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Context: The diagnosis of giant cell tumor of bone (GCTB) is difficult in small biopsies with unusual age of presentation, location, and extensive secondary changes. Most of the GCTBs harbor H3F3A G34W mutations with a subset of cases showing alternate G34V, G34R, and G34L mutations. Objectives: To analyze the expression of anti-histone H3.3G34W antibody in different cellular components of GCTB across different locations and presentations (including the unusual ones) and validate the utility of this antibody in the diagnosis of GCTB and differentiate it from the other osteoclast-like giant-cell-rich lesions. Design: Immunohistochemistry was performed using anti-histone H3.3G34W antibody in the diagnosed cases of GCTB (136 cases of GCTB from 133 patients, including two malignant GCTBs) and other giant cell-containing lesions (62 cases). The presence of unequivocal crisp nuclear staining was considered positive. Results: Immunohistochemistry revealed unequivocal nuclear positivity in the mononuclear cells in 87.3% of the cases of GCTB. Of these, most showed diffuse expression with moderate to strong intensity staining. The positive staining was restricted to the nuclei of mononuclear cells with the nuclei of osteoclastic giant cells being distinctly negative. In addition to conventional GCTBs, two cases each of multicentric and malignant GCTB showed positive staining. The other giant-cell containing lesions were distinctly negative. The present study showed a sensitivity of 87.3% with specificity and positive predictive value of 100%. Conclusion: The anti-histone G34W antibody is a highly sensitive and specific marker for the diagnosis of GCTB and differentiating it from its mimics. The positive staining is restricted to the mononuclear cell component of GCTB with sparing the osteoclastic giant cells further reiterating the fact that the mononuclear stromal cells are the true neoplastic component of GCTB.
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Objective:To study the expression and significance of neutrophil extracellular traps (NETs) in neonatal sepsis.Methods:Prospective research were used in this study. Term infants with neonatal sepsis hospitalized for the first time in the Department of Neonatology, Children's Hospital of Soochow University from June 2020 to November 2020 were selected as the sepsis group. According to a ratio of about 1∶1, term infants with mild hyperbilirubinemia who were admitted in the same period, with gestational age difference less than 1 week from those in the sepsis group, and whose parents agreed to participate in the study were selected as the control group. On admission, clinical data as well as blood samples of the two groups were collected. Levels of NETs marker citrulline histone H3-DNA (CitH3-DNA) were detected by Enzyme-linked immunosorbent assay, and circulating cell-free DNA (cfDNA) was tested by the fluorescence microplate reader. General data, white blood cell (WBC), neutrophil count (NE), platelet (PLT), C- reactive protein (CRP), blood culture, CitH3-DNA and cfDNA were compared between the two groups. The diagnostic value of CITH3-DNA and cfDNA in neonatal septicemia was analyzed by the receiver operating characteristic (ROC) curve.Results:A total of 74 infants were included in the study, including 39 cases in the sepsis group and 35 cases in the control group. CitH3-DNA and cfDNA in the sepsis group were significantly higher than those in the control group [CitH3-DNA (optical density): 0.85±0.05 vs. 0.48±0.03, cfDNA (mg/L): 0.90±0.05 vs. 0.56±0.03] ( P<0.01). There was no significant correlation between CitH3-DNA and cfDNA. The level of CitH3-DNA had no correlation with gender, gestational age, age, birth weight, WBC, NE, PLT and CRP ( P>0.05). cfDNA was positively correlated with age and NE ( P<0.05), and negatively correlated with PLT ( P<0.05). Combined with CRP, the area under the ROC curve of CitH3-DNA+CRP, cfDNA+CRP, and CitH3-DNA+cfDNA+CRP were 0.947, 0.947 and 0.970 respectively, and the sensitivity to predict neonatal sepsis were 92.3%, 84.6% and 94.9% respectively, the specificity were 94.3%, 97.1% and 100% respectively, all higher than the predictive value of each index alone. Conclusions:The plasma NETs levels increase significantly in neonatal sepsis patients, especially CitH3-DNA with a strong specificity, and can be considered as a biomarker for early diagnosis of neonatal sepsis. NETs together with CRP, could drastically improve the predictive value of neonatal sepsis.
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OBJECTIVE: To observe the effect of electroacupuncture (EA) on expression of histone deacetylase 2 (HDAC2), histone H3, bone formation related genes and proteins in osteoporosis rats, so as to reveal its mechanisms underlying improvement of osteoporosis. METHODS: Female SD rats were randomly divided into 4 groups: sham operation, model, EA and medication (n＝ 10 rats in each group). The osteoporosis model was established by castration. EA (2 Hz, 1 mA) was applied to bilateral "Shenshu" (BL23) and "Pishu" (BL20) for 10 min, once every other day for 8 weeks. Rats of the medication group received subcutaneous injection of 17 β-estradiol (100 µg/kg, 20 µg/mL). The bone quality and quantity including the cortical bone mineral density (CBMD), trabecular bone mineral density (TBMD), ratio of bone volume /total volume (BV /TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb. Sp), trabecular bone pattern factor (Tb.Pf), and structure model index (SMI) of the right thigh-bone were detected by using a micro-computed tomography. Serum alkaline phosphatase (ALP) and estrogen 2 (E2) contents were assayed by using colorimetry and ELISA, expression levels of HDAC2, histone H3 and Runx2 in the thigh-bone were detected using Western blot, and that of Runx2 mRNA was detected using quantitative real-time PCR, separately. The co-expression of Ac-histone H3/Runx2 and Runx2/ALP was observed by using immunofluorescence histochemical staining. RESULTS: After modeling, the levels of TBMD, BV/TV, Tb.Th, and Tb.N, serum E2 and ALP, and expression of Runx2 protein and mRNA, Ac-histone and ALP proteins were significantly lower (P0.05). The effects of EA were significantly superior to 17 β-estradiol in down-regulating the expression of HDAC2 and histone H3 proteins and in up-regulating expression of Ac-histone H3 protein (P<0.01，P<0.05)．. CONCLUSION: EA treatment can increase bone density, increase bone mass and trabecular bone, and promote trabecular bone rod-like changes in plate shape in osteoporosis rats, which is related to its effect in up-regulating the expression of Ac-histone H3 protein, and down-regulating the expression of bone formation-related proteins.
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Limitation in the number of insulin-producing pancreatic b-cells is a typical feature of diabetes. It has been indicated thatactivating pancreatic transcription factors can promote the transformation of hepatocytes into insulin-secreting b-like cells,indicating that direct hepatocyte differentiation seems promising as a treatment for diabetes. Nevertheless, the reprogramming efficiency still remains low. Our previous study found that the expression of c-fos-induced growth factor (FIGF)was increased in the pancreatic tissues in partial pancreatectomy mice compared to that in normal mice. Here, we observedthat treatment with Ad-FIGF was found to enhance MafA and Ngn3-induced reprogramming of BNL CL.2 cells to b-likecells with the ability of secreting insulin. And FIGF overexpression increased the levels of histone H3/H4 acetylation atMafA and Ngn3 promoter regions in BNL CL.2 cells. Importantly, in vivo study further confirmed that forced expression ofFIGF facilitated the insulin expression and decreased the blood glucose levels in STZ mice. These results strengthen thepossibility of developing cell-based therapies for diabetes through utilizing b-like cells derived from non-insulin-secretingcells.
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Objective To investigate the pathogenesis of the production of anti-neutrophil cytoplasmic antibodies (ANCA) in the rat models of chronic bronchitis (CB) with recurrent infections. Methods The CB models were made by double element of smoking and lipopolysaccharide (LPS) stimulation. The rats were divided into four groups, including normal control group (n=5), phorbol-12-myristate-13-acetate (PMA)-treated healthy rats control group (n=5), CB rats group (n=5) and PMA-treated CB rats group (n=6). Renal function of rats was detected. The histopathological lung and kidney tissues were observed by HE staining of paraffin section. Immunological markers, including myeloperoxidase anti-neutrophil cytoplasmic antibodies (MPO-ANCA), proteinase 3 anti-neutrophil cytoplasmic antibodies (PR3-ANCA) and citrullinated histone H3 (CitH3), were measured by enzyme-linked immune-sorbent assay (ELISA) at different time points. Correlation between CitH3 and MPO-ANCA was analyzed by the Spearman rank correlation. NETs components were further detected in lung and kidney tissue by confocal immunofluorescence and colocalization analysis. Results (1) The serum levels of CitH3 and MPO-ANCA in CB+PMA group showed an increased trend. Compared with those in the normal control group and CB rats group, the serum levels of CitH3 and MPO-ANCA in CB+PMA group increased significantly at the sixth week (both P＜0.05). Serum CitH3 levels in rats were positively correlated with serum MPO-ANCA levels (rs=0.490,P=0.024). (2) There were pathological manifestations of CB in the lung tissues of rats in CB group and CB+PMA group, and no obvious abnormalities in the lung tissues of rats in the normal control group and control group. In the rat kidney tissue of CB+PMA group, there were inflammatory cells infiltrated in the glomerular and around the renal tubules, but glomerular necrosis was not found. No obvious abnormalities were observed in the kidney tissues of rats in the normal control group, PMA-treated healthy rats control group and CB group. (3) In the lung and kidney tissues of CB+PMA group NETs could be detected by confocal immunofluorescence analysis. Conclusion CB rats with the recurrent infections can release large amounts of NETs, in which the exposure of MPO antigen will break the immune tolerance and result in the production of MPO-ANCA.
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Congenital heart disease is one of the main types of birth defect.The mammalian heart developmen-tal progress requires precise gene patterning in time and space.In addition to the gene sequence,recent research showed that the regulation of core cardiac gene expression has been proved to be closely related to cardiac transcription factors as well as the modification of genomic architecture of the histone.Methylation of histone might be the key nodes in the regula-tion of cardiac gene expression and chromatin structure.This review focuses on the role of histone H 3 methylation in heart development process,which may lay a foundation for the prediction of epigenetic modification of congenital heart disease.
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Objective Abnormal activation of mitogen-and stress-activated kinase (MSK1) plays an important role in the development of various cancers.This study was to explore the effect of small interfering RNA (siRNA)-mediated MSK1-silencing on the proliferation of human nasopharyngeal carcinoma (NPC) cells and its underlying mechanism.Methods The siRNA vector targeting MSK1 was constructed and transfected into CNE2 cells, and the NPC cell line stably expressing MSK1 was established.Then the cells were divided into a blank control (without transfection of the plasmid), a negative control (with stable transfection of the negative control plasmid), and an experimental group (with stable transfection of the positive recombinant plasmid).The expressions of MSK1 mRNA and protein were detected by real-time quantitative PCR and Western blot, respectively, the proliferation of the cells determined by CCK-8 and colony formation assays, the cell cycles analyzed by flow cytometry, the level of histone H3 phosphorylation at Ser10 examined by Western blot, and The transcriptional activity and expression of the c-jun protein measured by dual-luciferase reporter gene assay and Western blot.Results Compared with the blank control, the inhibition rates of cell proliferation at 48, 72 and 96 hours were significantly reduced in the experimental group (P<0.05), and so were the colony formation ability of the cells (P<0.01) and the expression and transcriptional activity of the c-jun protein (P<0.05).In comparison with the negative control, the experimental group showed significant decreases in the rate of cell growth after 24 hours, the inhibition rates of cell proliferation at 48, 72 and 96 hours (P<0.05), the number of formed colonies ([221.00±20.08] vs [99.67±15.57] / 300 cells, P<0.01), the proportion of S-phase cells (P<0.01), and the expression of the c-jun protein in the CNE2 cells ([100.00±0.00] vs [48.77±10.71] %, P<0.05), but a remarkable increase in the percentage of G0/G1-phase cells (P<0.01).Furthermore, histone H3 phosphorylation at Ser10 was markedly reduced (P<0.01) but no significant change was observed in the expression of the total c-jun protein in the experimental group.Conclusion Knockdown of MSK1 using siRNA can significantly inhibit the growth and proliferation of CNE2 cells, which may be closely related to the decreased phosphorylation of histone H3 and subsequently down-regulated transcriptional activity of c-jun.
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Objective To investigate the expression of phospho-histone H3 (PHH3) protein in pancreatic neuroendocrine neoplasms (PNENs) and explore its potential value for pathological grading of PNENs.Methods Clinical and pathological data of 283 patients with PNENs treated in Changhai Hospital from December 2000 to May 2016 were retrospectively analyzed.PNENs tissue chip was prepared,and immunohistochemistry was used to examine the expression of PHH3 and Ki67.The receiver operating characteristic curve for PHH3 was drawn and the area under the curve(AUC) was calculated to determine the cutoff value for PNENs grading.Ki67 was used for PNENs grading according to domestic and intemational criteria for PNENs classification.The relationship of PHH3 and Ki67 classification with clinical pathological features and prognosis of PNENs as well as the correlation between the two classification methods were analyzed.Results Of 283 patients,132 were male and 151 were female,aged from 16 to 78 years old.The average age was (50 ± 1) years old.There were 44 cases with functional PNENs and 239 with non-functional PNENs.The diameter of tumors ranged from 1.1 cm to 17 cm and the average diameter was (4.1 ± 1.2)cm.According to the Ki67 standard,there were 127,116,33 and 7 cases of Grade G1,G2,pancreatic neuroendocrine tumor(PNET) G3 and pancreatic neuroendocrine cancer(PNEC) G3.According to the PHH3 standard,there were 118,119,3 8 and 8 cases of Grade G1,G2,PNET G3 and PNEC G3.There was a positive correlation between the two grading criteria (r =0.941,P ＜0.001).PHH3 expression and PNENs grading were not correlated with age,gender,tumor functional or not and tumor locations (all P ＞ 0.05),but were both positively correlated with tumor size,histological grade,lymph node metastasis,TNM stage and prognosis (all P ＜0.05).The average time of observing PHH3 and Ki67 staining per case in the immunohistochemistry of tissue chip was 9 min and 45 min,respectively.Conclusions Detection of PHH3 expression in PNENs can be used for the pathological diagnosis,grading and prognostic evaluation,which was more accurate and convenient than Ki67 criteria.
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OBJECTIVE To analyze trimethylation of genome-wide histone H3 lysine 4(H3K4met3) induced by silicon dioxide(SiO2)through chromatin immunoprecipitation linked to microarrays(ChIP-chip)in lung fibroblast(LF)of rats. METHODS A primary co-culture model of rat alveolar macrophages (AM)and LF in vitro. AM were exposed to 100 mg · L-1 free SiO2 for 24 h,before LF were collected and the phenotype of LF was determined after transdifferentiation by immunohistochemistry. ChIP-chip was used to profile the variations of trimethylation in H3K4 of lung fibroblasts in CpG island regions. ChIP-qPCR was used to validate the microarray results. The mRNA expression of nfib and kpna3 was analyzed by qRT-PCR. RESULTS Totally 1815 (518 increased and 1297 decreased) genes of H3K4met3 displayed significant differences in SiO2 100 mg·L-1 group compared with control group(Cy3/Cy5 value>2.0 or <0.5,NimbleScan V2.5 software). The results of ChIP-qPCR were quite consistent with those of microarray. CONCLUSION There are significant differences in methylation of genome-wide H3K4 between SiO2 100 mg·L-1 group and control group. These novel candidate genes may become potential biomarkers or new interfered targets.
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Objective To explore the role of histone H3 acetylation modification of brain derived neurotrophic factor (BDNF) in the pathogenesis of Alzheimer's disease (AD).Methods 2 months and 8 months SAMP8 mice were used as AD model.Morris water maze was used to detect the impairment of learning and memory.Western blot was used to detect BDNF protein expression in the hippocampus,and chromatin immunoprecipitation (CHIP) was applied to study the changes of histone H3 acetylation in different BDNF promoters.Results The results of water maze test showed that the time across the target quadrant in 8 months SAMP8 mice(0.9±0.4) was significant declined compared with that of 2 months SAMP8 mice(3.7 ± ±0.9) and 8 months SAMR1 mice (3.3±0.6)(all P＜0.05).Meanwhile,compared with 2 months SAMP8 mice ((23.9±4.0) s) and 8 months SAMR1 mice ((21.5± 2.3) s),target quadrant time in the 8 months SAMP8 mice((11.7±2.8) s) was also significantly reduced(both P＜0.05).The western blot showed the expression of BDNF in the hippocampus of 8 months SAMP8 mice was significantly decreased compared with that of 2 months SAMP8 mice and 8 months SAMR1 mice(P＜0.05).Lastly,CHIP assays showed that histone H3 acetylation of BDNF exon Ⅳ and Ⅵ in the hippocampus of 8 months SAMP8 mice were remarkably decreased(P＜0.05) compared with that of 2 months SAMP8 mice and 8 months SAMR1 mice.There was no significant change of histone H3 acetylation of BDNF exon Ⅰ and Ⅲ among all groups(P＞0.05).Conclusion Histone H3 acetylation of BDNF exon Ⅳ and Ⅵ is reduced during the development of AD,which may be the mechanism underlying the impairment of learning and memory in AD.
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BACKGROUND: A long non-coding RNA hox transcript antisense intergenic RNA (HOTAIR) is involved in epigenetic regulation through chromatin remodeling by recruiting polycomb repressive complex 2 (PRC2) proteins (EZH2, SUZ12, and EED) that induce histone H3 trimethylation at lysine 27 (H3K27me3). Deregulation of c-MYC and interaction between c-MYC and EZH2 are well known in lymphomagenesis; however, little is known about the expression status of HOTAIR in diffuse large B-cell lymphomas (DLBCLs). METHODS: The expression status of PRC2 (EZH2, SUZ12, and EED), H3K27me3, c-MYC, and BCL2 was analyzed using immunohistochemistry (n = 231), and HOTAIR was investigated by a quantification real-time polymerase chain reaction method (n = 164) in DLBCLs. RESULTS: The present study confirmed the positive correlation among PRC2 proteins, H3K27me3, and c-MYC in DLBCLs. Expression level of HOTAIR was also positively correlated to EZH2 (p < .05, respectively). Between c-MYC and HOTAIR, and between c- MYC/BCL2 co-expression and HOTAIR, however, negative correlation was observed in DLBCLs (p < .05, respectively). High level of H3K27me3 was determined as an independent prognostic marker in poor overall survival (hazard ratio, 2.0; p = .023) of DLBCL patients. High expression of HOTAIR, however, was associated with favorable overall survival (p = .004) in the univariate analysis, but the impact was not significant in the multivariate analysis. The favorable outcome of DLBCL with HOTAIR high expression levels may be related to the negative correlation with c- MYC expression or c-MYC/BCL2 co-expression. CONCLUSIONS: HOTAIR expression could be one of possible mechanisms for inducing H3K27me3 via EZH2-related PRC2 activation, and induced H3K27me3 may be strongly related to aggressive DLBCLs which show poor patient outcome.
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Humans , B-Lymphocytes , Chromatin Assembly and Disassembly , Epigenomics , Histones , Immunohistochemistry , Lymphoma, B-Cell , Lymphoma, Large B-Cell, Diffuse , Lysine , Methods , Multivariate Analysis , Polycomb Repressive Complex 2 , Real-Time Polymerase Chain Reaction , RNA , RNA, Long NoncodingABSTRACT
AIM:To study the epigenetic mechanisms involved in the evolution of prostate cancer from an an-drogen-dependent state to an androgen-independent state , and the global difference of histone H 3 methylation between an-drogen-dependent and -independent prostate cancer cells .METHODS:The methylation sites and patterns of histone H 3 in androgen-dependent prostate cancer cell line LNCaP and androgen-independent prostate cancer cell line DU 145 were ana-lyzed by heavy methyl stable isotope labeling with amino acids in cell culture ( SILAC) coupled with 2D LC-MS/MS.West-ern blotting was used to verify the results from MS .The differential expression of related methylases and demethylases was tested by real-time PCR.RESULTS:Five methylation sites on histone H3 were found in both cell lines, the patterns of which were as follows: H3K14me2, H3R17me1, H3K36me1, H3K36me2, H3K36me3, H3R72me2, H3K79me1 and H3K79me2.There were 2 different peptides both containing methylated H 3K36,“KSAPATGGVKKPHR” and“KSAPSTG-GVKKPHR”, which were different from the 31th amino acid residue “A” and “S”.The former peptide belonging to his-tone H3 variants, H31T, H31 and H32, was mainly identified in DU145 cells, the total peptide counts of which were much more than that of the latter peptide belonging to histone H 3 variant H31T, suggesting that these 2 cell lines expressed differ-ent histone H3 variants.Mono-and dimethylation of H3K36 were not different between these 2 cell lines, but the trimethyl-ation was significantly higher in DU 145 cells than that in LNCaP cells .Many H3K36 demethyltransferases were decreased in DU145 cells compared with LNCaP cells .CONCLUSION: The differential expression of histone H 3 variants and H3K36 demethyltransferases may result in up-regulation of H3K36 tri-methylation during the evolution of prostate cancer from an androgen-dependent state to an androgen-independent state .
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PURPOSE: The purpose of the present study was to investigate the aberrance of histone H3 lysine 4 trimethylation (H3K4me3) in patients with IgA Nephropathy (IgAN). MATERIALS AND METHODS: In this study, H3K4me3 variations in peripheral blood mononuclear cells (PBMCs) from 15 IgAN patients and 15 healthy subjects were analyzed using chromatin immunoprecipitation linked to microarrays analysis (ChIP-chip). ChIP real-time PCR was used to validate the microarray results. Expression analysis by quantitative real-time PCR (qRT-PCR) revealed correlations between mRNA and H3K4me3 levels. DNA methylation status was analyzed by quantitative methylation-specific PCR. RESULTS: We found that 321 probes displayed significant H3K4me3 differences in IgAN patients compared with healthy controls. Among these probes, 154 probes displayed increased H3K4me3 and 167 probes demonstrated decreased H3K4me3. For further validation, we selected 4 key relevant genes (FCRL4, GALK2, PTPRN2 and IL1RAPL1) to study. The results of ChIP real-time PCR coincided well with the microarray data. Quantitative RT-PCR revealed the correlations between the mRNA expression and the methylation levels of H3K4me3. Different degrees of DNA methylation alterations appeared on the selected positive genes. CONCLUSION: Our studies indicated that there were significant alterations in H3K4me3 in IgAN patients. These findings may help to explain the disturbed immunity and abnormal glycosylation involved in IgAN patients.
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Adult , Female , Humans , Male , Young Adult , Case-Control Studies , Chromatin Immunoprecipitation , Glomerulonephritis, IGA/genetics , Histones/metabolism , Leukocytes, Mononuclear/metabolism , Lysine/metabolism , Methylation , Oligonucleotide Array Sequence Analysis/methods , Real-Time Polymerase Chain ReactionABSTRACT
BACKGROUND: Epigenetic alteration may affect a patient's prognosis by altering the development and progression of the tumor. Some recent reports have identified a correlation between histone modification and patient outcome. However, no studies have been conducted on global histone modification in osteosarcomas. METHODS: We investigated histone modification in 54 cases of osteosarcoma by performing immunohistochemical staining. The immunohistochemical expression of four histone modification markers, acetylated H4 lysine 12 (H4K12Ac), acetylated H3 lysine 18, trimethylated H3 lysine 27, and dimethylated H3 lysine 4 were evaluated. RESULTS: High H4K12Ac expression was correlated with patient age (p=0.011). However, the other histone modification markers showed no correlation with any of the clinicopathological data such as survival, tumor grade, tumor site, metastasis, age, or gender. CONCLUSIONS: Our study showed that all four histone modification markers are expressed in osteosarcoma (median expression rate, 40 to 60%). However, we did not find a correlation with the clinicopathological factors except for age. Further study to evaluate the reason for the association between H4K12Ac and patient age is needed.
Subject(s)
Humans , Epigenomics , Histones , Lysine , Neoplasm Metastasis , Osteosarcoma , PrognosisABSTRACT
Objective To study histone H3 lysine 4 trimethylation (H3K4me3) in peripheral blood mononuclear cells (PBMCs) of systemic lupus erythematosus(SLE) patients.Methods PBMCs were isolated by density gradient centrifugation from 10 active SLE patients,7 inactive SLE patients and 8 healthy volunteers.Chromatin immunopreeipitation linked to mieroarrays (ChIP-chip) was used to profile the variations in H3K4me3 in CpG island regions in PBMCs of SLE patients and controls.ChIP-qPCR was used to validate the mieroarray results.To confirm correlations between H3K4me3 and gene expression,expression analysis by qRT-PCR was performed on three randomly selected H3K4me3 candidates.Results 413 (137 increased and 276 decreased H3K4me3) and 393 genes (112 increased and 281 decreased H3K4me3) displayed significant differences in H3K4me3 between active and inactive SLE when compared with healthy SUhjeets.The results of ChiP-qPCR were consistent with microarray.ConclusiOn There are significant differences in H3K4me3 profiling between SLE and healthy subjects.These novel candidate genes may be potential biomarkers for future therapeutic targets.The ChIP-chip technology can help further reveal SLE molecular mechanisms and discover new therapeutic targets.
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Objective Applying a reliable precise method to assess the mitotic index of cardiomyocytes,to disclose of disclosure the mechanism implicated in cardiomyocytes proliferation. Methods H9c2(2-1) cardiomyocytes were originally developed from rat BD1X heart(ATCC).These cells were cultured on coverslips.Double immunofluorescence staining with monoclonal antibodies(1:100) against phospho-histone H3 and ?-sarcomeric actin was performed on the cultured cells.Anti-mouse IgG FITC was used as the secondary antibody for the H3P antibody,and anti-mouse IgM Cy3 was used as the secondary antibody for the ?-sarcomeric actin.DNA was visualized with Hochest 33342.All photographs were taken with an Olympus fluorescence microscope. Results The cytoplasm of cardiomyocytes appeared red,the mitotic chromosomes green with distinct shape,and Hochest 33342-stained nuclei blue.Conclusion Our method is the reliable and exact means to observe and assess cardiomyocytes mitosis.