硫化镍诱导转化人支气管上皮细胞基因组DNA甲基化的研究

目的对结晶型硫化镍诱导转化及成瘤的人支气管上皮细胞(16HBE)基因组DNA甲基化状况进行研究,以寻找DNA甲基化异常的基因片段,并探讨镍的外遗传致癌机制.方法抽提基因组DNA后,采用限制性内切酶MseI单独酶切或限制性内切酶MseI与BstuI双重酶切后,其酶切产物采用甲基化敏感的限制性指纹识别技术(MSRF)进行分析,显示的异常甲基化基因片断,采用TA克隆技术构建测序载体,对测序结果进行同源性分析比较.结果发现结晶型NiS诱导转化及成瘤的16HBE细胞基因组DNA存在高甲基化的DNA片段.其中一基因片段与位于16q24.3编码鼻咽癌易感性蛋白基因ANKRD11同源(99%),另一基因片段与HOXA3基因序列同源(99%).HOXA3在脊椎动物中编码转录因子,编码调控基因表达,形态发生和变异的DNA结合转录因子.结论结晶型NiS诱导转化及成瘤的16HBE细胞基因组DNA的高度甲基化可能导致基因表达抑制,这可能是结晶型NiS诱导16HBE细胞转化和成瘤的一种外遗传机制.     

硫化镍与苯并芘转化人支气管上皮细胞基因差异表达谱的研究

目的用cDNA微阵列技术探讨经硫化镍(NiS)与二羟环氧苯并芘(dihydroxyepoxy benzo pyrene,BPDE)转化后的人支气管上皮细胞(16HBE)基因的差异表达;以了解不同种类和性质的化合物在转化细胞及致癌分子机制方面的异同;为预防人类生存环境不同外来化合物对人类健康的危害和影响提供科学依据.方法取16HBE分为NiS处理组,BPDE处理组和16HBE(对照组)三组同步培养,传代至第35代;提取三种细胞的总RNA,逆转录合成cDNA并以Cy3-dCTP和Cy5-dCTP荧光素分别标记制作探针.探针混合后与含4000个人类基因的芯片杂交.以ScanArray 4000扫描仪扫描芯片,以GenPix Pro3.0软件分析荧光信号,对三组差异表达的基因进行生物学信息分析.结果 NiS转化的16HBE细胞与BPDE转化的16HBE细胞经分析比较,呈现差异表达的基因有22个,其中11个(50%)下调,另11个(50%)上调.结论 NiS与BPDE对16HBE细胞株的转化作用在抑癌基因,细胞周期蛋白相关基因,细胞凋亡和应激反应基因相关基因,DNA合成、修复和重组蛋白相关基因,DNA结合、转录和转录因子类基因,细胞受体相关基因,代谢相关基因,蛋白翻译和合成相关基因等多类基因上呈现差异表达.认为基因芯片技术在筛选不同种类和性质的化合物转化细胞相关基因的改变上,具有高通量、高敏感、快速等特点,对化合物在细胞、分子毒作用和致癌机制的研究中意义重大.     

硫酸镍诱导的人支气管上皮细胞系癌变细胞基因组不稳定性分析

目的　检测硫酸镍对基因组不稳定性的影响,从而进一步探讨镍化合物致癌的分子机制。方法　采用随机扩增多态性DNA(random amplified polymorphic DNA, RAPD)技术对经硫酸镍在诱导人支气管上皮细胞系(16HBE)的恶变细胞中的基因组不稳定性进行分析。结果　本实验所选用的7条随机引物均能扩增出清晰、明显的条带,条带数在1~6条之间。7条引物中第1、4、7的3条引物扩增的片段在实验组和对照组之间无差异。其余4条引物均有明显差异,对于同一随机引物它们都具有特异的带型。结论　硫酸镍能诱发16HBE细胞基因组不稳定。     

甲基丙烯酸环氧丙酯致人支气管上皮细胞恶性转化过程中P16基因的甲基化状态

目的 分析甲基丙烯酸环氧丙酯(glycidyl methacrylate,GMA)致人支气管上皮16HBE细胞恶性转化过程中不同时点P16基因甲基化状态的差异,探讨GMA诱导16HBE细胞发生恶性转化相关DNA甲基化机制.方法 收获GMA转化早期(染毒结束)、转化前期(第10代)、转化后期(第30代)的16HBE细胞,采用甲基化特异性聚合酶链反应(Methylation-specific PCR,MSP)检测该基因组P16基因启动子区的甲基化状态,并与未转化的16HBE细胞及同期培养的DMSO溶剂对照细胞进行比较.结果 转化早期及转化前期,正常对照组及DMSO溶剂对照细胞中该基因启动子区均表现为非甲基化,而GMA组细胞中表现为不同程度的甲基化,阳性对照表现为甲基化;转化后期,正常对照组细胞中该基因启动子区表现为非甲基化,而DMSO溶剂对照组及GMA组细胞中均表现为部分甲基化,阳性对照表现为甲基化.结论 在GMA诱导16HBE细胞恶性转化早期及前期P16基因的甲基化状态具有特异性,故可将其作为GMA诱导16HBE细胞发生恶性转化的一个早期敏感指标.     

DNA甲基转移酶1基因低表达的人支气管上皮细胞基因组甲基化水平的体外试验

目的 观察人支气管上皮细胞(16HBE)的DNA甲基转移酶1(DNMT1)基因低表达细胞株细胞周期和基因组整体甲基化水平的改变.方法 用慢病毒介导的RNA干扰方法将4个不同的短发夹RNA片段转染16HBE细胞,并用免疫印迹法(Western blotting)检测该细胞DNMT1蛋白表达水平,用流式细胞仪和5-甲基胞嘧啶(5-mC)免疫荧光法检测该细胞的细胞周期和细胞基因组整体甲基化水平.结果 16HBE-shDNMT1-4细胞株的DNMT1蛋白表达与对照组相比平均降低约44%,差异有统计学意义(P＜0.05),但细胞周期和基因组整体甲基化水平无明显改变.结论 成功建立DNMT1基因低表达的16HB细胞株,其细胞周期和基因组整体甲基化水平无明显改变.

Abstract：

Objective To construct DNA methyltransferase 1 (DNMT1) low expression 16HBE cell line and observe the variation of cell cycle and global genomic DNA methylation. Methods The method of Lenti-virus induced RNA interference was applied to introduce four different shRNA fragment into 16HBE cells. Flow cytometry and 5-mC immunofluorescence methods were used to observe the cell cycle and global DNA methylation status of DNMT1 low expression 16HBE cells. Results The DNMT1 protein relative expression level of 16HBE-shDNMT1-4 cell line was down regulated about 44%(P＜0.05 ) compared with the control. No obvious differences of cell cycle and global genome DNA methylation status were observed between the 16HBE and 16HBE-shDNMT1. Conclusion The DNMT1 gene low expression cell is successfully constructed, and there are no obvious changes happened on the cell cycle and global genomic DNA methylation.     

miR-17-5p与反式-7,8-二羟-9,10-环氧苯并芘诱导转化的人支气管上皮细胞生物学特性的关系研究

目的探讨miR-17-5p对反式-7,8-二羟-9,10-环氧苯并芘(anti-BPDE)诱导转化的人支气管上皮细胞(16HBE-T)的生物学特性方面的影响。方法运用逆转录定量聚合酶链反应(qRT-PCR)检测miR-17-5p成熟体在16HBE-T和非转化对照细胞(16HBE-N)的相对表达水平。通过瞬时转染miRNA模拟物及抑制剂,改变16HBE-T中miR-17-5p的表达,分析转染后16HBE-T的增殖能力、细胞周期、细胞凋亡和软琼脂集落形成率等。结果转染前,16HBE-T中miR-17-5p表达水平是16HBE-N的2.35倍;转染后,转染miR-17-5p抑制剂组和转染miR-17-5p模拟物组中的miR-17-5p表达水平分别是16HBE-T的0.45和1.71倍。与抑制剂阴性对照组相比,转染了miR-17-5p抑制剂的试验组出现增殖能力下降、细胞周期阻滞和凋亡上升、克隆形成率下降。相反,转染了miR-17-5p模拟物的试验组与模拟物阴性对照组相比出现了增殖能力上调、凋亡减少,克隆形成率升高。结论 anti-BPDE恶性转化细胞中miR-17-5p成熟体表达水平改变能够影响16HBE-T的增殖能力、细胞周期和凋亡、克隆形成率,推断miR-17-5p在anti-BPDE致癌过程中发挥类癌基因作用。     

苯并(a)芘代谢物反式二羟环氧苯并芘诱发人支气管上皮细胞恶性转化

以不同浓度的苯并 (a)芘代谢物反式二羟环氧苯并芘 (BPDE)多次处理人支气管上皮细胞 16HBE ,并观察转化细胞的恶性特征。发现BPDE可诱导 16HBE细胞恶性转化 ,形成转化灶。转化灶细胞失去接触抑制 ,排列紊乱 ,无方向性 ,交叉重叠生长。转化的细胞可在软琼脂上生长 ,各浓度处理组细胞集落形成率均显著高于对照组 ,有良好的剂量—反应关系。经BPDE处理的细胞在裸鼠体内成瘤 ,病理学诊断为鳞状细胞癌。本实验以反式BPDE成功地诱发了人支气管上皮细胞恶性转化 ,为后期进一步研究其致癌的分子机制、寻找致癌相关基因提供了理想的生物学材料。     

结晶型硫化镍诱发细胞恶性转化过程中基因组总体DNA甲基化的改变

目的 探讨结晶型硫化镍(NiS)对体外培养细胞基因组总体DNA甲基化水平的影响.方法 分别以0.25、0.50、1.00、2.00 μg/cm2结晶型NiS处理人支气管上皮细胞系(16HBE)24 h,隔天再次进行相同处理,共处理3次;以3μmol/L DNA甲基化转移酶抑制剂5-脱氧杂氮胞苷(DAC)处理72 h的正常细胞为阳性对照.应用免疫荧光法和SssI甲基转移酶法定性、定量分析结晶型NiS处理细胞和恶性转化细胞(NSTC)基因组DNA总体甲基化的变化.结果 结晶型NiS处理细胞DNA甲基化免疫荧光的强度均有不同程度降低,转化细胞DNA甲基化免疫荧光的强度明显降低.定量分析结果显示,对照组基因组总体甲基化率(mCpG%)为(81.9±7.3)%,0.25、0.50、1.00、2.00 μg/cm2的结晶型NiS处理3次的细胞(NiS0.25、NiS0.50、NiS1.00、NiS2.00)基因组总体mCpG%则分别为(77.9±6.2)%、(75.3±6.8)%、(59.5±4.9)%、(67.4±5.1)%.经单因素方差分析显示,不同组间总体mCpG%差异有统计学意义(F=124.95,P＜0.01),两两比较发现NiS1.00和NiS2.00组与对照组相比,差异均有统计学意义(t值分别为7.64、4.89,P值均＜0.01);恶性转化细胞(NSTC1、NSTC2)基因组总体mCpG%分别为(46.2 ±4.1)%、(43.6±4.3)%,低于对照组,差异有统计学意义(t值分别为12.79、13.56,P值均＜0.01).结论 结晶型NiS诱发细胞恶性转化过程中,整体基因组DNA甲基化水平降低.     

结晶型硫化镍诱导的人支气管上皮细胞系转化细胞基因组不稳定性分析

目的　检测结晶型硫化镍对基因组不稳定性的影响 ,从而进一步探讨镍化合物致癌的分子机制。方法　采用随机扩增多态性DNA(randomamplifiedpolymorphicDNA ,RAPD)技术对经结晶型硫化镍在诱导人支气管上皮细胞系 (16HBE)的恶变细胞中的基因组不稳定性进行分析。结果　本实验所选用的 7条随机引物均能扩增出清晰、明显的条带 ,条带数在 1～ 6条之间。 7条引物中第 4条、第 5条和第 7条两条扩增的片段在实验组和对照组之间无明显差异 ,其余 4条引物均有差异。对于同一随机引物他们都具有特异的带型。结论　结晶型硫化镍能诱发 16HBE细胞基因组不稳定     

结晶型硫化镍诱发细胞恶性转化过程中基因组总体DNA甲基化的改变

Objective To observe the effect of crystalline NiS on genome DNA methylation profile in in vitro cultured cells.Methods 16HBE Cells were treated with crystalline NiS at 0.25,0.50,1.00 and 2.00 μg/cm2 for 24 h and three times at total.DAC treatment was given at 3 μmol/L for 72 h.5-mC immunofluorescence and SssI emthyltrasferase assay methods were applied to investigate if the hypomethylation of genome DNA involved.Results The results of 5-mC immunofluorescence showed that the fluorescence intensity of NiS-treated cells were decreased in some degree, and transformed cells were decreased dramatically.By the SssI methylase assay, an average of (81.9 ± 7.3 )% methylated CpG were found in negative control cells.By contrast, ( 77.9 ± 6.2) %, ( 75.3 ± 6.8 ) %, ( 59.5 ± 4.9 ) %, ( 67.4 ±5.1 ) % methylated CpG were observed in cells treated with NiS for three times at dosage of 0.25,0.50,1.00and 2.00 μg/cm2 which were abbreviated as NiS0.25, NiS0.50, NiS1.00, NiS2.00 respectively.The ANOVA analysis results showed that there was a significant difference in the 5 groups above ( F = 124.95,P ＜0.01 ).The results of Dunnett-t test showed that the methylated CpG of both group NiS1.00 and NiS2.00were significantly decreased compared with the negative control group(t values were 7.64,4.89 respectively,P ＜0.01 ).For methylated CpG, (46.2 ±4.1 ) % and (43.6% ±4.3)% were observed in NiS-transformed cells (NSTC1 and NSTC2) which were dramatically decreased compared with the negative control group(t values were 12.79,13.56 respectively, P ＜ 0.01 ).Conclusion Genomic DNA methylation levels were decreased during NiS induced malignant transformation.     

镉转化细胞DNA异常甲基化对肿瘤相关基因表达的影响

目的 对镉转化细胞DNA异常甲基化及其对肿瘤相关基因表达的影响进行研究，探讨镉的外遗传致癌机制。方法 从CdCl2转化BAIB／c—3T3细胞中提取基因组DNA，经甲基化非敏感性酶(Mse1)单独消化或Mse1和甲基化敏感性酶(BstUl)联合消化，消化产物用甲基化敏感性内切酶指纹法(MSRF)进行分析，差异显示出异常甲基化基因片段，进一步以异常甲基化DNA为探针进行Southern分子杂交加以证实，并进行DNA序列测定，与基因文库中的基因进行类比分析。结果 发现镉转化细胞存在异常甲基化DNA，其中一个甲基化DNA片段为p16抑癌基因。结论 DNA高甲基化会导致基因表达抑制，因此，p16基因高甲基化会导致其抑癌功能减弱或丧失，这可能是镉诱导细胞转化及其致癌作用的一种外遗传机制。     

硅转化BALB/c-3T3细胞基因组DNA异常甲基化的研究

目的 对硅转化细胞基因组DNA异常甲基化进行研究，探讨硅的表遗传致癌机制。方法 从结晶型硅(Si)转化BALB/c-3T3细胞中提取基因组DNA，经Msel(甲基化非敏感性酶)单独消化或Msel和BstU1(甲基化敏感性酶)联合消化，消化产物用甲基化敏感性内切酶指纹法(MSRF)进行分析，差异显示出异常甲基化基因片段，进一步将异常甲基化DNA片段亚克隆和序列测定，再与基因文库中的基因进行类比分析。结果发现硅转化细胞存在6条异常甲基化DNA(其中1条为高甲基化，5条有低甲基化现象)，序列测定显示这些异常甲基化基因片段似乎来源于一些RNA转录和蛋白质翻译等基因家族。结论DNA异常甲基化会导致基因表达激活或抑制，因此硅转化细胞基因组某些功能基因DNA异常甲基化导致的异常表达，可能间接是硅诱导细胞转化及其致癌作用的一种表遗传机制。     

Alcohol consumption in relation to aberrant DNA methylation in breast tumors

The mechanism for the observed association of alcohol consumption breast cancer risk is not known; understanding that mechanism could improve understanding of breast carcinogenesis and optimize prevention strategies. Alcohol may impact breast malignancies or tumor progression by altering DNA methylation. We examined promoter methylation of three genes, the E-cadherin, p16, and retinoic acid-binding receptor-β₂ (RAR-β₂) genes in archived breast tumor tissues from participants in a population-based case–control study. Real time methylation-specific PCR was performed on 803 paraffin-embedded samples, and lifetime alcohol consumption was queried. Unordered polytomous and unconditional logistic regression were used to derive adjusted odds ratios (ORs) and 95% confidence intervals (CIs). RAR-β₂ methylation was not associated with drinking. Among premenopausal women, alcohol consumption was also not associated with promoter methylation for E-cadherin and p16 genes. In case–case comparisons of postmenopausal breast cancer, compared with lifetime never drinkers, promoter methylation likelihood was increased for higher alcohol intake for E-cadherin (OR = 2.39; 95% CI, 1.15–4.96), in particular for those with estrogen receptor-negative tumors (OR = 4.13; 95% CI, 1.16–14.72), and decreased for p16 (OR = 0.52; 95% CI, 0.29–0.92). There were indications that the association with p16 was stronger for drinking at younger ages. Methylation was also associated with drinking intensity independent of total consumption for both genes. We found alcohol consumption was associated with DNA methylation in postmenopausal breast tumors, suggesting that the association of alcohol and breast cancer may be related, at least in part, to altered methylation, and may differ by drinking pattern.     

DNA差异甲基化在结核病检测中的应用进展

结核分枝杆菌感染导致的结核病仍然是全球公共卫生的重大挑战。结核病诊断新技术是提高结核病控制水平的关键。本文对全球基于DNA 甲基化诊断结核病新技术的相关进展进行综述,主要包括可能作为诊断靶标基因的甲基化,以及这些差异甲基化基因涉及的信号通路。     

DNA甲基化与衰老研究进展

DNA甲基化与衰老的研究是近20a来分子生物学研究的热点之一。本文综述了近年来国内外文献，概括DNA甲基化理论研究进展，探讨影响甲基化与衰老的主要因素，以揭示两者之间可能存在的联系，并分析了与甲基化和衰老相关疾病的发生机制。     

离子交换色谱法检测人血白细胞基因组DNA整体甲基化水平

目的改进现有方法,能快速、稳定、简便地测定人基因组DNA整体甲基化水平。方法采用美国HPAgilent1100色谱工作站,利用离子交换液相色谱法对健康人外周血白细胞基因组DNA整体甲基化水平进行检测。色谱柱采用德国MN公司的强阳离子交换色谱柱（250mm×4．6mm5μm）;流动相为60mM醋酸＋15％乙腈,用氢氧化钠调节pH为4．6;流速为1．0ml／min;检测器波长为276nm;柱温为28℃;进样量为50μl。基因组DNA整体甲基化水平用5甲基脱氧胞嘧啶核苷占DNA样品中总脱氧胞嘧啶核苷的百分数来表示。结果在上述色谱条件下,约10min可以将DNA中的脱氧胞嘧啶核苷和5甲基脱氧胞嘧啶核苷完全分离。并测得健康成人血白细胞基因组DNA整体甲基化水平为（4．389±0．0159）％。结论本方法能快速、有效地对基因组DNA整体甲基化水平进行检测,可供广大实验室采用。     

DNA甲基化研究方法的回顾与评价

DNA甲基化是表观遗传学的重要组成部分,在维持正常细胞功能、遗传印记、胚胎发育以及人类肿瘤发生中起着重要作用,是目前新的研究热点之一.随着对甲基化研究的不断深入,各种各样甲基化检测方法被开发出来以满足不同类型研究的要求,这些方法概括起来可分为3类:基因组整体水平的甲基化检测、基因特异位点甲基化的检测和新甲基化位点的寻找.该文主要介绍目前应用的大部分DNA甲基化研究方法,并对其相关特性进行简要分析与总结.     

聚腺苷二磷酸核糖聚合酶1在苯并(a)芘诱导人支气管上皮细胞DNA甲基化改变中的作用

目的 观察苯并(a)芘[benzo(a)pyrene,B(a)P]诱导体外细胞DNA甲基化水平改变,探讨聚腺苷二磷酸核糖聚合酶1[poly(ADP-ribose)polymerase 1,PARP1]在该过程中的作用.方法 以1.0、2.0、5.0、10.0、15.0、30.0 μmol/L浓度B(a)P分别处理人支气管上皮细胞(16HBE)及其PARP1缺陷细胞(16HBE-shPARP1)72 h.采用免疫荧光和高效毛细管电泳检测其基因组DNA整体甲基化水平改变,同时动态监测PARP1和DNA甲基转移酶1(DNA methyltransferases 1,DNMT1)表达的变化.结果 16HBE和16HBE-shPARP1细胞基因组整体甲基化百分比(mCpG%)分别为(4.04±0.08)%和(9.69±0.50)%.经5-氮杂脱氧胞苷(DAC)处理72 h后,mCpG%值分别下降为(3.15±0.14)%、(6.07±0.54)%.经B(a)P染毒72 h后,16HBE细胞基因组mCpG%值[B(a)P浓度由低到高]分别为(5.10±0.13)、(4.25±0.10)、(3.91±0.10)、(4.23±0.27)、(3.70±0.15)、(3.08±0.07);16HBE-shPARP1细胞基因组mCpG%值(浓度由低到高)分别为(10.63±0.60)、(13.08±0.68)、(9.75±0.55)、(7.32±0.67)、(6.90±0.49)、(6.27±0.21).两种细胞不同处理组间mCpG%差异有统计学意义(F值分别为61.67、60.91,P值均＜0.01).16HBE细胞各剂量组[B(a)P浓度由低到高]PARP1基因mRNA相对表达水平分别为对照组的141.0%、158.0%、167.0%、239.0%、149.0%、82.9%,差异均有统计学意义(t值分别为11.45、17.32、32.24、33.44、20.21、9.87,P值均＜0.01);16HBE-shPARP1细胞各剂量组(浓度由低到高)PARP1基因mRNA相对表达水平分别为对照组的169.0%、217.0%、259.0%、323.0%、321.0%、256.0%,差异有统计学意义(t值分别为9.06、15.92、22.68、26.23、37.19、21.15,P值均＜0.01).当B(a)P染毒剂量达5.0 μmol/L后,16HBE细胞各剂量组(浓度由低到高)DNMT1基因mRNA相对表达水平分别为对照组的125.0%、162.0%、275.0%、233.0%,差异有统计学意义(t值分别为12.74、24.92、55.11、59.07,P值均＜0.01);当B(a)P染毒剂量达2.0 μmol/L后,16HBE-shPARP1细胞各剂量组(浓度由低到高)DNMT1基因mRNA相对表达水平分别为对照组的135.0%、151.0%、180.0%、229.0%、186.0%,差异有统计学意义(t值分别为23.82、40.17、32.69、74.85、46.76,P值均＜0.01).结论 B(a)P诱导的16HBE细胞基因组整体甲基化水平降低可能是其恶性转化过程中早期重要的分子事件,PARP1可通过抑制DNMT1的酶活性来调节B(a)P诱导的16HBE细胞DNA甲基化水平,这种效应可因PARP1的缺失而缓解.

Abstract：

Objective To investigate DNA methylation variation in human cells induces by B (a)P, and to explore the role of PARP1 during this process. Methods The changes of DNA methylation of 16HBE and its PARP1-deficient cells exposed to B ( a ) P ( 1.0, 2. 0, 5.0, 10. 0, 15.0, 30. 0 μ mol/L ) were investigated by immunofluorescence and high performance capillary electrophoresis, and simultaneously, the expression level of PARP1 and DNMT1 were monitored dynamically. Results The percentage of methylated DNA of overall genome ( mCpG% ) in 16HBE and 16HBE-shPARP1 cells were separately (4. 04 ±0. 08) %and (9. 69 ±0. 50)%. After being treated by 5-DAC for 72 hours,mCpG% decreased to (3.15 ±0. 14)%and (6. 07 ± 0. 54 ) %. After both being exposed to B (a) P for 72 hours, the mCpG% in 16HBE group ( ascending rank ) were separately ( 5. 10 ± 0. 13 ), ( 4. 25 ± 0. 10 ), ( 3.91 ± 0. 10 ), ( 4. 23 ± 0. 27 ),(3.70 ± 0. 15 ), ( 3.08 ± 0. 07 ); while the figures in 16HBE-shPARP1 group ( ascending rank ) were respectively (10.63 ±0.60), (13.08 ±0.68), (9.75 ±0.55), (7.32 ±0.67), (6.90 ±0.49) and (6. 27 ±0. 21 ). The difference of the results was statistically significant ( F values were 61.67 and 60. 91,P＜ 0.01 ) . For 16HBE group, expression of PARP1 and DNMT1 were 141.0%, 158.0%, 167.0%,239. 0%, 149. 0% ,82. 9% and 108. 0%, 117.0%, 125.0%, 162. 0% ,275. 0% ,233.0% comparing with the control group, whose difference also has statitical significance (t values were 11.45,17. 32,32. 24,33.44,20.21 and 9. 87 ,P ＜ 0. 01 ). For 16HBE-shPARP1 group, expression of PARP1 and DNMT1 were 169.0% ,217.0%, 259.0%, 323.0% , 321.0% , 256.0% and 86.0% , 135.0% , 151.0% , 180.0%,229. 0%, 186. 0% comparing with the control group,with statitical significance (t values were 9. 06,15. 92,22. 68,26. 23,37. 19 and 21.15, P ＜ 0. 01 ). When the dose of B (a) P reached 5.0 μmol/L, the mRNA expression of DNMTI in 16HBE group (ascending rank) were 125.0%, 162. 0% ,275.0% ,233.0% times of it in control group, with statistical significance ( t values were 12. 74,24.92,55. 11,59. 07, P ＜ 0. 01 );while the dose of B(a) P reached 2.0 μmol/L, the mRNA expression of DNMT1 in 16HBE-shPARP1 group were 135.0%, 151. 0%, 180. 0% ,229.0%, 186. 0% of the results in control group, and the differences were statistically significant ( t values were 23. 82,40. 17,32. 69,74. 85,46. 76, P ＜ 0. 01 ). Conclusion The hypomethylation of 16HBE cells induced by B(a)P might be one important molecular phenomenon in its malignant transformation process. It suggests that PARP1 could regulate DNA methylation by inhibiting the enzyme activity of DNMT1, and this effect could be alleviated by PARP1-deficiency.     

焦炉工外周血TH1/TH2型细胞因子表达与IFN-γ、IL-10 DNA甲基化分析

目的了解焦炉逸散物(coke oven emissions,COEs)暴露的焦炉工多环芳烃(polycyclic aromatic hydrocarbons,PAHs)内暴露水平及焦炉工机体外周血白细胞介素-2(interleukin-2,IL-2)、γ-干扰素(interferon-γ,IFN-γ)、白细胞介素-4(IL-4)、白细胞介素-10(IL-10)的表达,及IFN-γ、IL-10表观遗传机制在COEs暴露损伤中的表现。方法随机选取某焦化厂炼焦车间接触COEs的85名工人作为暴露组,同是焦化厂非COEs暴露的47名作业工人作为对照组,采集暴露组及对照组工人晨尿进行碱水解-高效液相色谱荧光法检测1-羟基芘(1-OHPyr)水平,尿肌酐校正;采集空腹外周静脉血,用酶联免疫吸附试验测定血清中IL-2、IFN-γ、IL-4和IL-10的表达;采用飞行时间质谱分析IFN-γ和IL-10的甲基化水平。结果焦炉工人尿1-OHPyr含量高于对照组(F=12.446,P<0.05),且炉侧工和炉顶工高于对照组(P<0.05)。与对照组相比,焦炉工血清IL-2含量降低(F=14.77...