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邻苯二甲酸酯类化合物(phthalate esters, PAEs)是邻苯二甲酸酐与醇反应生成的化合物,它是提高聚氯乙烯(polyvinyl chloride,PVC)弹性的重要添加剂,其中邻苯二甲酸二(2-乙基己基)酯(Di(2-ethylhexyl)phthalate,DEHP)是目前使用量最大的一种邻苯二甲酸酯,用量高达80%[1]. DEHP广泛应用于儿童玩具、塑料包装、化妆品及各类医疗器械[2-3]. DEHP的广泛使用导致了不可避免的环境释放以及人体摄入. 大量实验指出我国人群DEHP的暴露剂量约为11—116 μg·kg−1·d−1,接近DEHP的每日可耐受摄入量(TDI)(20—140 μg·kg−1·d−1),表明 DEHP对人群的健康构成重大危害[4].
DEHP具有内分泌干扰效应,可以诱发生殖发育毒性、肝脏毒性、胚胎毒性等多种毒性[5-6]. DEHP通过消化道进入人体后在胃肠道的脂肪酶作用下水解成初级代谢物乙基己醇(2-EH)和邻苯二甲酸单乙基己基酯(mono-ethylhexyl phthalate,MEHP),再通过尿液排出体外[7]. 通过对尿液的单酯类物检测发现,原尿和酶解后的尿液中 MEHP 的检出率最高,平均检出率超过60% [8]. 此外,试验结果表明DEHP的毒性主要来源于其代谢物 MEHP,其毒性作用高达DEHP的10倍[9]. 同时,MEHP的动物实验表明,其主要分布在肾脏、膀胱和肝脏,其中肝脏为MEHP最主要的靶器官[10]. Thomas等的研究中,大鼠口服500 mg·kg−1 DEHP,30 min后,肝脏MEHP水平为12.5 mg·g−1 [11]. MEHP毒性作用主要体现在增加肝脏亲脂活性,导致肝脏中出现脂肪堆积,继而引发肝细胞脂肪变性[12].
肝脏在体内发挥着外源物质解毒和代谢脂类物质的主要作用. 肝脏内游离脂肪酸增加和甘油三酯沉积是肝脏脂质代谢紊乱的主要表现. 近年来,全球肥胖病与非酒精性脂肪肝患病率快速上升. 体内体外实验提示脂肪代谢紊乱会增加肥胖、糖尿病、非酒精性脂肪肝病的患病危险度. 流行病学和毒理学研究报告称,接触DEHP会影响机体脂肪代谢,从而促进肥胖[13]. 体内实验表明大鼠用DEHP处理后,肝脏重量增加,主要机制可能与肝代谢酶改变有关[14]. 有体外实验表明,MEHP处理HepG2细胞后,激活了PPARα使脂肪酸的氧化分解受到抑制[9],导致肝细胞内的脂质堆积并造成肝脏损伤. 在本课题组的前期研究发现,MEHP 处理后的HepG2 细胞的乙酰辅酶 A 羧化酶(acetyl-CoA carboxylase, ACC)的亚型ACC1蛋白表达水平增加,最终使肝脏细胞中脂肪酸合成增加. 这些结果表明,MEHP 可能通过影响脂质合成相关基因或蛋白的表达,从而导致肝细胞脂肪代谢出现紊乱. 为此,本实验以HepG2细胞为实验对象,通过MEHP染毒,观察细胞内脂质代谢情况,并通过基因芯片高通量筛查差异基因,探讨MEHP对体外脂质合成的影响及其可能的作用机制.
邻苯二甲酸单乙基己基酯暴露对肝脏脂质代谢的影响及调控机制
Effects of mono- (2-ethylhexyl) phthalate exposure on liver lipid metabolism and its regulatory mechanism
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摘要: 邻苯二甲酸二(2-乙基己基)酯(Di(2-ethylhexyl) phthalate,DEHP)是一种重要的内分泌干扰物,对神经系统、生殖系统、内分泌系统等都具有特定的损伤作用. DEHP被机体摄入后,在肝脏和肠道中被消化酶代谢为邻苯二甲酸单(2-乙基己基)酯(mono-(2-ethylhexyl)phthalate,MEHP)以发挥毒性作用. 本研究采用体外HepG2暴露结合mRNA芯片以及生信分析的方法,探讨MEHP暴露对HepG2细胞脂质代谢的影响. 首先,采用油红O 染色法观察细胞内脂滴蓄积情况,发现MEHP暴露能够以浓度依赖性的方式增加HepG2细胞内脂质蓄积. 为了探究其可能的机制,采用Agilent(人)表达谱芯片分析并筛选差异基因,共筛选出93个差异表达基因,其中上调基因57个,下调基因36个. 在此基础上,通过DAVID在线数据库对差异表达基因进行基因功能富集分析,发现其主要参与细胞脂质代谢过程(cellular lipid metabolic process)、跨膜运输(transmembrane transport)、跨膜转运的调控(regulation of transmembrane transport)等生物学过程. 随后,通过qRT-PCR法对关键基因的mRNA表达水平进行验证. 最后,通过 GEPIA 在线验证上述基因在肝细胞癌(LIHC)中的表达. 综上,MEHP暴露能够影响细胞脂质代谢通路中的基因FADS6、MVD、SC5D、PLA2G4E在HepG2细胞中的表达,在一定程度上促进了肝脏细胞中的脂肪蓄积,从而使肝细胞内脂质代谢紊乱.
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关键词:
- 邻苯二甲酸单乙基己基酯 /
- HepG2 细胞 /
- 脂肪蓄积 /
- 生信分析.
Abstract: Di(2-ethylhexyl) phthalate (DEHP) is an important endocrine disruptor, which has specific damage to nervous system, reproductive system and endocrine system. DEHP is metabolized by digestive enzymes into mono-(2-ethylhexyl)phthalate (MEHP) in liver and intestine , so as to produce toxic effects on human health. HepG2 was exposed to MEHP and then mRNA microarray and bioassay was used to investigate the effects of MEHP exposure on lipid metabolism of HepG2. First, oil red O staining was used to observe the accumulation of lipid droplets in HepG2 cells. It was found that MEHP exposure could increase the accumulation of lipid droplets in HepG2 cells. In order to explore the possible mechanism, Agilent (human) expression microarray was used to analyze and screen differentially expressed genes. A total of 93 differentially expressed genes were screened, including 57 up-regulated genes and 36 down-regulated genes. In addition,gene function enrichment analysis of differentially expressed genes was conducted through DAVID online database. It was found that these differentially expressed genes were mainly involved in the regulation of cellular lipid metabolic process, transmembrane transport and transmembrane transport Transport and other biological processes. Then, the mRNA expression levels of key genes were verified by qRT-PCR. Finally, GEPIA was used to verify the expression of these genes in Liver hepatocellular carcinoma(LIHC). In conclusion, MEHP exposure can affect the expression of FADS6、MVD、SC5D and PLA2G4E genes in cellular lipid metabolism pathway in liver, and promote the accumulation of fat in HepG2 cells, thus leading to the disorder of lipid metabolism in liver cells.-
Key words:
- mono- (2-ethylhexyl) phthalate /
- HepG2 cells /
- fat storage /
- raw letter analysis.
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表 1 实时定量PCR引物序列
Table 1. Primer sequences for real-time quantitative PCR
基因
Gene引物
PrimerPDP2 S——GAAGATGAGGTGACAAGGAA F——GCCAGCACAAG MVD GAACTTA PLA2G4E S——TTCTGTCCTATGGCTCCTT F——GTTCTTCACTCGGCTCTG FADS6 S——CCTCAACCGCTATGTCTAC F——CGATGTGCTGGAAGATGT PTPRQ S——ATGTCTATATTGCGGCTGAA F——TTCTTACTTGCGTGGATTCT CD28 S——GCTCTTGGCTCTCAACTTA F——CCTGCTCCTCTTACTCCT SC5D S——CTTGCTGGAGATAAGAGGTT F——TATGGTGGTCTGTATGATGAG MVD S——CAAGGACTTCACCGAGGA F——GTAGGCTAGGCAGGCATA 表 2 差异基因的表达情况
Table 2. The expression of differentially expressed genes
Up Down symbol lgFC P.Value symbol lgFC P.Value DPPA3 0.743871 0.000032637 TTTY14 −0.75345 0.000683968 MYLIP 0.685596 0.000320629 XLOC_008352 −0.691 0.001413602 XLOC_000888 0.664559 0.000498704 LOC100129112 −0.83639 0.001799658 LOC100127994 0.609364 0.00056601 SC5D −0.92097 0.002090916 SYT17 0.650856 0.000986627 XLOC_004178 −0.72657 0.002192512 XLOC_008003 0.727595 0.00115933 C14orf119 −0.78947 0.003595082 LOC100506487 0.589801 0.001543009 XLOC_l2_012082 −0.61338 0.004605101 FADS6 0.784036 0.002373877 XLOC_012908 −0.92876 0.006283058 AQP10 0.603687 0.00355145 GABRG1 −1.01945 0.007994 XLOC_001387 0.675839 0.003916862 NUMB −0.57939 0.008775398 KCNE4 0.831973 0.003949384 FLJ21408 −0.61905 0.009899712 XLOC_005215 0.65671 0.004608305 LOC100505657 −0.89616 0.010826125 OR5L1 0.922639 0.004609802 ZNF554 −0.60913 0.011699612 C14orf180 0.649561 0.005207664 XLOC_012871 −0.78553 0.01385829 RHOXF1 0.622636 0.007822227 XLOC_007131 −0.7273 0.014006601 PTPRQ 0.739332 0.008548009 XLOC_l2_014785 −0.66169 0.01855114 LOC286382 0.586815 0.008726912 PRO0611 −0.78996 0.018748849 ACOD1 0.61305 0.008957375 CREG2 −0.70749 0.018758137 LOC100130744 0.85844 0.009748146 XLOC_l2_004857 −0.66225 0.023632428 ZAP70 0.625018 0.010183849 PDP2 −0.80976 0.023933353 PLA2G4E 1.427762 0.011394311 XLOC_l2_011207 −0.57923 0.026335268 LOC652586 0.614123 0.011818286 XLOC_007761 −0.58384 0.027416794 XLOC_006019 0.666199 0.012002598 GNL3LP1 −0.6286 0.027632132 SLAMF7 0.896594 0.013447959 XLOC_l2_013646 −0.89314 0.028992205 XLOC_005433 0.588529 0.014072632 MVD −0.63712 0.031037423 KRTAP13-2 0.681023 0.014111061 LOC728065 −0.58089 0.031598939 XLOC_008237 0.804098 0.01445258 XLOC_l2_011011 −0.60147 0.033573436 XLOC_008244 0.677154 0.01652238 XLOC_001687 −1.03697 0.037696163 LOC100505966 0.836326 0.01818776 XLOC_004804 −0.68571 0.039579631 XLOC_001422 2.454291 0.018227482 LOC100507110 −0.60107 0.041523521 CSN2 1.0231 0.020348553 CACNG8 −0.61314 0.041704333 XLOC_003782 0.639271 0.021116476 SNORD70 −0.92894 0.044259091 XLOC_005572 0.585382 0.02204518 LOC100128126 −1.05439 0.044856832 XLOC_003349 1.233979 0.022738339 NPPA-AS1 −0.59523 0.048087643 XLOC_l2_00770 0.610547 0.023106187 XLOC_001550 −0.64752 0.04894801 SNORD115-48 0.719119 0.024508996 INE1 −1.017 0.0496582 SNORD115-4 0.688309 0.026211298 XLOC_001755 0.605516 0.028061446 ANK2 0.906401 0.029169596 CD28 0.589859 0.029610282 LOC100506563 1.019834 0.029837404 ANXA10 0.626939 0.030222491 CYBB 1.151916 0.03336549 XLOC_012064 1.019834 0.0345969 XLOC_013649 0.624561 0.035226312 XLOC_010183 1.380268 0.035959747 XLOC_006983 0.862151 0.040708371 XLOC_004274 0.720596 0.040795527 XLOC_008690 0.686422 0.041491936 XLOC_007888 0.852148 0.04152697 XLOC_l2_011145 0.704819 0.042378972 SLC24A4 0.757531 0.043335006 XLOC_013458 0.71734 0.043999913 TREML1 0.800135 0.047577606 TRIM49 0.796312 0.048328175 ANKDD1B 0.683738 0.049447534 DUSP21 0.604901 0.049510457 表 3 DAVID分析中前5个通路(BP)与基因
Table 3. Top 5 GO terms (BP) of the genes with the DAVID analysis
GO Term
GO 条目Count
数量Genes
基因Fold Enrichment
富集倍数P Value
P值GO:0044255
cellular lipid metabolic process7 PDP2, PLA2G4E, FADS6,
PTPRQ, CD28, SC5D, MVD3.85628326776209 0.0068 GO:0045087
innate immune response5 TREML1, ZAP70, CYBB,
ACOD1, SLAMF73.042438312 0.0494 GO:0055085
transmembrane transport7 SLC24A4, CACNG8, KCNE4, AQP10,
CYBB, ANK2, GABRG13.024137931 0.0204 GO:0006629
lipid metabolic process7 PDP2, PLA2G4E, FADS6, PTPRQ,
CD28, SC5D, MVD5.660947851 0.0210 GO:0034762
regulation of transmembrane transport4 CACNG8, KCNE4, CYBB, ANK2 3.43652037617554 0.0300 -
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