蛋白质去乙酰化酶抑制剂对炎症反应的调控及其对糖皮质激素的作用

组蛋白尾端不同的化学修饰往往和基因的表达调控有着密切的关系,乙酰化就是常见的一种修饰形式。研究表明,组蛋白的乙酰化与去乙酰化状态,可作用于T、B细胞的发育、分化、成熟、分泌及表面因子的表达等多个方面。随着研究的深入,发现蛋白质乙酰化状态对细胞因子的分泌、NO的产生等方面均有较大的影响。乙酰化及去乙酰化作为一种影响炎症反应的重要因素,其机理及药用价值逐渐为人们所重视。     

曲古菌素A对HL-60细胞组蛋白乙酰化水平和凋亡的作用

本研究旨在探讨曲古菌素A(trichostatinA ,TSA)高效低毒的抗癌机理。应用细胞培养、MTT法 ,免疫细胞化学技术和Annexin V FITC PI双标流式细胞术观察TSA对HL 6 0细胞和正常人外周血单个核细胞 (normalhumanperipheralbloodmononuclearcell,NPBMNC)的生长抑制 ,组蛋白乙酰化水平以及诱导凋亡的影响。结果表明 :TSA能够以时间、剂量依赖性方式抑制HL 6 0细胞增殖 ,36小时IC50 为 10 0ng ml。TSA能够诱导HL 6 0细胞凋亡 ,其作用也呈时间、剂量依赖性。TSA在显著诱导HL 6 0细胞凋亡的浓度和时间范围内对NPBMNC无明显的诱导凋亡作用。TSA处理 4小时后 ,HL 6 0细胞和NPBMNC组蛋白乙酰化水平上调显著高于未用TSA各组 (P<0 .0 5 ) ,但两者之间无显著性差异 (P >0 .0 5 )。结论 :TSA对HL 6 0细胞有明确的抑制增殖能力 ;TSA有明确的诱导HL 6 0细胞凋亡的能力 ,这可能是TSA体外抑制白血病细胞系HL 6 0生长和发挥抗白血病作用的主要机理之一 ;与NPBMNC相比较 ,TSA能够选择性诱导HL 6 0细胞凋亡 ;TSA选择性抑制HL 6 0细胞的机理与TSA调控HL 6 0细胞和NPBMNC组蛋白乙酰化水平的差异无关。     

BCL11A基因对γ-珠蛋白基因转录的影响

本研究旨在探讨B细胞淋巴瘤/白血病11A（B-cell lymphoma/leukemia 11A,BCL11A）基因对γ-珠蛋白基因转录的影响。通过小干扰RNA（small interfering RNA,siRNA）技术沉默人红白血病细胞（K562细胞）的BCL11A基因的表达,用实时荧光定量RT-PCR法定量细胞内γ-珠蛋白基因mRNA水平,分析BCL11A基因对γ-珠蛋白基因转录的调控作用。结果表明,所构建的4个siRNA表达载体对BCL11A基因表达的沉默率分别为49.7%、55.4%、78.2%和84.1%。将沉默率为84.1%的siRNA表达载体转导K562细胞,K562细胞的γ-珠蛋白基因的转录水平较转导对照载体质粒升高了2.4倍。结论：BCL11A基因表达被沉默后γ-珠蛋白基因mRNA水平升高显示BCL11A基因对γ-珠蛋白基因表达可能存在负调控。     

组蛋白去乙酰化酶抑制剂对乳腺癌细胞株MCF-7增殖周期的影响

目的 研究组蛋白去乙酰化酶抑制剂对乳腺癌细胞株增殖周期的影响.方法 培养乳腺癌细胞株MCF-7,用TSA、SAHA、CS055、MS-275 4种不同类型的组蛋白去乙酰化酶抑制剂分别作用于细胞,于24、48、72、96 h用MTT比色法观察细胞生长情况,初筛出高效的抑制剂.用所选出的抑制剂的不同浓度处理细胞,流式细胞术检测S期细胞的比例和周期素Cyclin D1、Cyclin A2,并进行统计分析.结果 在TSA、SAHA、CS055和MS-275 4种不同类型的组蛋白去乙酰化酶抑制剂中,SAHA显示出较强的抑制能力(P＜0.05);在一定时间和浓度范围内,其抑制能力有明显的时效关系;在抑制剂作用下,S期细胞比例和周期素Cyclin A2表达下降,Cyclin D1表达上升(P均＜0.05).结论 组蛋白去乙酰化酶抑制剂SAHA是乳腺癌细胞增殖的高效抑制剂,其抑制作用有一定的时效关系;周期素Cyclin D1、Cyclin A2参与了抑制剂对细胞增殖周期的调控.     

异丙酚暴露对老年大鼠的认知功能和组蛋白去乙酰化酶的影响

目的 探讨异丙酚暴露对老年大鼠的认知功能和组蛋白去乙酰化酶(HDAC)的影响。方法 选取健康雄性Wistar大鼠30只,通过随机数表法将大鼠分为对照组、实验组及空白组,每组10只大鼠。空白组于正常环境下喂养,对照组给予异丙酚注射后注入90 mg/kg等渗溶媒,实验组给予异丙酚注射后注入90 mg/kg HDAC抑制剂。用Morris水迷宫实验记录逃避潜伏期评估大鼠认知功能,干预结束后分离脑组织使用免疫荧光法检测HDAC蛋白的表达水平,并采用酶联免疫吸附试验测定脑细胞因子[肿瘤坏死因子α(TNF-α)、缺氧诱导因子1α(HIF-1α)、脑源性神经营养因子(BDNF)、血管内皮生长因子(VEGF)]的含量。结果 干预后1、3、5 d,实验组及对照组潜伏期均高于空白组,但实验组潜伏期低于对照组,差异均有统计学意义(P<0.05)。实验组及对照组跨格数、直立次数及中央区域停留时间均高于空白组,但实验组低于对照组,差异均有统计学意义(P<0.05)。实验组及空白组HDAC含量均低于对照组,且实验组低于空白组,差异均有统计学意义(P<0.05)。实验组、对照组大鼠脑组织中TNF-...     

组蛋白去乙酰化酶抑制剂SAHA对树突状细胞成熟的影响及其机制

目的:本研究探讨新型组蛋白去乙酰化酶抑制剂SAHA(Suberoylanilide hydroxamic acid)对健康人外周血单个核细胞(PBMNC)来源树突状细胞(DC)成熟的影响及其机制。方法:PBMNC取自健康志愿者的外周血,经贴壁处理后培养于含100 ng/ml rh GM-CSF、500 U/ml rh IL-4的RPMI 1640完全培养液中,用脂多糖(LPS)刺激经SAHA处理和未经处理的DC为实验组,并将不经LPS和SAHA处理的细胞设为对照组,在倒置显微镜下观察各组细胞形态学改变;流式细胞术检测各组DC表面抗原的表达情况;采用混合淋巴细胞培养法(MLC)观察各组DC对同种异体T淋巴细胞的刺激作用;应用凝胶电泳迁移变动测定法(EMSA)检测各组DC NF-κB通路的活化情况。结果:SAHA能够有效抑制LPS诱导的DC成熟,主要表现在经SAHA+LPS处理的DC具有未成熟DC的形态学特征;经SAHA+LPS处理组和对照组的DC表面抗原CD80、CD83、HLA-DR的表达量均低于单用LPS组(P0.01);SAHA+LPS处理组和对照组的DC刺激同种异体淋巴细胞增殖的能力明显低于单用LPS刺激组(P0.01);电泳迁移变动检测(EM SA)实验结果提示,SAHA+LPS处理的DC NF-κB活性显著下降。结论:SAHA能够有效抑制DC的成熟和对异基因T淋巴细胞的刺激作用,该作用与SAHA抑制DC NF-κB信号通路活化有关。     

孕妇血清亲环素A和组织型转谷氨酰胺酶水平检测与子痫前期发生不良妊娠的相关性研究

目的　探讨孕妇血清亲环素A(cyclophilin A,CYPA)和组织型转谷氨酰胺酶(tissue transglutaminase,tTG)水平的变化与子痫前期(preeclampsia,PE)发生不良妊娠的相关性。方法　选择 2018年1月~2020年1月在咸阳市第一人民医院和西安市第一医院就诊的112例PE孕妇作为研究对象,依据妊娠结局分为正常妊娠组(85例)和不良妊娠组(27例),另选取51例同期体检健康孕妇作为对照组。采用酶联免疫吸附法（ELISA）检测血清CYPA和tTG水平,采用免疫比浊法检测24h尿蛋白水平,通过彩色多普勒超声检查获得所有研究对象的子宫动脉血流阻力参数,包括搏动指数(pulsatility index,PI)和阻力指数(resistance index, RI)。收集所有研究对象的血压等临床资料,比较分析以上指标的变化与PE孕妇发生不良妊娠的相关性。结果　对照组、正常妊娠组和不良妊娠组血清CYPA和tTG水平分别为59.16±9.71,79.50±13.91,96.18±17.25ng/L和7.21±1.13,10.68±1.67,13.35±2.35ng/ml。与对照组比较,正常妊娠组和不良妊娠组的tTG和CYPA水平明显增高,其中不良妊娠组增高更显著,差异均有统计学意义（F=59.286~100.061,均P=0.000)。相关性分析显示,正常妊娠组和不良妊娠组的tTG和CYPA水平分别呈正相关性(r=0.793,0.812,均P<0.01)。正常妊娠组的tTG和CYPA水平分别与收缩压、24h尿蛋白水平、RI及PI呈正相关性(rtTG=0.802,0.816,0.823,0.799,均P<0.01; rCYPA=0.823,0.806,0.813,0.805,均P<0.01)。不良妊娠组的tTG和CYPA水平分别与收缩压、 24h尿蛋白水平、RI及PI呈正相关性(rtTG=0.815,0.827,0.833,0.807,P均<0.01;rCYPA=0.831,0.812,0.824,0.817,均P<0.01)。结论　tTG和CYPA可能参与PE的发病机制。高水平的tTG和CYPA可能诱导妊娠高血压和蛋白尿的发生,进而增加PE孕妇发生不良妊娠的风险。     

血小板衍化生长因子A基因转染成骨细胞生长特性的影响

背景:采用基因转染技术加强或改造种子细胞的功能是当今组织工程研究中的热点问题,用血小板衍化生长因子A转染成骨细胞用于牙周组织再生少见报道。目的:观察血小板衍化生长因子A基因转染成骨细胞后对其生长特性的影响。方法:构建血小板衍化生长因子A真核表达质粒,分离培养3T3细胞并进行不同方式的转染,RT-PCR检测血小板衍化生长因子A基因在成骨细胞中的表达;MTT法检测细胞的增殖活性;体外细胞划痕实验观察细胞的迁移情况。结果与结论:RT-PCR检测结果显示,血小板衍化生长因子A真核表达质粒转染的细胞表达量明显增加、生长、迁移速度明显增快(P<0.05)。说明脂质体介导血小板衍化生长因子A基因成功转染成骨细胞,并能促进细胞的增殖和迁移,为血小板衍化生长因子A基因在牙周组织中的治疗奠定基础。     

银杏叶提取物对糖尿病大鼠肾脏组织型转谷氨酰胺酶表达的影响

目的观察银杏叶提取物对糖尿病大鼠肾脏组织型转谷氨酰胺酶（tTG）表达的影响。方法链脲佐菌素（STZ）复制糖尿病动物模型,将糖尿病动物随机分为,糖尿病组（DM）,银杏叶提取物组（GBE）;另设正常对照组（Con-trol）。逆转录聚合酶链式反应（RT-PCR）检测肾皮质tTG mRNA的表达、Western blot检测肾皮质tTG蛋白表达,PAS染色评估ECM含量。结果与正常对照组比较,糖尿病大鼠肾脏细胞外基质（ECM）积聚,tTG mRNA及蛋白表达均明显增高（P〈0.01）;给予银杏叶提取物可明显减轻ECM积聚,同时tTG表达量也明显降低（P〈0.01）。结论银杏叶提取物可以明显减轻糖尿病大鼠肾脏ECM积聚,提示银杏叶提取物可能通过降低tTG的表达,改善糖尿病肾脏ECM积聚。     

组蛋白去乙酰化酶抑制剂对内毒素所致血管内皮细胞损伤的影响

目的 探讨组蛋白去乙酰化酶(HDAC)抑制剂(HDACI)对内毒素所致血管内皮细胞损伤的影响,为临床脓毒症相关血管内皮细胞损伤后的保护提供实验依据.方法 体外培养血管内皮细胞特性的EAhy926细胞株.采用四甲基偶氮唑盐(MTT)比色法检测不同剂量脂多糖(LPS,50、100、500、1 000 ng/ml)对血管内皮细胞的增殖作用.选择细胞存活率较高的LPS剂量,加入100 μ g/ml HDACI曲古抑霉素(TSA),采用蛋白质免疫印迹法(Western blotting)检测LPS刺激内皮细胞3、6、9、12、24 h时细胞内Toll样受体4(TLR4)和HDAC2的蛋白表达.结果 与空白对照组比较,100 ng/ml LPS刺激内皮细胞9、12、24 h后,TLR4蛋白表达明显上调(1.01±0.14、1.25±0.16、1.20±0.19比0.34±0.05,均P＜0.01);刺激12h、24h时HDAC2蛋白表达有所上调(1.14±0.10、1.20±0.04比0.17±0.02,均P＜0.01).与LPS组相比,TSA组刺激12 h时TLR4蛋白表达明显下调(0.37±0.07比1.25±0.16,P＜0.05),24 h时TLR4蛋白表达无明显差异(0.37土0.10比1.20±0.19,P＞0.05);TSA组各时间点HDAC2蛋白表达下调,未见条带显现.结论 LPS刺激血管内皮细胞后可上调内皮细胞TLR4及HDAC2的蛋白表达;而TSA可以下调LPS刺激内皮细胞后的TLR4表达.     

Alterations in peptidoglycan of Neisseria gonorrhoeae induced by sub-MICs of beta-lactam antibiotics.

Exposure of Neisseria gonorrhoeae to sub-MICs of selected beta-lactam antibiotics caused distortion of normal cell morphology. Analysis of the peptidoglycan indicated that the cells were accumulating increased quantities of disaccharide pentapeptide in their cell walls. The O-acetylated form of the disaccharide pentapeptide was not detected among the major peaks. The correlation of antibiotic binding to gonococcal penicillin-binding protein 2 and accumulation of non-O-acetylated disaccharide pentapeptide suggested an explanation for the previously observed relationship of penicillin-binding protein 2 and O-acetylation of peptidoglycan.     

Effects of steroid hormones on Neisseria gonorrhoeae.

Various steroids were tested for their effects upon gonococcal O2 consumption and glucose catabolism. The ability to inhibit gonococcal O2 uptake appeared to be related to the molecular configuration of the steroid. The presence of lipophilic groups enhanced inhibition, whereas the addition of hydrophilic groups markedly diminished inhibition. Steroid inhibition decreased with an increasing number of polar groups. Glucose catabolism was inhibited by steroid hormones, and the degree of inhibition was influenced by pH and medium composition. Changes in growth medium and pH also resulted in differential steroid inhibition of O2 uptake. Under certain conditions, lactate partially relieved this inhibition. Gonococci that were grown in one environment and shifted to a new environment were inhibited by steroids to the same extent as if they had been originally grown in the new environment. The differential effects of medium and pH upon steroid inhibition may be due to structural rearrangements involving membrane phase transitions or to altered receptor affinity.     

A DNA sequence for the discrimination of Neisseria gonorrhoeae from other Neisseria species.

A 350 base pair Neisseria gonorrhoeae DNA restriction fragment was cloned after subtractive hybridization to Neisseria meningitidis DNA. This restriction fragment hybridized to 105 out of 106 N. gonorrhoeae strains tested. While three N. meningitidis strains did not hybridize to this probe, Neisseria mucosa DNA exhibited cross-hybridization. This particular clone was used to screen a N. gonorrhoeae genomic DNA library. A positive 2.4 kilobase pair clone was shown by DNA sequencing to contain two long open reading frames. One open reading frame did not hybridize to N. mucosa and other Neisseria species, while it retained specificity for the original 105 N gonorrhoeae strains. This open reading frame also showed significant homology to cytosine DNA methyltransferases.     

Involvement of a change in penicillin target and peptidoglycan structure in low-level resistance to beta-lactam antibiotics in Neisseria gonorrhoeae.

A penicillin-susceptible gonococcus and its low-level resistant penA transformant were examined with regard to their penicillin-binding proteins (PBPs) and their peptidoglycan structures. Treatment of the susceptible strain with its MIC of penicillin (0.01 microgram/ml) led to significant binding to PBPs 2 and 3 and a substantial decrease in the O-acetyl modification on the peptidoglycan. Peptidoglycan synthesis gradually ceased over an extended time. When the penA strain was treated with the same concentration of penicillin, only binding to PBP 3 was observed and there was no O-acetylation decrease, with continued peptidoglycan synthesis. This suggested that PBP 2 was the primary target in penicillin-susceptible gonococci and that this protein participated in the O-acetylation of peptidoglycan. Penicillin concentrations representing the MIC for the penA transformant (0.06 microgram/ml) caused significant binding to PBPs 1, 2, and 3 in the susceptible strain and PBPs 1 and 3 in the penA strain. In both strains the rate of peptidoglycan synthesis and the cross-linkage of the peptidoglycan made declined sharply, suggesting that significant inhibition of PBP 1 interfered with transpeptidation. A model for low-level resistance is proposed in which a decreased PBP 2 affinity leads to assumption of the role of primary target in the resistant transformant by PBP 1. The differences observed in peptidoglycan metabolism are a direct consequence of this change.     

Effect of penicillin G on release of peptidoglycan fragments by Neisseria gonorrhoeae: characterization of extracellular products

The effect of penicillin G (penG) on the turnover and release of peptidoglycan (PG) by Neisseria gonorrhoeae RD5 was investigated. We previously showed that exponentially growing gonococci (labeled in the glycan moiety with glucosamine and muramic acid and in the peptide with diaminopimelic acid) turn over ca. 35% of their PG per generation. In current studies, addition of penG accelerated the rate of PG hydrolysis by more than twofold and resulted in a corresponding increase in the amount of soluble PG fragments found in the medium. This increase of soluble PG was accounted for mainly by the release of nonreducing (anhydro-muramyl-containing) disaccharide peptide dimers (molecular weight, about 2,000) and trimers that were composed of subunits, linked not by peptide cross-linking bonds, but probably only by glycosidic bonds. The enhanced release of these products suggested that penG, directly or indirectly, stimulates the activity of a glycan-splitting, gonococcal PG:PG-6 muramyl transferase (transglycosylase). PG monomers that were released in the presence of penG were identical, both qualitatively and quantitatively, to the monomers released as a result of turnover in the absence of penG and consisted of anhydro-muramyl-containing disaccharide tripeptides and tetrapeptides. PenG-treated bacteria consistently released slightly less free disaccharide and free peptides than did control cultures, implying a penG-associated depression in the activity of the gonococcal N-acetylmuramyl-L-alanine amidase. In addition to stimulating the release of PG fragments, penG was also associated with greatly enhanced release from cells of glucosamine-containing non-PG macromolecule(s).     

Effects of Thiamphenicol and Chloramphenicol in Inhibiting Neisseria gonorrhoeae Isolates

Thiamphenicol was compared with penicillin, tetracycline, and chloramphenicol for its ability to inhibit 530 isolates of Neisseria gonorrhoeae, including 13 penicillinase-producing isolates. Thiamphenicol proved to be as active as chloramphenicol in inhibiting all of the isolates.     

Beta-lactam susceptibility of Neisseria gonorrhoeae isolates from pelvic inflammatory disease.

Gonococci isolated in France from urethral and endocervical cultures in 12 women with pelvic inflammatory disease were similar, in their susceptibility to six beta-lactam antibiotics as determined by an agar dilution procedure, to isolates from patients with uncomplicated anogenital infections (83 patients). These data contrast with earlier study done in the United States.