细胞周期蛋白D1在支气管哮喘小鼠肺组织中的表达及其与气道重塑的关系

目的 研究细胞周期蛋白D1(Cyclin D1)在支气管哮喘(简称哮喘)小鼠肺组织中的表达,探讨Cyclin D1在哮喘及气道重塑中的作用.方法 将40只SPF级BALB/c小鼠按随机数字表法分为正常对照组(A组)、哮喘雾化2周组(B组)、哮喘雾化4周组(C组)、哮喘雾化8周组(D组)4组,每组10只.用10%鸡卵白蛋白(OVA)致敏和1%OVA激发小鼠建立哮喘模型.分析支气管肺泡灌洗液(BALF)中嗜酸粒细胞(EOS)计数及分类;用动物肺功能仪检测各组小鼠肺功能状况;用苏木精-伊红(HE)染色观察气道炎症及细胞浸润情况;用图像分析软件观察气道壁及平滑肌层变化情况;用逆转录-聚合酶链反应(RT-PCR)及实时定量(Real-time)PCR测定肺组织中Cyclin D1 mRNA水平表达变化;用Western blot法观察肺组织中Cyclin D1的蛋白表达变化.结果 BALF分析结果提示,B、C、D组EOS计数分别为(42.6±0.9)×104/L、(54.7±1.4)×104/L、(44.8±2.4)×104/L,与A组[(3.4±0.5)×104/L]比较差异有统计学意义(q值分别为79.75、91.42、84.82,P均＜0.01);对小鼠呼气阻力检测发现,乙酰胆碱浓度为45 μg/kg时B、C、D组分别为(5.27±0.16)cm·L-1·min-1、(6.68士0.20)cm·L-1·min-1、(7.14±0.41)cm·L-1·min-1,与A组[(4.11±0.15)cm·L-1·min-1]比较差异有统计学意义(q值分别为5.58、6.39、7.11,P均＜0.05);支气管平滑肌面积/管腔内周长(Wam/Pi)B组为2.8±0.6,C组为4.8±0.6,D组为6.4±0.7,与A组(2.4±0.4)比较差异有统计学意义(q值分别为6.40、8.28、9.27,P＜0.05);管壁面积/管腔内周长(Wat/Pi)B组为6.4±0.8,C组为8.3±1.2,D组为9.3±1.0,与A组(5.6±1.0)比较差异有统计学意义(q值分别为2.80、4.83、6.37,P均＜0.05);Western blot检测发现Cyclin D1在B、C、D组表达量分别为0.587±0.015、0.808±0.029、0.826±0.022,与A组(0.404±0.016)比较差异有统计学意义(q值分别为5.87、8.08、8.26,P均＜0.01);相关性分析结果提示呼气阻力和Cyclin D1水平表达呈正相关(r=0.83,P＜0.05).结论 Cyclin D1在哮喘小鼠肺组织中表达增加,其表达与气道反应性呈正相关,Cyclin D1可能通过细胞外信号调节激酶(ERK)信号通路参与气道重塑过程.     

亚砷酸钠对HaCaT细胞MGMT基因启动子区甲基化CpG结合蛋白-2、DNA甲基转移酶1、组蛋白去乙酰化酶1结合的影响

目的 观察亚砷酸钠(NaAsO2)对人肤角质形成细胞株(HaCaT细胞)MGMT基因启动子区甲基化CpG结合蛋白-2(MeCP2)、DNA甲基转移酶1(DNMT1)及组蛋白去乙酰化酶1(HDAC1)结合情况的影响,为深化阐释砷毒作用机制提供依据.方法 分别以0.00(空白对照)、3.13、6.25、12.50、25.00 μmol/L NaAsO2重复间隔处理HaCaT细胞72 h(NaAsO2处理24h,隔天再次相同处理,重复3次),以人表皮鳞癌细胞株(A431)作为阳性对照,定量染色质免疫共沉淀技术(Q-ChIP)检测MGMT基因转录调控区ChIP1、ChIP2区域及MGMT基因编码区ChIP3区域MeCP2、DNMT1、HDAC1结合情况.结果 各组HaCaT细胞MGMT基因转录调控区ChIP1、ChIP2区域MeCP2、DNMT1、HDAC1蛋白结合水平比较,差异有统计学意义(F值分别为7.387、84.634、78.442和19.263、69.649、26.546,P均＜0.05);其中各NaAsO2处理组ChIP1、ChIP2区域MeCP2、DNMT1、HDAC1蛋白结合水平[3.13 μmol/L NaAsO2处理组:(136.00±16.97)％、(145.00±2.83)％、(88.50±19.09)％和(106.50±37.48)％、(112.34±8.73)％、(59.71±8.49)％;6.25 μmol/L NaAsO2处理组:(130.00±42.43)％、(154.50±4.95)％、(101.00±1.27)％和(88.50±3.54)％、(134.32±2.82)％、(102.75±19.91)％;12.50 μmol/LNaAsO2处理组:(141.50±23.33)％、(161.50±7.78)％、(125.00±11.31)％和(119.50±24.75)％、(171.59±3.54)％、(167.61±10.61)％;25.00 μmol/NaAsO处理组:(134.50±43.13)％、(472.50±50.20)％、(383.50±30.41)％和(180.09±12.73)％、(348.50±27.58)％、(158.45±12.02)％]均高于空白对照组[(51.50±9.19)％、(82.00±12.73)％、(25.03±2.91)％和(37.02±4.24)％、(91.56±26.16)％、(19.09±2.90)％,P均＜0.05].各组HaCaT细胞MGMT基因编码区ChIP3区域MeCP2蛋白结合水平比较,差异无统计学意义(F=1.670,P＞0.05),而DNMT1、HDAC1蛋白结合水平比较,差异有统计学意义(F值分别为4.404、9.863,P均＜0.05),其中25.00 μmol/L NaAsO2处理组DNMT1、HDAC1蛋白结合水平[(615.85±29.63)％、(306.09±59.40)％]与空白对照组[(99.70±12.02)％、(92.45±48.79)％]比较,差异有统计学意义(P均＜0.05).结论 MeCP2可结合于砷所致高甲基化MGMT基因转录调控区,通过招募DNMT1及HDAC1使组蛋白去乙酰化,同时DNMT1可结合于MGMT基因编码区,以非甲基化DNA结合蛋白(MBD)依赖的方式招募HDAC1,通过染色质重塑方式导致MGMT基因沉默,可能是砷毒性表现的早期分子事件.     

Developmental changes in DNA methylation and covalent histone modifications of chromatin associated with the epsilon-, gamma-, and beta-globin gene promoters in Papio anubis

The baboon is a suitable and relevant animal model to study the mechanism of human globin gene switching. This investigation addresses the role of DNA methylation and histone coding in globin gene switching in the baboon, Papio anubis. Bisulfite sequencing and chromatin immunoprecipitation studies were performed in erythroid cells purified from fetuses of varying gestational ages and from adult bone marrow to analyze the manner that changes in DNA methylation of the epsilon-, gamma-, and beta-globin promoters and association of ac-H3, ac-H4, H3-dimeK4, H3-dimeK36, and H3-dimeK79 with the epsilon-, gamma-, and beta-globin promoters occur during development. Changes in DNA methylation of the epsilon- and gamma-globin gene promoters during transitional stages of globin gene switching were consistent with the stochastic model of methylation and a role of DNA methylation in gene silencing. Enrichment of ac-H3, ac-H4, and pol II at the promoters of developmentally active genes was observed, while the pattern of distribution of H3-dimeK4 and H3-dimeK79 suggests that these modifications are found near both currently and formerly active promoters. Enrichment of H3-dimeK36 at the silenced epsilon-globin gene promoter was observed. These studies demonstrate that coordinated epigenetic modifications in the chromatin structure of the beta-like globin gene promoters accompany the highly regulated changes in expression patterns of these genes during development.     

肝癌中cyclinD1扩增与HBV感染及病理学的关系

目的探讨癌基因ｃｙｃｌｉｎＤ１在肝癌发生发展中的作用以及与ＨＢＶ感染和临床病理之间的关系．方法取手术切除的肝细胞癌标本,用Ｓｏｕｔｈｅｒｎ杂交的方法对ｃｙｃｌｉｎＤ１和ＣＤＫ４癌基因以及ＨＢＶ的感染状态进行了检测,切片同时行病理检查．结果肝癌３９例中９例ｃｙｃｌｉｎＤ１存在２～３０倍的扩增（２３７％）,其中１例检出重组杂交带;３７例中５例（１３５％）有ＣＤＫ４的扩增;ＨＢＶＤＮＡ在肝癌组织中检出的阳性率为９４７％,ＨＢＶＤＮＡ整合率为７４４％．相关分析发现,ｃｙｃｌｉｎＤ１的扩增与ＣＤＫ４扩增,ＨＢＶＤＮＡ整合,肿瘤的分化高低和肿块大小等之间有相关关系,相关系数ｒ分别为０５１,０３１,０３９和０５５,全部Ｐ＜００５．结论ｃｙｃｌｉｎＤ１的扩增与ＨＢＶＤＮＡ的整合密切相关,并在肝癌的恶化进展中起着重要作用．     

SREBP-1 binds to multiple sites and transactivates the human ApoA-II promoter in vitro : SREBP-1 mutants defective in DNA binding or transcriptional activation repress ApoA-II promoter activity.

-Screening of an expression human liver cDNA library resulted in the isolation of several cDNA clones homologous to sterol regulatory element-binding protein-1 (SREBP-1) that recognize the regulatory element AIIAB and AIIK of the human apoA-II promoter. DNaseI footprinting of the apoA-II promoter using SREBP-1 (1 to 460) expressed in bacteria identified 5 overall protected regions designated AIIAB (-64 to -48), AIICD (-178 to -154), AIIDE (-352 to -332), AIIHI (-594 to -574), and AIIK (-760 to -743). These regions contain inverted E-box palindromic or direct repeat motifs and bind SREBP-1 with different affinities. Transient cotransfection experiments in HepG2 cells showed that SREBP-1 transactivated the -911/29 apoA-II promoter 3.5-fold as well as truncated apoA-II promoter segments that contain 1, 2, 3, or 4 SREBP binding sites. Mutagenesis analysis showed that transactivation by SREBP was mainly affected by mutations in element AIIAB. Despite the strong transactivation of the apoA-II promoter by SREBP-1 we could not demonstrate significant changes on the endogenous apoA-II mRNA levels of HepG2 cells after cotransfection with SREBP-1 or in the presence or absence of cholesterol and 25-OH-cholesterol. An SREBP-1 mutant lacking the amino-terminal activation domain bound normally to its cognate sites and repressed the apoA-II promoter activity. Repression was also caused by specific amino acid substitutions of Leu, Val, or Gly for Lys359, which affected DNA binding. Repression by the DNA binding-deficient mutants was abolished by deletion of the amino-terminal activation domain (1 to 90) of SREBP-1. Overall, the findings suggest that the wild-type SREBP-1 can bind and transactivate efficiently the apoA-II promoter in cell culture. SREBP-1 mutants lacking the activation domain bind to their cognate sites and directly repress the apoA-II promoter whereas mutants defective in DNA binding indirectly repress the apoA-II promoter activity, possibly by a squelching mechanism.     

Examination of ATM,BRCA1, and BRCA2 promoter methylation in patients with pancreatic cancer

BackgroundPancreatic cancer is a lethal disease with a poor 5-year survival rate. Pathogenic germline variants in the coding regions of ATM, BRCA1, and BRCA2 are found in up to 4.8% of pancreatic cancer patients. Germline promoter methylation and gene silencing arising from a germline variant or through other mechanisms have been described as a cause of tumor suppressor gene inactivation.MethodsWe measured the level of promoter methylation of the ATM, BRCA1, and BRCA2 genes in peripheral blood lymphocytes from 655 patients with pancreatic cancer using real-time PCR.ResultsNo evidence of germline promoter methylation of any of these genes was found. Promoter methylation levels were minimal with no patient having promoter methylation greater than 3.4%, 3.3%, and 7.6% for ATM, BRCA1 and BRCA2, respectively, well below levels found in patients who have inherited promoter methylation (∼50%).ConclusionsWe found no evidence of germline promoter methylation for the pancreatic susceptibility genes ATM, BRCA1 and BRCA2 in patients with pancreatic cancer. This study reveals that constitutive germline methylation of promoter CpG islands is rare in pancreatic cancer.     

Age-dependent response of lymphocytes in the induction of the linker histone variant, H1 degrees and histone H4 acetylation after treatment with the histone deacetylase inhibitor, trichostatin A

In the present study we investigated the age-related response of Phytohemaglutinin (PHA)-activated S phase human lymphocytes isolated from peripheral blood from donors of four different age groups, namely young (25-30 years), mid-aged (40-45 years), senior (60-65 years) and elderly (80-95 years) on the induction of the linker histone variant, H1 degrees and histone H4 acetylation after treatment with the very specific histone deacetylase (HDAC) inhibitor, trichostatin A (TSA). The cell system of peripheral blood lymphocytes is ideal for the study of H1 degrees induction since they do not synthesize this particular linker histone variant. Lymphocytes isolated from peripheral blood were activated with PHA (5 microg/10(6) cells/ml medium) and placed in culture for a duration of 72 h at which time cells are in the S phase. Forty-eight hours after inoculation, TSA (250 ng/10(6) cells/ml medium) was added to the cell cultures for a period of 24 h. Assays were performed 72 h after initiation of cultures. The results showed that the induction of H1 degrees after TSA treatment increased to a statistically significant degree in the elderly age group with respect to both the young and the mid-aged age groups. Moreover histone H4 acetylation was found to increase as a function of increasing donor age. A hyperacetylation pattern was observed even in the youngest age group analyzed. Specifically, the tetra-acetylated (H4.4) H4 form increased to a statistically significant degree with the concomitant decrease in the non-acetylated H4 for (H4.0) as a function of donor age. The other acetylated H4 forms (H4.1, H4.2, and H4.3) remained more or less constant, irrespective of donor age. These results show that the sensitivity of lymphocytes to TSA is enhanced with increasing donor age. Since to date, 11 class I and II HDACs have been isolated that have been found by other investigators to have differential responses to HDAC inhibitors, these findings may indicate that there is also a differential age-related response of certain HDACs or perhaps a senescent-specific HDAC. This line of research warrants further study.