甲基丙烯酸环氧丙酯诱导的恶性转化16HBE细胞中泛素化过程抑制对蛋白质CD44和MMP14的影响  

Inhibition of GMA-induced ubiquitination process in 16HBE malignantly transformed cells on protein CD44 and MMP14

作　　者：金惠萍 王全凯[1,2] 顾轶婷 乌瀚宝栎尔 李昕苇 崔旭芳 张林媛[1,2] 许建宁[1,2] Jin Huiping;Wang Quankai;Gu Yiting;Wuhan Baolier;Li Xinwei;Cui Xufang;Zhang Linyuan;Xu Jianning(National Institute of Occupational Health and Poison Control,Chinese Center for Disease Control and Prevention,Beijing 100050,China;State Key Laboratory of Trauma and Chemical Poisoning,Chinese Center for Disease Control and Prevention,Beijing 100050,China)

机构地区：[1]中国疾病预防控制中心职业卫生与中毒控制所,北京100050 [2]中国疾病预防控制中心创伤与化学中毒全国重点实验室,北京100050

出　　处：《卫生研究》2023年第6期871-876,共6页Journal of Hygiene Research

基　　金：国家自然科学基金(No.81673221)。

摘　　要：目的观察甲基丙烯酸环氧丙酯(glycidyl methacrylate,GMA)诱导的恶性转化人支气管上皮(human bronchial epithelial,16HBE)细胞中泛素化过程抑制对I型跨膜糖蛋白(CD44 antigen,CD44)和基质金属蛋白酶-14(matrix metalloproteinase-14,MMP14)表达的影响。方法成功复苏的16HBE细胞使用终浓度为8μg/mL GMA染毒作为处理组,1μg/mL二甲基亚砜作为溶剂对照组,每次染毒72 h,间隔24 h后再次染毒,重复染毒3次后,分别进行传代培养。取第40代(P40)GMA处理组和同代龄溶剂对照组细胞进行软琼脂克隆形成实验和刀豆凝集素A(concanavalin A,ConA)凝集实验以确定GMA诱导的第40代16HBE细胞已具备恶性转化细胞特征;使用5、10、20、40和60μmol/L漆树酸抑制GMA诱导的恶性转化16HBE细胞的泛素化过程,采用蛋白质免疫印迹技术(Western blotting)检测CD44、MMP14蛋白表达的变化,实时荧光定量PCR技术(qPCR)检测CD44、MMP14、TFAP2A转录水平的变化。结果(1)克隆形成实验中,溶剂对照组细胞形成的克隆数为22个,GMA处理组恶性转化细胞形成的克隆数为208个;ConA凝集实验中,溶剂对照组细胞在ConA溶液中均匀散布,30 min内未发生明显凝集,而GMA处理组细胞在5 min时即发生明显凝集,凝集细胞团块较大且凝集速度较快,凝集敏感性增加;(2)不同程度抑制GMA诱导的恶性转化16HBE细胞泛素化过程后,与未处理组相比,GMA诱导的恶性转化16HBE细胞中CD44、MMP14蛋白表达水平均呈降低趋势(P<0.05);MMP14、CD44的转录水平随抑制剂浓度增加而降低(P<0.05),上游转录因子TFAP2A的转录水平也同时降低(P<0.05)。结论抑制细胞泛素化过程可抑制GMA诱导的恶性转化16HBE细胞中CD44和MMP14的蛋白表达。OBJECTIVE To observe the effect of the ubiquitination process on the expression of CD44 antigen(CD44)and matrix metalloproteinase-14(MMP14)in human bronchial epithelial(16HBE)malignantly transformed cells induced by glycidyl methacrylate(GMA).METHODS Successfully resuscitated 16HBE cells were cultured using a final concentration of 8μg/mL GMA as the treatment group and 1μg/mL dimethyl sulfoxide as the solvent control group,each time stained for 72 h,and then stained again after an interval of 24 h.After repeating the staining three times,the cells were cultured in passages respectively.The 40th generation(P40)GMA-treated group and the samegeneration solvent control group were subjected to soft agar colony formation assay and concanavalin A(ConA)agglutination test to confirm that the 40th generation of GMAinduced malignant transformed 16HBE cells possessed malignant transformed cell characteristics.5,10,20,40,60μmol/L anacardic acid were used to inhibit the ubiquitination process of GMA-induced malignant transformed 16HBE cells.The protein expression of CD44 and MMP14 were detected by western blotting,while the transcript levels of CD44,MMP14,and TFAP2A were assessed by real-time fluorescence quantitative PCR(qPCR).RESULTS(1)In the soft agar colony formation assay,the number of clones formed by the cells in the solvent control group was 22,and the number of clones created by the malignantly transformed cells in the GMA-treated group was 208.In the ConA agglutination test,the cells in the solvent control group were uniformly dispersed in ConA solution,and no obvious agglutination occurred for 30 min,whereas the cells in the GMA-treated group were agglutinated in the 5th min,and the agglutinated cells were larger and more rapidly agglutinated.The agglomerates were more significant and faster,and the sensitivity of agglutination was increased.(2)After differential inhibition of GMA-induced ubiquitination in malignantly transformed 16HBE cells,the expression levels of CD44 and MMP14 were reduced in GMA-induced mali

关 键 词：甲基丙烯酸环氧丙酯 16HBE细胞 恶性转化 泛素化过程 

分 类 号：R994.6[医药卫生—毒理学] R114[医药卫生—药学] R730.2