苯并芘%DNA甲基化%聚ADP核糖聚合酶类%DNA(胞嘧啶5-)-甲基转移酶
苯併芘%DNA甲基化%聚ADP覈糖聚閤酶類%DNA(胞嘧啶5-)-甲基轉移酶
분병비%DNA갑기화%취ADP핵당취합매류%DNA(포밀정5-)-갑기전이매
Benzo(a) pyrene%DNA methylation%Poly(ADP-ribose) polymerases%DNA ( cytosine-5 - ) -methyltransferase
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.
目的 觀察苯併(a)芘[benzo(a)pyrene,B(a)P]誘導體外細胞DNA甲基化水平改變,探討聚腺苷二燐痠覈糖聚閤酶1[poly(ADP-ribose)polymerase 1,PARP1]在該過程中的作用.方法 以1.0、2.0、5.0、10.0、15.0、30.0 μmol/L濃度B(a)P分彆處理人支氣管上皮細胞(16HBE)及其PARP1缺陷細胞(16HBE-shPARP1)72 h.採用免疫熒光和高效毛細管電泳檢測其基因組DNA整體甲基化水平改變,同時動態鑑測PARP1和DNA甲基轉移酶1(DNA methyltransferases 1,DNMT1)錶達的變化.結果 16HBE和16HBE-shPARP1細胞基因組整體甲基化百分比(mCpG%)分彆為(4.04±0.08)%和(9.69±0.50)%.經5-氮雜脫氧胞苷(DAC)處理72 h後,mCpG%值分彆下降為(3.15±0.14)%、(6.07±0.54)%.經B(a)P染毒72 h後,16HBE細胞基因組mCpG%值[B(a)P濃度由低到高]分彆為(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細胞基因組mCpG%值(濃度由低到高)分彆為(10.63±0.60)、(13.08±0.68)、(9.75±0.55)、(7.32±0.67)、(6.90±0.49)、(6.27±0.21).兩種細胞不同處理組間mCpG%差異有統計學意義(F值分彆為61.67、60.91,P值均＜0.01).16HBE細胞各劑量組[B(a)P濃度由低到高]PARP1基因mRNA相對錶達水平分彆為對照組的141.0%、158.0%、167.0%、239.0%、149.0%、82.9%,差異均有統計學意義(t值分彆為11.45、17.32、32.24、33.44、20.21、9.87,P值均＜0.01);16HBE-shPARP1細胞各劑量組(濃度由低到高)PARP1基因mRNA相對錶達水平分彆為對照組的169.0%、217.0%、259.0%、323.0%、321.0%、256.0%,差異有統計學意義(t值分彆為9.06、15.92、22.68、26.23、37.19、21.15,P值均＜0.01).噹B(a)P染毒劑量達5.0 μmol/L後,16HBE細胞各劑量組(濃度由低到高)DNMT1基因mRNA相對錶達水平分彆為對照組的125.0%、162.0%、275.0%、233.0%,差異有統計學意義(t值分彆為12.74、24.92、55.11、59.07,P值均＜0.01);噹B(a)P染毒劑量達2.0 μmol/L後,16HBE-shPARP1細胞各劑量組(濃度由低到高)DNMT1基因mRNA相對錶達水平分彆為對照組的135.0%、151.0%、180.0%、229.0%、186.0%,差異有統計學意義(t值分彆為23.82、40.17、32.69、74.85、46.76,P值均＜0.01).結論 B(a)P誘導的16HBE細胞基因組整體甲基化水平降低可能是其噁性轉化過程中早期重要的分子事件,PARP1可通過抑製DNMT1的酶活性來調節B(a)P誘導的16HBE細胞DNA甲基化水平,這種效應可因PARP1的缺失而緩解.
목적 관찰분병(a)비[benzo(a)pyrene,B(a)P]유도체외세포DNA갑기화수평개변,탐토취선감이린산핵당취합매1[poly(ADP-ribose)polymerase 1,PARP1]재해과정중적작용.방법 이1.0、2.0、5.0、10.0、15.0、30.0 μmol/L농도B(a)P분별처리인지기관상피세포(16HBE)급기PARP1결함세포(16HBE-shPARP1)72 h.채용면역형광화고효모세관전영검측기기인조DNA정체갑기화수평개변,동시동태감측PARP1화DNA갑기전이매1(DNA methyltransferases 1,DNMT1)표체적변화.결과 16HBE화16HBE-shPARP1세포기인조정체갑기화백분비(mCpG%)분별위(4.04±0.08)%화(9.69±0.50)%.경5-담잡탈양포감(DAC)처리72 h후,mCpG%치분별하강위(3.15±0.14)%、(6.07±0.54)%.경B(a)P염독72 h후,16HBE세포기인조mCpG%치[B(a)P농도유저도고]분별위(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-shPARP1세포기인조mCpG%치(농도유저도고)분별위(10.63±0.60)、(13.08±0.68)、(9.75±0.55)、(7.32±0.67)、(6.90±0.49)、(6.27±0.21).량충세포불동처리조간mCpG%차이유통계학의의(F치분별위61.67、60.91,P치균＜0.01).16HBE세포각제량조[B(a)P농도유저도고]PARP1기인mRNA상대표체수평분별위대조조적141.0%、158.0%、167.0%、239.0%、149.0%、82.9%,차이균유통계학의의(t치분별위11.45、17.32、32.24、33.44、20.21、9.87,P치균＜0.01);16HBE-shPARP1세포각제량조(농도유저도고)PARP1기인mRNA상대표체수평분별위대조조적169.0%、217.0%、259.0%、323.0%、321.0%、256.0%,차이유통계학의의(t치분별위9.06、15.92、22.68、26.23、37.19、21.15,P치균＜0.01).당B(a)P염독제량체5.0 μmol/L후,16HBE세포각제량조(농도유저도고)DNMT1기인mRNA상대표체수평분별위대조조적125.0%、162.0%、275.0%、233.0%,차이유통계학의의(t치분별위12.74、24.92、55.11、59.07,P치균＜0.01);당B(a)P염독제량체2.0 μmol/L후,16HBE-shPARP1세포각제량조(농도유저도고)DNMT1기인mRNA상대표체수평분별위대조조적135.0%、151.0%、180.0%、229.0%、186.0%,차이유통계학의의(t치분별위23.82、40.17、32.69、74.85、46.76,P치균＜0.01).결론 B(a)P유도적16HBE세포기인조정체갑기화수평강저가능시기악성전화과정중조기중요적분자사건,PARP1가통과억제DNMT1적매활성래조절B(a)P유도적16HBE세포DNA갑기화수평,저충효응가인PARP1적결실이완해.
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.
