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 acu...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 acuminata.The 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 cysts.Results showed that AZA inhibited the growth of S.acuminata significantly,and displaced a clear dose-dependent inhibition trend with the 96h EC50 of 146.77μmolL^(-1)(35.84mgL^(-1)).Pellicle cysts of S.acuminata were less sensitive to AZA than the vegetative cells,and the EC50 value of AZA to the germination of pellicle cysts of S.acuminata was 8.08mmolL^(-1)(1.97g L^(-1)).After exposed to AZA,the antioxidant activities in S.acuminata responded rapidly and significantly.Among them,soluble pro-tein and superoxide dismutase(SOD)were more sensitive to AZA,and significant promotions occurred after exposed to 10μmolL^(-1)AZA for 24h.Meanwhile,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 AZA.The glutathione(GSH)levels in S.acu-minata increased significantly under high concentrations of AZA,and remained unchanged at low concentrations of AZA.The results suggested that the enhanced protein level and SOD activity of S.acuminata eliminated reactive oxygen species(ROS)to a certain ex-tent,and thus protected algal cells against damages of ROS caused by AZA.展开更多
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 ...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. )展开更多
DNA methylation is widespread in plants and associated with plant development and defense mechanisms.However,the relationship between DNA methylation and plant secondary metabolism has rarely been reported.Here,when b...DNA methylation is widespread in plants and associated with plant development and defense mechanisms.However,the relationship between DNA methylation and plant secondary metabolism has rarely been reported.Here,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 changed.For studying triterpenoid accumulation,0.1 mM azaC was optimal.A qRT-PCR assay revealed increased expression of genes encoding key triterpenoid biosynthetic enzymes.Evaluation of methylation polymorphisms at CCGG sites showed that the methylation level was lower in cells treated with 5-azaC.These 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 enzymes.The study provides evidence of a relationship between DNA methylation and regulation of secondary metabolism.展开更多
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 ...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.展开更多
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 clini...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 hearts treated with control. Conclusion The 5-azacytidine has a unique capacity to induce myogenesis in cardiac fibroblasts in vitro and transplantation of cardiac-like muscle cells into ventricular scar tissue improves myocardial function.展开更多
目的探讨甲基转移酶5(methyltransferase-like 5,METTL5)在三阴乳腺癌(triple-negative breast cancer,TNBC)中的作用和潜在机制。方法采用免疫组织化学方法和Western blot检测TNBC肿瘤组织和细胞系中METTL5的表达情况。用靶向METTL5的s...目的探讨甲基转移酶5(methyltransferase-like 5,METTL5)在三阴乳腺癌(triple-negative breast cancer,TNBC)中的作用和潜在机制。方法采用免疫组织化学方法和Western blot检测TNBC肿瘤组织和细胞系中METTL5的表达情况。用靶向METTL5的shRNA(shRNA-METTL5)转染TNBC细胞后,用CCK-8、集落形成、伤口愈合以及Transwell实验分别检测细胞增殖活性、迁移与侵袭,Western blot检测Wnt/β-catenin信号关键蛋白的表达。构建异种移植瘤模型,验证敲降METTL5对TNBC细胞在体内生长以及Wnt/β-catenin信号活性的影响。结果METTL5在TNBC肿瘤组织和细胞系中表达上调(P<0.01)。敲降METTL5可抑制TNBC细胞的增殖、迁移和侵袭并降低了Wnt/β-catenin信号分子β-catenin、细胞周期蛋白(Cyclin)D1、基质金属蛋白酶(MMP)-2和MMP-7的表达(均P<0.01)。体内实验显示,敲降METTL5减缓了移植瘤生长和Wnt/β-catenin信号活性。结论敲降METTL5能抑制TNBC细胞的增殖、迁移与侵袭,其作用可能与抑制Wnt/β-catenin信号通路有关。展开更多
基金supported by the National Natural Science Foundation of China(No.42076141).
文摘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 acuminata.The 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 cysts.Results showed that AZA inhibited the growth of S.acuminata significantly,and displaced a clear dose-dependent inhibition trend with the 96h EC50 of 146.77μmolL^(-1)(35.84mgL^(-1)).Pellicle cysts of S.acuminata were less sensitive to AZA than the vegetative cells,and the EC50 value of AZA to the germination of pellicle cysts of S.acuminata was 8.08mmolL^(-1)(1.97g L^(-1)).After exposed to AZA,the antioxidant activities in S.acuminata responded rapidly and significantly.Among them,soluble pro-tein and superoxide dismutase(SOD)were more sensitive to AZA,and significant promotions occurred after exposed to 10μmolL^(-1)AZA for 24h.Meanwhile,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 AZA.The glutathione(GSH)levels in S.acu-minata increased significantly under high concentrations of AZA,and remained unchanged at low concentrations of AZA.The results suggested that the enhanced protein level and SOD activity of S.acuminata eliminated reactive oxygen species(ROS)to a certain ex-tent,and thus protected algal cells against damages of ROS caused by AZA.
基金supported by research grants from State 863 high technology R&D Project of China(2002AA205051and 2003AA205160)the National Key Rroject for Basic Research of China(2001CB509906)
文摘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. )
基金The work was supported by The Fundamental Research Funds for the Central Universities(2572017EA05)the National Natural Science Foundation of China(31870588).
文摘DNA methylation is widespread in plants and associated with plant development and defense mechanisms.However,the relationship between DNA methylation and plant secondary metabolism has rarely been reported.Here,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 changed.For studying triterpenoid accumulation,0.1 mM azaC was optimal.A qRT-PCR assay revealed increased expression of genes encoding key triterpenoid biosynthetic enzymes.Evaluation of methylation polymorphisms at CCGG sites showed that the methylation level was lower in cells treated with 5-azaC.These 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 enzymes.The study provides evidence of a relationship between DNA methylation and regulation of secondary metabolism.
文摘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.
文摘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 hearts treated with control. Conclusion The 5-azacytidine has a unique capacity to induce myogenesis in cardiac fibroblasts in vitro and transplantation of cardiac-like muscle cells into ventricular scar tissue improves myocardial function.
