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Journal of Forestry Research 2020年第5期31卷 1843-1850页

作者： Fansuo Zeng Xiaoyi Li Rui Qie Leilei Li Minghao Ma Yaguang Zhan State Key Laboratory of Tree Genetics and Breeding Northeast Forestry UniversityHarbin 150040People’s Republic of China College of Life Science Northeast Forestry UniversityHarbin 150040People’s Republic of China First Affiliated Hospital Heilongjiang University of Chinese MedicineHarbin 150040People’s Republic of China

DNA methylation is widespread in plants and associated with plant development and defense ***,the relationship between DNA methylation and plant secondary metabolism has rarely been ***,when birch suspension cells were treated with 5-azacytidine(5-azaC),which blocks DNA methylation,triterpenoid accumulation was significantly promoted and antioxidant and defense enzymatic activity *** studying triterpenoid accumulation,0.1 mM azaC was optimal.A qRT-PCR assay revealed increased expression of genes encoding key triterpenoid biosynthetic *** of methylation polymorphisms at CCGG sites showed that the methylation level was lower in cells treated with *** results demonstrated that 5-azaC treatment led to an increase in the production of triterpenoids in cell cultures through a mechanism that involved in DNA methylation,which resulted in the induction of genes encoding the key *** study provides evidence of a relationship between DNA methylation and regulation of secondary metabolism.

关键词： 5-azacytidine Methylation Suspension cells Triterpenoids biosynthesis

Effects of 5-azacytidine(AZA)on the Growth,Antioxidant Activities and Germination of Pellicle Cysts of Scrippsiella acuminata(Dinophyceae)

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Journal of Ocean University of China 2023年第6期22卷 1660-1668页

作者： WANG Zhaohui ZHANG Jianneng TANG Tao ZHANG Yuning HU Ren College of Life Science and Technology Jinan UniversityGuangzhou 510632China

In this study,we investigated the effects of different concentrations of 5-azacytidine(AZA),a DNA methyltransferase in-hibitor,on the growth,antioxidant activities and germination of pellicle cysts of Scrippsiella *** purpose of this study is to understand the toxic effects of AZA on marine microalgae,and to demonstrate the effect of DNA methyltransferase inhibitors on the germination of pellicle *** showed that AZA inhibited the growth of *** significantly,and displaced a clear dose-dependent inhibition trend with the 96h EC50 of 146.77μmolL^(-1)(35.84mgL^(-1)).Pellicle cysts of *** were less sensitive to AZA than the vegetative cells,and the EC50 value of AZA to the germination of pellicle cysts of *** was 8.08mmolL^(-1)(1.97g L^(-1)).After exposed to AZA,the antioxidant activities in *** responded rapidly and *** them,soluble pro-tein and superoxide dismutase(SOD)were more sensitive to AZA,and significant promotions occurred after exposed to 10μmolL^(-1)AZA for ***,malondialdehyde(MDA)contents in algal cells did not change significantly after exposed to low concen-trations of AZA,but increased firstly and then decreased under high concentration of *** glutathione(GSH)levels in ***-minata increased significantly under high concentrations of AZA,and remained unchanged at low concentrations of *** results suggested that the enhanced protein level and SOD activity of *** eliminated reactive oxygen species(ROS)to a certain ex-tent,and thus protected algal cells against damages of ROS caused by AZA.

关键词： 5-azacytidine Scrippsiella acuminata pellicle cysts toxicity superoxide dismutase glutathione malondialdehyde

5-azacytidine induces changes in electrophysiological properties of human mesenchymal stem cells

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Cell Research 2006年第12期16卷 949-960页

作者： Bartosz Balana Cecilia Nicolett Ihor Zahanich Eva M Graf Torsten Christ Sabine Boxberger Department of Pharmacology and Toxicology Medical Clinic I Medical FacultyDresden University of TechnologyFetscherstr.74D-01307 DresdenGermany

Previously,mouse bone marrow-derived stem cells（MSC）treated with the unspecific DNA methyltransferase inhibi-tor 5-azacytidine were reported to differentiate into *** aim of the present study was to investigate theefficiency of a similar differentiation strategy in human mononuclear cells obtained from healthy bone marrow *** 1-3 passages,cultures were exposed for 24 h to 5-azacytidine（3μM）followed by 6 weeks of further *** treatment did not induce expression of myogenic marker MyoD or cardiac markers Nkx2.5 and GATA-4 and didnot yield beating cells during *** patch clamp experiments,approximately 10-15% of treated and untreatedcells exhibited L-type Ca2+ *** all cells showed outwardly rectifying K+currents of rapid or slow *** current amplitude at +60 mV doubled after 6 weeks of treatment compared with time-matched *** capacitance of treated cells was significantly larger than in controls 2 weeks after treatment and remained highafter 6 *** levels ofmRNAs for the K+channels Kv1.1,Kv1.5,Kv2.1,Kv4.3 and KCNMA1 and for theCa2+ channel Cav1.2 were not affected by *** with potassium channel blockers tetraethylammoniumand clofilium at concentrations shown previously to inhibit rapid or slowly activating K+currents of hMSC inhibitedproliferation of these *** results suggest that despite the absence of differentiation of hMSC into cardiomyocytes,treatment with 5-azacytidine caused profound changes in current density.

关键词： human mesenchymal stem cells 5-azacytidine cardiac differentiation outward K~+ currents

The Effect of 5-azacytidine (5-Aza-CR) And Its Analogue on Cell Differentiation and DNA Methylation of HL-60 Cells

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Science China Chemistry 1993年第4期36卷 430-438页

作者：何忠效饶泓 Department of Biology Beijing Normal University National Laboratory of Biomacromolecule Beijing 100875 PRC

The effect of 5-aza-CR and 5-aza-2'-deoxycytidine(5-aza-CdR)on cell differentiation and DNA methylation of HL-60 cells was studied. The differentiation index of HL-60 cells was measured after being treated with drugs by using the NBT stain method. DNA methylase activities of HL-60 cells treated with the drugs were assayed by using ~3H-methyl-S-adenosylmethionine(~3H-SAM) as a methyl donor. The DNA methylation level of HL-60 cells treated with the drugs was measured by HPLC. The resuhs showed that the HL-60 cell differentiation index was increased after being treated with 5-aza-CR or 5-aza-CdR at a certain concentration for 4 days. But, at the same time, DNA methylase activity and the DNA methylation level were decreased. And all these changes were related to the concentration of the drugs. 5-Aza-CdR was more efficient than 5-aza-CR. We also assayed the E. coli RNA polymerase activity in vitro by using different DNA templets different in DNA methylation level. We found that the transcriptional activity of RNA polymerase was increased with the decrease of the DNA methylation level of HL-60 cells.

关键词： 5-azacytidine cell differentiation DNA mcthylation DNA methylase RNA polymerase.

Cardiomyocyte-like differentiation of human bone marrow mesenchymal stem cells after exposure to 5-azacytidine in vitro

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Journal of Geriatric Cardiology 2004年第2期1卷 101-107页

作者： Feng CAO Lili NIU Ling MENG Lianxu ZHAO Dongmei Wang Ming ZHENG Cixian BAI Guoliang JIA Xuetao PEI Beijing Institute of Transfusion Medicine Chinese Academy of Military Medical Sciences Beijing 100850 China Department of Cardiology Xijing Hospital Fourth Military Medical University Xi''an Shaan''xi China Beijing Institute of Transfusion Medicine Chinese Academy of Military Medical Sciences Beijing 100850 China Beijing Institute of Transfusion Medicine Chinese Academy of Military Medical Sciences Beijing 100850 China Beijing Institute of Transfusion Medicine Chinese Academy of Military Medical Sciences Beijing 100850 China Beijing Institute of Transfusion Medicine Chinese Academy of Military Medical Sciences Beijing 100850 China Beijing Institute of Transfusion Medicine Chinese Academy of Military Medical Sciences Beijing 100850 China Beijing Institute of Transfusion Medicine Chinese Academy of Military Medical Sciences Beijing 100850 China Department of Cardiology Xijing Hospital Fourth Military Medical University Xi''an Shaan''xi China Beijing Institute of Transfusion Medicine Chinese Academy of Military Medical Sciences Beijing 100850 China

Objective To investigate the potential of adult mesenchymal stem cells (MSCs) derived from human bone marrow to undergo cardiomyogenic differentiation after exposure to 5-azacytidine (5-aza) in vitro. Methods A small bone marrow aspirate was taken from the iliac crest of human volunteers, and hMSCs were isolated by 1.073g/mL Percoll and propagated in the right cell culturing medium as previously described. The phenotypes of hMSCs were characterized with the use of flow cytometry. The hMSCs were cultured in cell culture medium (as control) and medium mixed with 5-aza for cellular differentiation. We examined by immunohistochemistry at 21 days the inducement of desmin, cardiac-specific cardiac troponin I (cTnI), GATA 4 and connexin-43 respectively. Results The hMSCs are fibroblast-like morphology and express CD44+ CD29+ CD90+ / CD34- CD45- CD31- CD11a. After 5-aza treatment, 20-30% hMSCs connected with adjoining cells and coalesced into myotube structures after 14days. Twenty-one days after 5-aza treatment, immunofluorescence showed that some cells expressed desmin,GATA4, cTnI and connexin-43 in 5,10 μmol/L 5-aza groups, but no cardiac specific protein was found in neither 3μmol/L 5-aza group nor in the control group. The ratio of cTnI positively stained cells in 10 μmol/L group was higher than that in 5 μmol/L group (65.3 ± 4.7% vs 48.2 ± 5.4%, P ＜ 0.05). Electron microscopy revealed that myofilaments were formed. The induced cells expressed cardiac-myosin heavy chain (MyHC) gene by reverse transcription-polymerase chain reaction (RT-PCR). Conclusions Theses findings suggest that hMSCs from adult bone marrow can be differentiated into cardiac-like muscle cells with 5-aza inducement in vitro and the differentiation is in line with the 5-aza concentration. (J Geriatr Cardiol 2004;1(2) :101-107. )

关键词： human bone marrow mesenchymal stem cells cardiomyocytes differentiation 5-azacytidine

Transplantation of 5-azacytidine treated cardiac fibroblasts improves cardiac function of infarct hearts in rats

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Chinese Medical Journal 2010年第18期123卷 2586-2592页

作者： TANG Cheng-chun MA Gen-shan CHEN Ji-yuan Department of Cardiology Zhongda Hospital Southeast University Nanjing Jiangsu 210009 China

Background Cellular cardiomyoplasty by transplantation of various cell types has been investigated as potential treatments for the improvement of cardiac function after myocardial injury. A major barrier for the clinical application of cell transplantation is obtaining sufficiently large quantities of suitable cells. AIIogeneic cellular cardiomyoplasty may provide an alternative source of abundant, transplantable, myogenic cells by in vitro manipulation of cardiac fibroblasts using chemicals including 5-azacytidine. This study evaluated cardiomyogenic differentiation of cardiac fibroblasts, their survival in myocardial scar tissue, and the effect of the implanted cells on heart function. Methods Primary cardiac fibroblasts from neonatal rats were treated with 5-azacytidine （10 pmol/L） or control. Treatment of 5-azacytidine caused myogenic differentiation of cultured cardiac fibroblasts, as defined by elongation and fusion into multinucleated myotubes with sarcomeric structures as identified by electron microscopy, and positive immunostaining for cardiac specific proteins, troponin I and 13-myosin heavy chain （13-MHC） and the gap junction protein connexin 43. The myogenic cells （1.0x106） were transplanted into the infarcted myocardium 2 weeks after coronary artery occlusion. Results By 1 month after transplantation, the converted fibroblasts gave rise to a cluster of cardiac-like muscle cells that in the hearts occupied a large part of the scar with positive immunostaining for the myogenic proteins troponin I and 13-MHC. Engrafted cells also expressed the gap junction protein connexin 43 in a disorganized manner. There was no positive staining in the control hearts treated with injections of culture medium. Heart function was evaluated at 6 weeks after myocardial injury with echocardiographic and hemodynamic measurements. Improvement in cardiac function was seen in the hearts transplanted with the 5-azacytidine-treated cardiac fibroblasts which was absent in the hear

关键词： cardiac fibroblasts myocardial infarction transplantation 5-azacytidine

5-azacytidine诱导黄曲霉突变株的筛选及鉴定

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5-azacytidine诱导黄曲霉突变株的筛选及鉴定

作者：杨坤龙福建农林大学

学位级别：硕士

黄曲霉毒素(Aflatoxins，AF)是黄曲霉(Aspergillus flavus)和寄生曲霉(Aspergillus parasiticus)等产毒菌种产生的一类次生代谢产物，是目前发现的毒性和致癌性最强的天然污染物之一。本研究通过DNA甲基化抑制剂5-azacytidine诱导黄曲... 详细信息

黄曲霉毒素(Aflatoxins，AF)是黄曲霉(Aspergillus flavus)和寄生曲霉(Aspergillus parasiticus)等产毒菌种产生的一类次生代谢产物，是目前发现的毒性和致癌性最强的天然污染物之一。本研究通过DNA甲基化抑制剂5-azacytidine诱导黄曲霉标准菌株133，获得了一株可以稳定遗传而且不产毒的黄曲霉突变菌株NT，通过ITS序列鉴定发现NT与野生型菌株的ITS序列完全匹配。通过对突变菌株的生长特性分析发现，5-azacytidine诱导得到的黄曲霉突变菌株，较野生型菌株具有较快的生长速率，菌落呈白色;突变菌的菌丝尖端长度为331.9±131.2μm，而野生型菌为141.7±31.7μm;突变菌菌丝隔膜变长，其中突变菌的隔膜长度为66.7±19.9μm，野生型黄曲霉则为49.1±14.1μm;产孢延迟且产孢量急剧下降，在形态上有较大的差异。采用薄层析法(TLC)对黄曲霉突变菌株的代谢产物合成情况进行分析，结果发现突变菌株在培养基中并没有检测到产毒素，但在其菌内却可以检测到少量的AFB2，可能是5-azacytidine诱导后除了AF合成变少外，AF分泌到孢外可能受到了抑制。为了进一步了解突变菌产毒及其形态是如何建成的，本研究通过荧光定量PCR检测黄曲霉突变菌株中和产毒相关和产孢相关的基因的表达情况，突变体中的产毒结构基因aflD在96h时下降了79.51倍，产毒调控基因aflR下调了4.55倍;而突变菌中产孢特异基因abaA和brlA在96h的时候，分别比野生型下调了48.26倍和66.99倍，mRNA表达水平检测结果与产毒和产孢检测情况一致。为了进一步研究产毒受抑制的机理，本研究对突变菌株的氧化应激水平进行了研究，结果表明，黄曲霉突变菌对过氧化氢相对比较敏感，在HO浓度为10μmol/L时，5-azacytidine诱变的突变菌的生长就受到了抑制;而菌内的脂质体氧化水平和谷胱甘肽含量检测实验表明，突变菌内的氧化水平比野生型菌株相对较低，可能是由于突变菌体内的氧化应激系统遭到了破坏。本研究旨在从一个全新的角度认识黄曲霉的产毒机理，探究5-azacytidine对黄曲霉的生理影响，进一步揭示丝状真菌次生代谢产物的调控及形态的建成。

关键词：黄曲霉 AFB1 5-azacytidine 氧化应激

Role of 5-azacytidine in differentiation of human mesenchymal stem cell sinto cardiomyocytes in vitro

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Journal of Geriatric Cardiology 2009年第3期6卷 182-188页

作者： Fang-Ge Deng Yu-Lin Li Xiu-Ying Zhang Key Laboratory of Pathobiology Jilin University Changchun 130021 China National Key Laboratory of Respiratory Diseases Guangzhou Institute of Respiratory Diseases First Affiliated Hospital of Guangzhou Medical College Guangzhou 510120 China

Objective 5-azacytidine could induce the differentiation of stem cells into cardiomyocytes （CMs）. The aim of this study was to screen the optimal condition for 5-azacytidine inducing differentiation of human mesenchumal stem cells （hMSCs） into CMs, and the effect of 5-azacytidine on adherence, cell vigor and chromosome karyotype of hMSCs. Methods hMSCs were isolated from human bone marrow and cultured in vitro. The phenotypes ofhMSCs were identified by flow cytometric analyses. MTT test was used to investigate the effect of different concentrations of 5-azacytidine on proliferation ofhMSCs. Four weeks after 5-azacytidine induction, semi-quantitative RT-PCR, transmission electron microscopy （TEM）, single-cell action potentials, detection of cardio-enzyme AST and LDH, cell adherence, cell viability and chromosome karyotype test were performed. Results The typical morphological features of hMSCs were fibroblast-like in shape, hMSCs expressed CD44 and CD105,and did not express CD34, CD45 and CD31. The optimal concentration of 5-azacytidine was 10μ mol/L. The shape of hMSCs treated with 5-azacytidine changed from fusiform to polygon or astrocyte gradually, and passaged cells were evenly arranged as polarity structure. Indueed-hMSCs connected with neighbouring cells, fbrming myotube-like structures 4 weeks later. It was confirmed that induced hMSCs shaped myotubule-like structure and had some of micro-histologic structures of CMs by TEM. RT-PCR showed that induced hMSCs expressed cardiac specific product BNNP and early cardio-myogenesis specific transcription factor NKX2.5mRNA. Besides, induced-MSCs led to the weak action potential and secreted cardio-enzyme AST and LDH. There was no significant difference in cell adherence and viability before and after induction. Both hMSCs and induced-hNSCs kept stable normal diploid nucleus. Conclusion The optimal condition for inducing effect of 5-azacytidine is 10 la mol/L and 24-hour incubation; and under this condition, the adhere

关键词： Human mesenchymal stem cells 5-azacytidine differentiation cardiomyocytes

Effect of 5-azacytidine on the Protein Expression of Porcine Bone Marrow Mesenchymal Stem Cells in vitro

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Genomics, Proteomics & Bioinformatics 2006年第1期4卷 18-25页

作者： Neng-Sheng Ye Rong-Li Zhang Yan-Feng Zhao Xue Feng Yi-Ming Wang Guo-An Luo Institute of Biomedicine Tsinghua University Beijing 100084 China

Bone marrow-derived mesenchymal stem cells （MSCs） are pluripotent stem cells that show a vital potential in the clinical application for cell transplantation. In the present paper, proteomic techniques were used to approach the protein profiles associated with porcine bone marrow MSCs and investigate the regulation of MSC proteins on the effect of 5-azacytidine （5-aza）. Over 1,700 protein species were separated from MSCs according to gel analysis. Compared with the expression profiling of control MSCs, there were 11 protein spots up-regulated and 26 downregulated in the protein pattern of 5-aza-treated cells. A total of 21 proteins were successfully identified by MALDI-TOF-MS analysis, among which some interesting proteins, such as alpha B-crystallin, annexin A2, and stathmin 1, had been reported to involve in cell proliferation and differentiation through different signaling pathways. Our data should be useful for the future study of MSC differentiation and apoptosis.

关键词： bone marrow mesenchymal stem cells 5-azacytidine two-dimensional gel electrophoresis mass spectrometry proteome

DNA甲基化抑制剂5-azacytidine对猪LLC-PK1细胞GDF11表达的影响及猪GDF11的原核表达

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DNA甲基化抑制剂5-Azacytidine对猪LLC-PK1细胞GDF11表达的影响及...

作者：孙亿山东农业大学

学位级别：硕士

生长分化因子11(GDF11)是转化生长因子β(TGF-β)超家族中的一种分泌型信号分子,又称为骨形态发生蛋白11(BMP11)。从1999年发现至今,对GDF11的研究主要集中于其在早期胚胎发育中的调控作用,特别是在中轴骨骼的图式形成以及肾发生中的作... 详细信息

生长分化因子11(GDF11)是转化生长因子β(TGF-β)超家族中的一种分泌型信号分子,又称为骨形态发生蛋白11(BMP11)。从1999年发现至今,对GDF11的研究主要集中于其在早期胚胎发育中的调控作用,特别是在中轴骨骼的图式形成以及肾发生中的作用。体长是猪育种中的一个重要的经济性状,其长短与胸腰椎数密切相关。因此,研究DNA去甲基化对猪GDF11表达的影响,对于了解猪体长变异的表观遗传机制,具有重要科学意义和潜在的应用价值。本研究可以分为两个部分:第一部分:用DNA甲基化抑制剂5-azaC处理猪肾近端小管上皮细胞系LLC-PK1,提取细胞总RNA,运用RT-PCR和半定量PCR的方法分析不同的处理浓度和处理时间对猪GDF11表达的影响。结果表明:处理24h后0.5、1和3μM 5-azaC处理组之间GDF11的相对表达量差异显著(P5);处理48h后差异不显著;处理72h后,0.05、0.1、0.3、0.5、1和3μM 5-azaC处理组之间差异显著(P5)。在每个处理浓度下,处理24、48和72h后GDF11的相对表达量差异极显著(P5cm猪胚肾cDNA作为模板,通过PCR的方法克隆编码猪GDF11蛋白羧基末端327个氨基酸的cDNA序列,将此序列连接入原核表达载体pGEX-6p-1中,测序验证表明重组质粒pGEX-6p-1-GDF11构建成功。将重组的表达质粒pGEX-6p-1-GDF11转化到大肠杆菌BL21(DE3)中,使用IPTG诱导目的蛋白的表达,经SDS-PAGE验证表明,融合蛋白GST-GDF11在大肠杆菌中得到成功表达。利用电洗脱的方法对GST-GDF11融合蛋白进行初步纯化,SDS-PAGE结果表明,得到纯度较高的融合蛋白GST-GDF11。

关键词：猪 GDF11 DNA甲基化 5-azacytidine LLC-PK1 原核表达