波形蛋白与肝细胞癌的关系及健脾益气方的干预作用  

Correlation Between Vimentin and Hepatocellular Carcinoma and Intervention Effect of Jianpi Yiqi Prescription

作　　者：王超 音金萍 蒋筱 卓少元 WANG Chao;YIN Jinping;JIANG Xiao;ZHUO Shaoyuan(School of Basic Medical Science,Guangxi University of Chinese Medicine,Nanning 530200,China;Guangxi Key Laboratory of Translational Medicine for Treating High-incidence Infectious Diseases with Integrative Medicine,Guangxi University of Chinese Medicine,Nanning 530200,China)

机构地区：[1]广西中医药大学基础医学院,南宁530200 [2]广西中医药大学广西高发传染病中西医结合转化医学重点实验室,南宁530200

出　　处：《中国实验方剂学杂志》2023年第10期123-133,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金地区科学基金项目(81860824);广西中医药大学“桂派杏林拔尖人才”项目(2022C004);广西中医药大学2018年博士科研启动基金项目(04B1803616)。

摘　　要：目的:研究波形蛋白(Vimentin)与肝细胞癌(HCC)的关系及健脾益气方通过Vimentin调控肝癌的分子机制。方法:利用基于肿瘤基因组图谱计划(TCGA)和临床蛋白质组肿瘤分析协作组(CPTAC)数据库在线分析平台、STRING数据库及Cytoscape软件分析Vimentin与肝细胞癌的关系。随机将SD大鼠分为正常组、模型组、健脾益气方低、中、高剂量组(5.25、10.5、21 g·kg^(-1))、信号转导蛋白及转录激活子3(STAT3)抑制组(C_(18)8-9,4.5 mg·kg^(-1))和糖蛋白130(gp130)抑制组(SC144,4.5 mg·kg^(-1)),每组10只。除正常组外,其余各组大鼠按照70 mg·kg^(-1)体质量腹腔注射二乙基亚硝胺(DEN)复制肝癌模型。造模成功后,正常组和模型组大鼠生理盐水灌胃,健脾益气方低、中、高剂量组分别给予对应剂量中药灌胃,2个抑制组分别腹腔注射对应抑制剂,每日1次,连续4周。末次处置后,采用实时荧光定量聚合酶链式反应(Real-time PCR)法检测大鼠肝组织中Vimentin m RNA水平;比色法检测肝组织胱天蛋白酶-3(Caspase-3)酶活性;蛋白免疫印迹法(Western blot)检测肝组织中Rho相关含卷曲螺旋蛋白激酶1(ROCK1)、Rho相关含卷曲螺旋蛋白激酶2(ROCK2)、aurora激酶B(AURKB)、锌指蛋白148(ZNF148或者ZBP-89)、STAT3、磷酸化STAT3(p-STAT3)、总Vimentin、磷酸化Vimentin(p-Vimentin)及肝组织细胞核中Vimentin蛋白含量;酶联免疫吸附测定法(ELISA)检测大鼠血清中Vimentin的含量。结果:VIM m RNA在肝癌患者不同病理分期(Ⅰ~Ⅳ期)、不同病理分级(G_(1)~G_(3)级)、无区域淋巴结转移患者(N0)和肝癌不同亚型患者组织中表达水平均显著升高(P<0.01);Vimentin蛋白水平在肝癌组织中显著降低(P<0.01);VIM基因有4处突变可以导致Vimentin蛋白一级结构的改变,这些遗传突变可降低患者无病生存期(P<0.01);VIM m RNA的表达水平与HCC纯度呈负相关(P<0.01),而与微环境中肿瘤相关成纤维细胞、M2型巨噬细胞和髓系树突�Objective:To study the correlation between Vimentin and hepatocellular carcinoma(HCC)and the mechanism of Jianpi Yiqi prescription against HCC through Vimentin.Method:Correlation between Vimentin and HCC was analyzed based on the cancer genome atlas(TCGA),clinical proteomic tumor analysis consortium(CPTAC),STRING,and Cytoscape.SD rats were randomized into normal group(normal saline,ig,once/day,4 weeks),model group(normal saline,ig,once/day,4 weeks),low-dose,medium-dose,and high-dose(5.25,10.5,21 g·kg^(-1),ig,once/day,4 weeks)JianpiYiqi prescription groups,signal transducer and activator of transcription 3(STAT3)inhibition group(C_(18)8-9,4.5 mg·kg^(-1),ip,once/day,4 weeks),and glycoprotein 130(gp130)inhibition group(SC144,4.5 mg·kg^(-1),ip,once/day,4 weeks),10 rats in each group.Diethylnitrosamine(DEN,70 mg·kg^(-1) body weight,ip)was injected in rats except the normal group to induce HCC.After the modeling,administration began.After last administration,Real-time polymerase chain reaction(Real-time PCR)was performed to determine Vimentin mRNA level in rat liver tissue.Caspase-3 activity in liver tissue was detected by colorimetry,and expression of Rho kinase(ROCK)1,ROCK2,aurora kinase B(AURKB),Zinc-finger protein 148(ZNF148)/zinc-binding protein-89(ZBP-89),STAT3,p-STAT3,total Vimentin,and phosphorylated(p)-Vimentin in liver tissue and Vimentin in liver tissue nucleus detected by Western blot.Serum Vimentin concentration was measured by enzyme-linked immunosorbent assay(ELISA).Result:Vimentin mRNA level was high in tissues from HCC patients with different cancer stages(stageⅠ-Ⅳ),different pathological grades(G1-G3),no regional lymph node metastasis(N0),and different subtypes(P<0.01).Vimentin mRNA expression was higher in tissues from patients with lymph node metastasis than in patients without lymph node metastasis and normal samples.Vimentin protein level was decreased in HCC tissues(P<0.01).Vimentin gene has 4 mutations which can induce change in the primary structure of Vimentin protein and patients with

关 键 词：健脾益气方 波形蛋白(Vimentin) 信号转导蛋白及转录激活子3(STAT3) 生物信息学 肝细胞癌 

分 类 号：R285[医药卫生—中药学] R289[医药卫生—中医学] R22R2-031R33