甲醛对人肝癌细胞株HepG2内质网应激相关Bip和CANXmRNA及蛋白表达水平的影响

Effects of formaldehyde on expression of endoplasmic reticulum stress-related Bip and CANX mRNA and proteins of HepG2 cell line

目的:研究甲醛(FA)染毒是否会诱导人肝癌细胞株Hep G2内质网应激相关基因Bip和CANX m RNA和蛋白表达水平的改变,引起内质网应激反应。方法:用Hoechst 33258进行核染色,以观察不同浓度(0、0.02、0.1、0.5、2.5、12.5 mmol/L)FA分别染毒肝细胞株24和48 h后的细胞核形态学变化。再分别用0.004、0.02、0.1 mmol/L的FA处理人肝癌细胞株Hep G2细胞24 h和48 h后,采用荧光定量PCR(q PCR)和Western blot检测内质网应激相关基因免疫球蛋白重链结合蛋白(Bip)及钙连蛋白(CANX)m RNA和蛋白的表达水平。结果:Hoechst 33258染色结果表明,染毒24和48 h后,0.5、2.5、12.5 mmol/L FA组出现大量核呈固缩状态或颗粒状荧光死亡细胞,阴性对照组和0.1 mmol/L及以下剂量组细胞核荧光染色较浅,细胞核未固缩,故后续试验采用0.1 mmol/L及以下剂量。经0.004、0.02和0.1 mmol/L FA处理细胞24和48 h后,q PCR检测结果显示,甲醛各剂量染毒组Bip和CANX的m RNA表达量与阴性对照组比较,差异均无统计学意义(P>0.05)。Western blot结果表明,染毒24 h,不同剂量甲醛处理组与阴性对照组相比,Bip和CANX蛋白表达量均无显著差异(P>0.05);染毒48 h,与阴性对照组相比,各FA处理组Bip蛋白表达量升高,0.02和0.1 mmol/L FA处理组CANX蛋白表达明显升高,差异均有统计学意义(P<0.05)。结论:甲醛染毒可诱导Hep G2细胞内质网应激相关蛋白Bip和CANX表达水平升高,引起肝细胞发生内质网应激反应。

OBJECTIVE:To investigate whether formaldehyde can induce endoplasmic reticulum stress inhepatocytes.METHODS:Hoechst33258was used for nuclear staining to observe morphological changes of hepatocytesin different formaldehyde concentration groups.The human hepatocellular carcinoma cell line HepG2was treated withformaldehyde(FA)solution at0.004mmol/L,0.02mmol/L and0.1mmol/L for24h and48h respectively.Expression ofBip and CANX mRNA in stress-related genes was detected by fluorescence quantitative PCR(qPCR).Western blot wasused to detect the expression of stress-related immunoglobulin heavy chain binding protein(Bip)and connexin(CANX)inendoplasmic reticulum.RESULTS:Cells in the negative control group and the low dose group were significantly higherwith Hoechst33258staining than in the control group(P<0.05).Results of qPCR show that expression of Bip and CANXmRNA in each dose group was not significantly different from that in the negative control group.Results of Western blotshow that there was no significant difference in the expression of Bip and CANX proteins among the different doses of FAgroup and the negative control group(P>0.05).Compared with the negative control group(P<0.05),expression of Bipprotein was increased in0.02mmol/L and0.1mmol/L FA group,the difference was statistically significant(P<0.05).CONCLUSION:Formaldehyde exposure may induce endoplasmic reticulum stress of hepatocytes.