA表达变�Objective To explore the relationship between human umbilical cord mesenchymal stem cells(hUCMSCs) and platelet homeostasis based on biological information analysis and animal experiments. Methods DisGeNET, GeneCards and STRING 11.5databases were used to analyze the targets and pathways of hUCMSCs induced thrombocytopenia. SD rats were randomly divided into five groups: Negative control group(0.9% sodium chloride injection), solvent control group(80 mg·kg^(-1)albumin) and hUCMSCs high, medium and low dose groups(1.0 × 10^(7), 5 × 10^(6), 2.5 × 10^(6)·kg^(-1), which were 2.5, 5.0 and 10.0 times of the clinical equivalent dose, respectively) with 6 in each group, half male and half female. 10 mL·kg^(-1)tail iv was applied once a week for four times, which was consistent with the proposed clinical method. Platelet count(PLT), platelet pressure(PCT), platelet distribution width(PDW), and mean platelet volume(MPV) were analyzed by automatic hematology analyzer. The rat sternum was dissected and bone marrow was extracted. Bone marrow smears were prepared and stained with Reiter-Giemsa composite dye solution. The total number of megakaryocytes and platelet-producing megakaryocytes in the whole film were counted under the optical microscope. Total spleen length was measured. The spleen was stained with HE and PAS for histopathological examination. Realtime fluorescence quantitative PCR(qRT-PCR) was used to analyze the mRNA changes of liver thrombopoietin(TPO),thrombopoietin receptor(Mpl), Kruppel-like factor 1(Klf1), Friend leukemia integrin 1(FLI1) and GATA binding protein 1(GATA1). Results hUCMSCs had 209 targets related to thrombocytopenia, acting on 158 signal pathways. The most closely related pathway to platelet formation was the hematopoietic cell lineage, which contains 20 targets. Based on the average connectivity, 14core targets were screened: IL6, TNFRSF11A, CD34, KIT, IL4, CSF2, CSF3, IL3, IL2RA, TPO, EPO, TFRC, CD44 and IL11.CD44 was both a core target and a positive surface marker of hUCMSCs used in t

关 键 词：人脐带间充质干细胞 血小板减少症 网络药理学 表面标志物 CD44 脾脏 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]