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.     

焦炉逸散物诱导人支气管上皮细胞O6-甲基鸟嘌呤DNA甲基转移酶基因高甲基化

目的 通过观察细胞遗传损伤指标和O6-甲基鸟嘌呤DNA甲基转移酶(MGMT)甲基化改变,探讨焦炉逸散物暴露诱导的损伤效应机制.方法 采用1 μmol/L苯并[a]芘[B(a)P]诱导人支气管上皮细胞16HBE 48 h后,分别用1‰二甲基亚砜(DMSO)和2.5、5.0、10.0、20.0 μg/ml浓度的焦炉逸散物有机提取物对16HBE细胞连续染毒5 d,构建细胞损伤模型;采用甲基化特异性PCR(MSP-PCR)检测细胞MGMT甲基化改变,并用RT-PCR检测细胞MGMT mRNA改变,免疫印迹法检测MGMT蛋白改变;采用碱性单细胞凝胶电泳技术检测细胞DNA损伤水平.结果 与DMSO组相比,MGMT基因在各处理组均有高甲基化改变,并随剂量的增加,其mRNA和蛋白表达水平都呈下降趋势.DMSO组及2.5、5.0、10.0、20.0μg/ml各剂量组MGMT mRNA及其蛋白的灰度比值分别为1.0、0.96、0.96、0.85、0.32和1.0、1.0、1.1、0.41、0.52.焦炉逸散物有机提取物处理后,细胞出现不同程度的DNA损伤,DMSO组及2.5、5.0、10.0、20.0μg/ml各剂量组彗星Olive尾距分别为(2.98±1.43)、(4.76±1.79)、(10.09±1.75)、(11.38±1.77)、(11.67±1.88),损伤呈明显的剂量-效应关系(F=41.22,P＜0.05);进一步分析发现,细胞的遗传损伤指数与MGMT蛋白及mRNA表达水平呈负相关.结论 焦炉逸散物引起的DNA损伤与MGMT高甲基化所致的MGMT基因表达水平降低有关.

Abstract：

Objective To elucidate the mechanism of carcinogenesis induced by coke oven emissions by investigating the cell genetic damage index and the methylation of O6-methylguanine-DNA methyltransferase (MGMT). Methods The human bronchial epithelial cell 16HBE was treated by 1 μmol/L B(a) P for 48 h,and then was exposed continuously to either 1% dimethyl sulfoxide (DMSO) or organic extracts of coke oven emission ( OE-COE ) for five days at the concentrations of 0,2. 5 ,5. 0,10. 0 and 20. 0 μg/ml. The methylation-specific PCR ( MSP-PCR), RT-PCR and immunoblotting were applied to detect the methylation status, changes of mRNA and protein of MGMT, respectively. Single cell gel electrophoresis was used to detect DNA damage induced by OE-COE. Results Compared with the control group ( DMSO),there was a significant hypermethylation in all study groups, along with the suppression of mRNA and protein in a dose-dependent manner,and the gradation ratio of them was 1. 0,0. 96,0. 96,0. 85,0. 32 and 1.0,1.0,1. 1 ,0. 41,0. 52 ,separately. There was a significant DNA damage with a dose-effect relationship in all study groups ( F = 41.22, P ＜ 0. 05 ), and the comet Olive tail moment was ( 2. 98 ± 1. 43 ), (4. 76 ± 1.79 ),( 10. 09 ± 1.75), ( 11.38 ± 1.77), ( 1 1. 67 ± 1. 88). The further study found that the index of DNA damage was negatively correlated to the expression of MGMT mRNA and its protein. Conclusion The DNA damage induced by COE might be associated with the suppression of MGMT caused by its hypermethylation.     

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.     

细胞体外恶性转化过程中差异甲基化基因的筛选

目的 筛选细胞体外恶性转化过程中差异甲基化基因,为毒作用效应以及肿瘤化学预防、生物监测寻找潜在的表观遗传生物标志物.方法 利用苯并[a]芘[B(a)P]诱导水生化人支气管上皮细胞(HBER)转化模型和猿猴空泡病毒小T抗原(SV40 ST)诱导HBER转化模型,采用甲基化DNA免疫沉淀-全基因组扩增法富集基因组甲基化DNA,应用甲基化芯片技术和信息学分析方法,寻找不同作用因素诱导细胞转化的共同差异甲基化基因,通过RT-PCR法检测这些基因在B(a)P诱导HBER细胞转化过程中mRNA表达水平的变化.结果 芯片检测发现,HBER、染毒后末转化细胞株(HBERNT)和转化细胞株(HBERT)中甲基化基因的数目分别为733、661、738个,83个基因在染毒前呈非甲基化状态,而在转化前和转化后呈甲基化状态,在这83个基因中,有25个基因在SV40ST诱导HBER转化细胞株(HBERST)中同样发生高甲基化.其中,序列相似性家族178A(family with sequence similarity 178,member A,FAM178A)、视黄酸受体应答子(他扎罗汀诱导)[retinoic acid receptor responder(tazarotene induced),RARRES1]、泛素特异性肽酶28(ubiquitin specific peptidase 28,USP28)、Scm-like with four mbt domains 2(SFMBT2)、序列相似性家族59A(family with sequence similarity 59,member A,FAM59A)和核受体亚家族4组A3(nuclear receptor subfamily 4,group A,member 3,NR4A3)等6个基因在B(a)P诱导HBER细胞转化过程中mRNA表达水平下降.结论 细胞体外转化过程中筛查出来的特定基因高甲基化,可能成为化学毒物暴露监测及肿瘤预测的生物标志物.

Abstract：

Objective To explore potential epigenetic biomarkers for toxic effects, tumor-related chemical prevention and biological monitor by a genome-wide screening for differential DNA methylation during human cell malignant transformation in vitro. Methods The two in vitro cell transformation models included B( a)P-induced human bronchial epithelial cell introduced by H-Ras (HBER) cell transformation and simian vacuolating virus 40 small T antigen induced (SV40 ST-induced ) HBER cell transformation. Methylated genes were collected by methylated DNA immunoprecipitation and whole genome amplification (MeDIP-WGA) at three time points during cell transformation which represented different transformation stage. Then, CpG island microarray was used to screen differentially methylated genes. The mRN A levels of hypermethylated genes were also observed by RT-PCR. Results The CpG island microarray showed that the number of hypermethylated genes in HBER,HBERNT,HBERT cells were 733,661 and 738 respectively. 83 genes were hypermethylated in pre-transformed cell and transformed cell. Moreover, 25 of 83 genes were also hypermethylated in SV40 ST-transformed cell (HBERST). We further confirmed that the mRNA expression of six of these 25 genes, namely family with sequence similarity 178, member A ( FAM178A ), retinoic acid receptor responder ( tazarotene induced ) ( RARRES1 ), ubiquitin specific peptidase 28 (USP28) ,Scm-like with four mbt domains 2 (SFMBT2), family with sequence similarity 59,member A(FAM59A) and nuclear receptor subfamily 4 ,group A,member 3 (NR4A3) were supressed during B(a) P-induced transformation. Conclusion The abnormal hypermethylation of specific genes was a common event in the two kinds of human cell transformation models, which shed light on the study for chemical exposure monitor and tumor-related epigenetic biomarkers.     

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.     

叶酸与DNA甲基转移酶1在宫颈癌及癌前病变中的作用

目的 探讨叶酸和DNA甲基转移酶1(DNMT1)与宫颈癌发生发展的关系及其在宫颈癌变中的相互作用.方法 选择经病理学确诊的宫颈鳞状细胞癌(SCC)患者100例、宫颈上皮内瘤样变(CrN)患者101例和宫颈炎(CI)患者109例为研究对象.在收集全部对象人口学特征、生殖因素等相关资料的同时,检测血清叶酸水平(RIA法)、人乳头瘤病毒16的感染状况(PCR法)和DNMT1蛋白的表达量(Westernblot法).采用SPSS 16.0分析软件,进行相关资料的t检验、x2检验、因素与疾病之间关联强度指标(OR值95%CI)的计算及交互作用分析.结果 在SCC、CIN和CI组,血清叶酸含量分别为(2.60±1.61)、(3.14±2.08)和(3.32±1.74)ng/ml,DNMT1蛋白表达水平分别为2.40±0.99、1.88±0.33和0.89±0.29,随着宫颈病变的加重叶酸含量逐渐降低,DNMT1蛋白表达水平逐渐增高,两者呈负相关关系(r=-0 186,P=0001).交互作用分析显示,叶酸水平低和DNMTl蛋白高表达在CIN和SCC组均存在正相加交互作用,OR值分别为2.50(95%CI:1.21～9.22)和6.03(95%CI:2.79～21.72),交互作用指标超额危险度、交互作用归因危险比和交互作用指数在CIN组分别为0.92、0.36和2.59,在SCC组分别为2.47、0.41和1.96.结论 血清叶酸水平低及DNMT1蛋白高表达均是导致宫颈癌和癌前病变发生的危险因素,两者存在协同作用.

Abstract：

Objective To explore the effect of folic acid and DNA methyltransferase 1 (DNMT1) on cervical cancer and cervix precancerous lesion. Methods 100 patients with cervix squamouscell carcinoma (SCC), 101 patients with cervical intraepithelial neoplasm (CIN) and 109 patients with cervix inflammation (CI) diagnosed by histology were included in this study. Radioimmunoassay (RIA), polymerase chain reaction (PCR) and Western blot were used to detect the levels of serum folate, HPV16 infection and the expression of DNMT1 protein,respectively. Results The average levels of serum folate were (2.60 ± 1.61) ng/ml, (3.14 + 2.08) ng/ml and (3.32+1.74) ng/ml,and the expression of DNMT1 protein were 2.40 + 0.99,1.88 + 0.33 and 0.89 ± 0.29 in the group of SCC, CIN and CI, respectively.The relationship of folate levels and DNMT1 protein expression showed inverse correlation (r=-0.186, P=0.00l). The results in our study indicated that there was an additive interaction between low-level of serum folate and high-expressionof DNMT1 protein related to the risk of CIN and SCC, with OR value as 2.50(95%C/: 1.21-9.22) and 6.03 (95%C/: 2.79-21.72) respectively. The relative excessrisk of interaction (RERI) , attributableproportion of interaction (API) and synergy index (S) were 0.92, 0.36 and 2.59 in the CIN group while 2.47, 0.41 and 1.96 in the SCC group. Conclusion The low level of serum folate and high expression of DNMT1 protein seemed to be associated with high risk of cervical cancer and its precancerous lesion. It suggested that there might be a synergistic action between serum folate and DNMT1 in the progression of cervix carcinogenesis.     

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.     

微囊藻毒素-LR对小鼠肝细胞DNA甲基化的影响

目的研究微囊藻毒素-LR(MC-LR)暴露对小鼠肝细胞DNA总体甲基化水平及DNA甲基转移酶(DNMTs)mRNA表达的影响。方法将40只健康SPF级昆明小鼠随机分为4组,分别为对照组(含0.02%DMSO)和5、10、20μg/kg MCLR染毒组,每组10只,雌雄各半。采用腹腔注射方式进行染毒,连续染毒20 d。采用高效液相色谱(HPLC)法检测小鼠肝细胞DNA中的脱氧胞苷和5-甲基脱氧胞苷含量,并计算DNA的总体甲基化水平;采用实时荧光定量PCR法检测DNA甲基转移酶DNMT1、DNMT3A和DNMT3B m RNA的表达水平。结果 10、20μg/kg MC-LR染毒组小鼠肝细胞DNA总体甲基化水平,DNMT1、DNMT3A及DNMT3B mRNA的表达水平均低于对照组,差异有统计学意义(P0.05)。小鼠肝细胞DNA总体甲基化水平与DNMT1、DNMT3A、DNMT3B mRNA表达水平均呈正相关(P0.05)。结论 MC-LR可抑制小鼠肝细胞DNMT1、DNMT3A和DNMT3B mRNA的表达,降低DNA总体甲基化水平。     

DNA依赖性蛋白激酶在石英诱导的细胞周期蛋白E和细胞周期蛋白依赖激酶2蛋白表达及细胞周期改变中的作用

Objective To study the roles of DNA dependent protein kinase (DNA-PK)in silicainduced cell cycle changes and expressions of CyclinE and CDK2 in human embryo lung fibroblasts (HELF).Methods The expressions of Ku80 and DNA-PKcs proteins were inhibited by siRNA plasmids, respectively.Flow cytometry was used to detect the distributions of cell cycle and western blot assay was used to determine the expression levels of CyclinE and CDK2 after cells were exposed to 200 μg/ml silica for 0,3,6,12,24 h.Results The proportion of G1 phases in negative control cells decreased from 83.53%±2.24% to 69.11%±3.12%after exposure to silica; the proportion of G1 phases in H-Ku80 and H-PKcs cells exposed to silica decreased from 85.16%±3.73% to 59.92%±3.31% and from 75.06%±2.23% to 58.32%±1.35%, respectively (P＜0.05).The exposure to silica resulted in the increasing protein expression levels of CyclinE and CDK2 in negative control cells, and the expression levels of CyclinE were obviously suppressed in H-Ku80 and H-PKcs as compared with control cells. However, the expression level of CDK2 protein did not change significantly.Conclusion DNA-PK might play a role in silica-induced alternations of cell cycle and regulate silica-induced overexpression of CyclinE in human embryo lung fibroblasts.     

DNA甲基化及其影响因素

在人类基因组中,DNA甲基化是一种重要的表遗传修饰,它与多种疾病发生密切相关。DNA甲基化在基因转录过程中起着重要的作用,与组蛋白修饰、染色质构型重塑共同参与转录调控。目前研究发现,基因组中DNA甲基化的水平与DNA甲基转移酶(DNMT)、组蛋白甲基化、饮食与环境、RNA干扰和病毒感染等多种因素有关。     

氢醌对人支气管上皮细胞DNA损伤及细胞周期的影响

目的探讨氢醌(HQ)对体外培养人支气管上皮细胞DNA损伤及细胞周期的影响。方法将HBE细胞用不同浓度(0、5、10、20、40、80、160、320μmo/lL)的氢醌作用24 h,采用噻唑蓝(MTT)比色法测定16HBE细胞的相对存活率,用单细胞凝胶电泳(SCGE)检测细胞DNA的损伤情况,流式细胞术检测细胞周期分布。结果在0～40μmo/lL范围内HQ作用24 h后,16HBE细胞的存活率未见明显变化(P>0.05);当染毒剂量超过80μmo/lL时,细胞存活率明显下降(P<0.01)。SCGE显示随着HQ浓度的升高,16HBE细胞的DNA断裂程度加重。HQ作用浓度在10～320μmo/lL范围内,16HBE细胞的细胞周期表现为G2期阻滞,G1期比例下降。结论 HQ会对16HBE细胞的DNA产生损害,并且引起G2期细胞阻滞。     

恶性转化人支气管上皮细胞基因组甲基化观察

目的对反式二氢二醇环氧苯并芘（anti-BPDE）和结晶型硫化镍（NiS）恶性转化人支气管上皮细胞（Hu-man bronchial epithelia,16HBE）基因组DNA甲基化状况进行研究,寻找DNA甲基化异常的基因片段,探讨反式-BPDE和NiS的表遗传致癌机制.方法采用限制性指纹识别技术（MSRF）,对反式-BPDE和NiS分别诱导转化及裸鼠成瘤的4种人支气管上皮细胞株基因组进行分析;对异常甲基化基因阳性片断采用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恶性转化人支气管上皮细胞基因组存在高甲基化的DNA片段,其中一基因片段与编码鼻咽癌易感性蛋白ANKRD11基因序列99%同源.另一基因片段与HOXA3基因序列99%同源;未发现反式BPDE恶性转化人支气管上皮细胞基因组DNA异常甲基化基因片段.结论结晶型NiS恶性转化人支气管上皮细胞DNA的高度甲基化可能导致基因表达抑制.可能是结晶型NiS致癌的一种表遗传机制;反式BPDE致癌过程可能与基因组磷酸胞苷酰（CpG）岛甲基化异常关系不明确.     

肥胖小鼠DNA甲基化改变以及n-3多不饱和脂肪酸的影响作用

【目的】 探讨肥胖状态下脂肪组织基因组DNA甲基化及甲基转移酶表达改变以及n-3多不饱和脂肪酸(n-3 PUFAs)的影响作用。 【方法】 使用30只3~4周龄C57BL/6J雄性小鼠,随机分为3组,每组10只。小鼠分别给予两种高脂饲料(脂肪含量为34.9%,供能比为60%)-n-6 PUFAs(脂肪来源于葵花籽油)饲料和n-3 PUFAs(脂肪来源于鱼油)饲料喂养14周;以正常脂饲料(脂肪含量为4.3%,供能比为10%)(脂肪来源于葵花籽油)为对照。喂养结束后对小鼠脂肪组织基因组DNA总甲基化以及甲基转移酶(DNMTs)进行测定。 【结果】 与正常脂饲料喂养小鼠相比,两组高脂饲料诱导肥胖小鼠的脂肪组织DNA总甲基化程度均明显升高,但n-3 PUFAs高脂饲料组升高的程度显著低于n-6 PUFAs组。n-6 PUFAs高脂饲料诱导肥胖小鼠的脂肪组织DNMT1、DNMT3a和DNMT3b的表达均显著高于正常脂饲料组小鼠,而n-3 PUFAs高脂饲料喂养小鼠脂肪组织中这3种酶的表达无变化。 【结论】 肥胖状态下脂肪组织基因组DNA甲基化程度升高;鱼油n-3 PUFAs具有抑制DNA甲基化的作用。     

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。     

短期暴露于纳米SiO_2对HaCaT细胞基因组DNA甲基化的影响

目的探讨纳米SiO2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2.5、5、10μg/ml纳米SiO2溶液和10μg/ml微米级SiO2处理人皮肤表皮细胞系(HaCaT)24h;以3μmol/L DNA甲基化转移酶抑制剂5-脱氧杂氮胞苷(DAC)处理48 h的10μg/ml组为阳性对照,并设立溶剂对照组。应用高效毛细管电泳(HPCE)定量分析纳米SiO2处理细胞24h后,基因组DNA总体甲基化的变化,用Q-PCR和Western Blot方法检测甲基化相关蛋白mRNA和蛋白的表达变化,并用DNA甲基化转移酶活性试剂盒检测DNA甲基化转移酶的活性。结果 HPCE定量分析结果显示纳米SiO2可引起HaCaT细胞总体甲基化程度降低,呈剂量依赖性关系;与正常细胞相比,微米SiO2以及2.5、5和10μg/ml纳米SiO2组分别降低37.8%、43.9%、54.9%和52.8%,差异有显著性(P<0.05);对照组5-aza-dC处理后使HaCaT细胞甲基化程度减少27.3%。各组酶的蛋白表达与mRNA表达水平及DNMTs活性变化具有相同的变化趋势,经纳米SiO2处理的HaCaT细胞,DNMT1、DNMT3a及MBD2表达随着纳米SiO2剂量的上升而下降,DAC组表达水平最低。结论纳米SiO2能引起HaCaT细胞整体基因组DNA甲基化水平降低,可能与DNA甲基化转移酶的酶活性降低有关。     

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。     

氢醌对DNA甲基转移酶表达的影响

目的探索氢醌(hydroquinone,HQ)致DNA整体低甲基化的分子机制。方法以磷酸盐缓冲液(PBS)溶解HQ,以PBS处理组为对照组,分别以2.5、5.0、10.0和20.0μmo/lL HQ染毒TK6细胞为处理组。应用实时荧光定量-聚合酶链反应检测DNA甲基转移酶DNMT1、DNMT3a和DNMT3b的表达水平。结果与对照组相比,DNMT1、DNMT3a和DNMT3b的mRNA表达量在各HQ处理组细胞中均下降,其中以20.0μmo/lL组细胞的下降最为明显,分别下降46%(P0.05)、83%(P0.05)和48%(P0.05),且DNMT1和DNMT3a的表达量随着HQ剂量的增加而下降。结论 HQ致DNA整体低甲基化下调机制可能与DNMT1、DNMT3a和DNMT3b表达异常有关。