肝星状细胞通过胱硫醚γ-裂解酶/硫化氢(CSE/H2S)调控肝细胞癌细胞凋亡的作用及其机制  

Role and mechanism of hepatic stellate cells in regulating the apoptosis of hepatocellular carcinoma cells through cystathionineγ-lyase/hydrogen sulfide

作　　者：尚宏伟[1] 马亚楠 路欣[1] 吕翎娜 丁惠国 SHANG Hongwei;MA Yanan;LU Xin;LYU Lingna;DING Huiguo(School of Basic Medical Sciences,Capital Medical University,Beijing 100069,China;Department of Hepatology and Gastroenterology,Beijing YouAn Hospital,Capital Medical University,Beijing 100069,China;Oncology Center,Shanxi Bethune Hospital,Taiyuan 030000,China)

机构地区：[1]首都医科大学基础医学院,北京100069 [2]首都医科大学附属北京佑安医院肝病消化中心,北京100069 [3]山西白求恩医院肿瘤中心,太原030000

出　　处：《临床肝胆病杂志》2024年第11期2238-2245,共8页Journal of Clinical Hepatology

基　　金：中德研究小组项目(GZ1517)。

摘　　要：目的肝星状细胞(HSC)和硫化氢(H2S)信号分子作为肝细胞癌(HCC)微环境中重要的组分,参与调控HCC的发生发展等多种生物学进程。本研究通过HSC与肝癌细胞系共培养,探讨HSC通过分泌H2S参与调控肝癌细胞凋亡的作用及其机制。方法以HSC细胞系(LX-2)及肝癌细胞系(Hep G2、PLC/PRF/5)为研究对象。RT-q PCR和Western Blot(WB)法检测LX-2中H2S关键合成酶——胱硫醚γ-裂解酶(CSE)m RNA及表达水平;ELISA测定上清液LX-2产生的H2S浓度;二代测序、细胞免疫荧光、染色质免疫沉淀(Ch IP)及WB检测H2S(内源性和外源性)作用Hep G2和PLC/PRF/5细胞后,JNK/Jun B-TNFSF14信号通路基因、结合位点及相关蛋白。Transwell小室将LX-2分别与Hep G2和PLC/PRF/5共培养,CCK-8和流式细胞术检测肝癌细胞的细胞活力、凋亡,WB测定H2S-TNFSF14信号通路相关蛋白。所有细胞实验均重复3次。计量资料两组间比较采用成组t检验;多组间比较采用单因素方差分析或重复测量方差分析,进一步两两比较采用Dunnett-t检验。结果LX-2主要通过CSE合成H2S,LX-2培养上清液中H2S浓度随着时间延长逐渐增加[(22.89±0.08)pg/m L vs(28.29±0.15)pg/m L vs(36.19±1.90)pg/m L,F=79.63,P<0.05]。LX-2中CSE m RNA水平显著高于CBS m RNA和MPST m RNA(1.008±0.13 vs 0.320±0.014 vs 0.05±0.02,F=80.84,P<0.05)。当CSE被炔丙基甘氨酸(PPG)抑制之后,随着PPG浓度增加,H2S浓度下降(P<0.05)。LX-2分别与Hep G2、PLC/PRF/5共培养,随着培养时间延长,Hep G2(87.48%±0.82%vs70.48%±0.641%vs 52.89%±0.57%vs 45.20%±0.69%,F=1517.13,P<0.001)和PLC/PRF/5(92.41%±0.48%vs 74.10%±0.73%vs 53.70%±0.60%vs 44.00%±0.27%,F=2626.21,P<0.001)细胞活力降低;凋亡增加(Hep G2:12.88%±0.64%vs15.5%±0.16%vs 18.43%±0.37%vs 13.01%±0.58%,F=142.15,P<0.001;PLC/PRF/5:8.51±0.05 vs 12.80±0.33 vs 15.59±0.21 vs 10.72±0.30,F=676.40,P<0.001),第3天最显著。二代测序显示,内源性H2S(LX-2产生)和Na HS(外源性H2S供体)参与调控Hep G2中多�Objective As important components in the microenvironment of hepatocellular carcinoma(HCC),hepatic stellate cells(HSCs)and hydrogen sulfide(H2S)participate in various biological processes that regulate the development and progression of HCC.Through the co-culture of HSCs and HCC cells,this article aims to investigate the role and mechanism of HSCs in regulating the apoptosis of HCC cells by secreting H2S.Methods The HSC cell line(LX-2)and HCC cell lines(HepG2 and PLC/PRF/5)were used for experiment.RT-qPCR and Western Blot(WB)were used to measure the mRNA and protein expression levels of cystathionineγ-lyase(CSE),a key synthase for H2S;ELISA was used to measure the concentration of H2S in supernatant;next-generation sequencing,cell immunofluorescence assay,chromatin immunoprecipitation(ChIP),and WB were used to measure the JNK/JunB-TNFSF14 signaling pathway genes,binding sites,and related proteins after HepG2 cells were treated by H2S.LX-2 cells were co-cultured with HepG2 or PLC/PRF/5 cells in a Transwell chamber;CCK-8 assay and flow cytometry were used to measure the viability and apoptosis of HCC cells,and WB was used to measure the H2S-TNFSF14 signaling pathway-related proteins.All cell experiments were repeated three times.The independent-samples t test was used for comparison of continuous data between two groups;a one-way analysis of variance or the analysis of variance with repeated measures was used for comparison between multiple groups,and the Dunnett-t test was used for further comparison between two groups.Results LX-2 cells synthesized H2S mainly through CSE,and the concentration of H2S in supernatant of LX-2 cells gradually increased over time(22.89±0.08 pg/mL vs 28.29±0.15 pg/mL vs 36.19±1.90 pg/mL,F=79.63,P<0.05).In LX-2 cells,the mRNA expression level of CSE was significantly higher than that of CBS and MPST(1.008±0.13 vs 0.320±0.014 vs 0.05±0.02,F=80.84,P<0.05).When CSE was inhibited by PPG,the concentration of H2S decreased with the increase in the concentration of PPG(P<0.05).LX-2 cells

关 键 词：癌 肝细胞 肝星状细胞 硫化氢 

分 类 号：R735.7[医药卫生—肿瘤]