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The eukaryotic genome is folded into higher-order conformation accompanied with constrained dynamics for coordinated genome functions. However, the molecular machinery underlying these hierarchically organized three-dimensional (3D) chromatin architecture and dynamics remains poorly understood. Here by combining imaging and sequencing, we studied the role of lamin B1 in chromatin architecture and dynamics. We found that lamin B1 depletion leads to detachment of lamina-associated domains (LADs) from the nuclear periphery accompanied with global chromatin redistribution and decompaction. Consequently, the inter-chromosomal as well as inter-compartment interactions are increased, but the structure of topologically associating domains (TADs) is not affected. Using live-cell genomic loci tracking, we further proved that depletion of lamin B1 leads to increased chromatin dynamics, owing to chromatin decompaction and redistribution toward nucleoplasm. Taken together, our data suggest that lamin B1 and chromatin interactions at the nuclear periphery promote LAD maintenance, chromatin compaction, genomic compartmentalization into chromosome territories and A/B compartments and confine chromatin dynamics, supporting their crucial roles in chromatin higher-order structure and chromatin dynamics.
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Humanos , Cromatina , Cromossomos , Genoma , Lamina Tipo B/genéticaRESUMO
Background: Enteroscopy-based biopsy pathology has high diagnostic value for suspected small bowel diseases. Retrograde single-balloon enteroscopy (SBE) is difficult to operate due to the influence of colonic segment. Transparent cap has been widely used in the diagnostic and therapeutic endoscopic procedure, which is conducive for inserting the enteroscope and stabilizing the intestinal cavity. Aims: To explore the role of transparent cap in retrograde SBE. Methods: A total of 64 cases of patients who were hospitalized for retrograde SBE in Suzhou Wuzhong People's Hospital were recruited and allocated into 2 groups according to the random number table method, with 32 cases in each group. Patients in experimental group received cap-assisted SBE and those in control group received routine SBE. All procedures were performed by an experienced senior endoscopist. Outcomes measured included time to reach the ileocecus, terminal ileum intubation rate, depth of insertion, diagnostic yield, and the related complications. Results: The mean time to reach the ileocecus was shorter in experimental group than in control group [(11.8±2.0) min vs. (13.6±2.8) min, P0.05). No severe complications were observed in all the cases. Conclusions: Cap-assisted retrograde SBE is an efficient method for optimizing the intubation rate, insertion depth and procedure time, and is suitable for promotion in clinical practice.
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Objective To investigate the application value of Multi-Latex polygranular technique joint detection of kidney injury-related urinary microproteins in noninvasive diagnosis after renal transplantation. Methods Clinical data of 72 recipients undergoing renal transplantation were retrospectively analyzed. According to the level of serum creatinine (Scr), the recipients were divided into normal renal function group (group A, n=14), mild kidney injury (group B, n=37), and severe kidney injury group (group C, n=21). 20 healthy volunteers were selected as the healthy control group (HC group). The contents of urinary retinol binding protein (RBP), microalbumin (mAlb), IgG, transferrin (TRF), α1-microglobulin (MG), and β2-MG of subjects in each group were detected using the Multi-Latex polygranular technique. The correlation between urinary microproteins and Scr, blood urea nitrogen (BUN) was analyzed. The differences of urinary microproteins in each group were compared. And the diagnostic value of single and joint detection of urinary microproteins was evaluated. Results Six kinds of urinary microproteins in HC group and group A were significantly lower than those in group B and group C, and six kinds of urinary microproteins in group B were significantly lower than those in group C (all P < 0.01). Six kinds of urinary microproteins in renal transplant recipients were positively correlated with BUN. RBP, mAlb, α1-MG, and β2-MG were positively correlated with Scr. The correlations were statistically significant (P < 0.001-0.05). The diagnostic value of joint detection of urinary microproteins is better than the detection of single index, among which TRF+mAlb+RBP+α1-MG quadruple detection had the highest diagnostic value. Conclusions Six kinds of urinary microproteins can be used as specific indicators to reflect graft renal function. The polygranular technique can simultaneously detect its contents and achieve noninvasive diagnosis. The diagnosis based on TRF+mAlb+RBP+α1-MG quadruple detection is expected to further improve the noninvasive diagnosis system after renal transplantation.
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Objective To investigate the dynamic changes of peripheral blood lymphocyte subsets and their correlation with renal function in recipients with stable graft status after renal transplantation. Methods Forty-five recipients who underwent renal transplantation for the first time and had stable graft function within postoperative 6 months were selected. The proportion and absolute value of lymphocyte subsets were detected by flow cytometry (FCM) in 180 peripheral blood samples from recipients at 15 d, 1, 3 and 6 months after renal transplantation. The dynamic changes of lymphocyte subsets with the extension of postoperative time and their correlation with serum creatinine (Scr) and blood urea nitrogen (BUN) were analyzed. Results The Scr levels did not significantly differ at 4 time points after renal transplantation (all P > 0.05). The BUN levels significantly differed between 15 d and 1 month after renal transplantation, and between 1 and 3 months after renal transplantation (P=0.002, P=0.001). The proportion of CD3+CD8+T cells, CD3+CD4+T cells, natural killer (NK) cells and CD4/CD8 ratio at postoperative 15 d significantly differed from those at 1 month after operation (P=0.009, P=0.004, P < 0.001, P=0.004). The proportion of B cells significantly differed between 15 d and 1 month, and between 1 and 3 months after renal transplantation (both P < 0.001). The absolute values of CD3+T cells, CD3+CD8+T cells, CD3+CD4+T cells and NK cells at postoperative 15 d significantly differed from those at 1 month after renal transplantation (P=0.001, P=0.002, P=0.003, P < 0.001). The absolute values of CD3+CD8+T cells significantly differed between 3 and 6 months after operation (P=0.015). The absolute value of B cells at 1 month after renal transplantation significantly differed from that at 3 months after renal transplantation (P=0.001). The proportion and absolute value of lymphocyte subsets were not significantly correlated with the Scr level (both P > 0.05). The proportion and absolute value of CD3+CD8+T cells and NK cells were negatively correlated with BUN (P < 0.001-0.05), whereas the proportion of CD3+CD4+T cells and B cells was positively correlated with the BUN level (P < 0.001-0.05). The absolute value of CD3+T cells was negatively associated with the BUN level (P < 0.05). Conclusions T cells and NK cells in the lymphocyte subsets of stable recipients raise to the stable state within 1 month after renal transplantation, whereas B cells decrease to stable state within 3 months renal transplantation. The dynamic changes of lymphocyte subsets are correlated with the BUN level.
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Objective To investigate the role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in the pathogenesis of psoriasis by detecting the level of PCSK9 in the plasma of patients with psoriasis and evaluating its effect on the secretion of interferon gamma (IFN-γ) and interleukin-17A (IL-17A) by peripheral CD4+ T cells.Methods Totally,30 outpatients with psoriasis vulgaris and 30 healthy volunteers (controls) were enrolled from Hospital for Skin Diseases,Chinese Academy of Medical Sciences between February 2016 and December 2017.Of the 30 patients,16 were males,and 14 were females.Their age varied from 18 to 66 years,and the course of disease ranged from 1 month to 15 years.Peripheral venous blood samples were obtained from the patients and controls,and the plasma and was performed to measure mRNA expression of PCSK9 in the PBMC,and enzyme-linked immunosorbent assay (ELISA) to determine the concentration of PCSK9 in the plasma.Peripheral CD4+ T cells were isolated from the PBMC by magnetic bead method,and divided into 2 groups to be co-cultured with (experiment group) or without PCSK9 protein (control group).After 24-hour treatment,ELISA was conducted to detect the levels of IFN-γ and IL-17A in the culture supernatant.Statistical analysis was carried out by using two-sample t test for the comparison between the two groups,and Pearson correlation analysis for analyzing correlations between the plasma level of PCSK9 and psoriasis area and severity index (PASI) score in the patients with psoriasis.Results PCSK9 mRNA expression was undetected in the PBMC of the patients with psoriasis and controls.The plasma level of PCSK9 was significantly higher in the patients with psoriasis ([243.58 ± 11.91] μg/L) than in the healthy controls ([199.74 ± 31.09] μg/L,t =5.761,P ＜ 0.001).After co-culture of the peripheral CD4+ T cells from patients with PCSK9 protein,the levels of IFN-γ and IL-17A both significantly increased ([6 150.00 ± 212.13] ng/L,[1 532.00 ± 11.31] ng/L,respectively) compared with the control group co-cultured without PCSK9 protein ([4 650.00 ± 212.13] ng/L,[698.5 ± 266.58] ng/L,respectively;t =7.071,4.418 respectively,both P ＜ 0.05).IFN-γand IL-17A were undetected in the culture supernatant of CD4+ T cells from the healthy controls in the experiment group or control group.Conclusion The plasma level of PCSK9 increases in patients with psoriasis,which may be involved in the pathogenesis of psoriasis by activating peripheral CD4+ T cells.
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Objective To assess the value of flow cytometry in the diagnosis of postoperative infection following renal transplantation. Methods According to postoperative imaging findings and laboratory examination outcomes, 51 recipients undergoing the first renal transplantation were divided into the bacteria (n=33), fungus (n=9) and BK virus (n=9) groups. Twenty-eight recipients with stable conditions after renal transplantation were assigned into the stable group. Flow cytometry was adopted to detect the percentage and absolute counting of lymphocyte subpopulation in the peripheral blood of recipients in each group. Renal function, percentage and absolute counting of lymphocyte subpopulation in the peripheral blood were statistically compared among different groups. Receiver operating characteristic (ROC) curve was drawn to evaluate the diagnostic value of the percentage and absolute counting of lymphocyte subpopulation in infectious diseases after renal transplantation. Results Compared with the stable group, the serum creatinine (Scr) and blood urea nitrogen (BUN) levels in the bacteria, fungus and BK virus groups were significantly up-regulated, respectively (P=0.035, 0.007, 0.024; 0.037, 0.006, 0.032). Compared with the stable group, the percentage of CD16+CD56+natural killer (NK) cells was significantly declined in the bacterial (P=0.036) and fungus groups (P=0.015), and the proportion of CD4+/CD8+T cells was dramatically decreased in the fungus group (P=0.004). Compared with the bacterial group, the percentage of CD3+CD8+T cells was significantly elevated (P=0.013 and 0.008), the proportion of CD3+CD4+T cells was considerably declined (P=0.003 and 0.010), and the percentage of CD4+/CD8+T cells was significantly declined (P=0.003 and 0.005) in the fungus and BK virus groups. Compared with the stable group, the quantity of CD3+T cells, CD3+CD8+T cells and CD16+CD56+NK cells was significantly declined in the bacterial, fungus and BK virus groups, respectively (P=0.025, 0.002, 0.003; 0.015, 0.005, 0.006; 0.001, 0.001, 0.031). In addition, the quantity of CD3+CD4+T cells was considerably decreased in the fungus and BK virus groups (P=0.001, 0.003). The quantity of CD19+B cells was significantly reduced in the BK virus group (P=0.019). Compared with the bacterial group, the quantity of CD3+CD4+T cells was considerably lower in the fungus group (P=0.023). ROC curve analysis revealed that the quantity of CD3+CD4+T cells [area under curve(AUC)=0.8492] and CD16+CD56+NK cells (AUC=0.8889) yielded relatively high accuracy in the diagnosis of fungal infection. The quantity of CD3+T cells (AUC=0.8472), CD3+CD4+T cells (AUC=0.8452) and CD19+B cells (AUC=0.8115) yielded relatively high accuracy in the diagnosis of BK virus infection. Conclusions Flow cytometry detection of the lymphocyte subpopulation in peripheral blood can evaluate the immune function of patients. Absolute counting of lymphocyte subpopulation can directly assess the degree of immunity. These two combined parameters provide guiding significance for the diagnosis and differential diagnosis of infectious diseases in recipients after renal transplantation.
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Acupuncture is developing rapidly in the world, and more attention is paid on acupuncture in various countries. Because of the cultural differences, there are different views on acupuncture between China and the west, which has brought influence and challenge to the development of acupuncture in the world. Acupuncture-related research is becoming increasingly extensive and complex, but the definition of acupuncture is lack of unified standards. The definition of acupuncture is in urgent need. Based on the analysis of acupuncture definition in the 201 international organizations of 48 countries on five continents and legislation of representative countries, this paper summarized the development status of acupuncture in foreign countries, and put forward that the definition of acupuncture should adopt the model of small connotation and large extension, integrate discipline superiority, expand the scope of acupuncture, and focus on the overall situation.
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Objective To evaluate the effect of γ-aminobutyric acid (GABA) and its receptors upon the proliferation of CD8+T cells.Methods The splenic CD8+T cells of Balb/c mice were obtained by CD8+f cell magnetic bead sorting kit.Under the effect of CD3/CD28-activated magnetic bead,CD8+T cells were stimulated by different concentrations of GABA.5-bromo-2-deoxyuridine (BrdU) labeling and flow cytometry were performed to detect the proliferation of CD8+T cells.The expression levels of GABA-A and GABA-B receptor before and after CD8+T cell activation were compared by fluorescent quantitative real-time polymerase chain reaction (PCR).Result Flow cytometry result revealed that GABA could inhibit the proliferation of activated CD8+T cells,manifested as significant decrease in the quantity of CD152+CD8+T cells.Fluorescent quantitative real-time PCR demonstrated that GABA-A receptor subtypes α2,α6 and GABA-B receptor subtype 1a were expressed only when the CD8+T cells were activated.After CD8+T cell activation,the quantity of GABA-A receptor subtypes α3,αs,β2,β3,γ1,γ2 and θ were significantly increased,whereas the quantity of GABA-B2R and GABA-B1b did not significantly differ before and after CD8+T cell activation.Conclusions GABA can suppress the proliferation of activated CD8+T cells.The activation of CD8+T cells is regulated by GABA receptors.However,the underlying mechanism remains to be elucidated.
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Objective To establish a novel method for detecting circulating tumor cells (CTCs) in phripheral blood of lung cancer patients with high sensitivity and specificity.Methods Experimental study.42 cases of initial treatment patient who underwent resection and diagnosed to be non-small cell lung cancer by biopsy were studied,including 7 patients at stage Ⅰ,9 patients at stage Ⅱ,16 patients at stage Ⅲ and 10 patients at stage Ⅳ.As a control group,20 cases of healthy volunteers were selected.A series of experiments was conducted to determine the efficiency of tumor cells isolation,in which varied concentration (50,100,200,500,1000 cells) of A549 cells spiked into 2 ml peripheral blood drawn from healthy donors.The blood was removed of unwanted erythrocytes by lysis buffer,and made the rest of nucleated cells incubate with anti-EpCAM magnetic beads,then separated and enriched by a specific detector.All epithelia cells were retained on a slide because of a magnetic force and identified by H&E staining protocol.On the basis of cell recovery rate we calculated the sensitivity of tumor cells isolation.20 blood samples taken from healthy individuals were also detected to validate the specificity of this method.Samples of 42 patients with lung cancer were assayed for CTCs detection by above method.The correction of CTCs quantity with the patients' clinical features,for example,ages,gender,clinical stage,tumor size was analyzed in lung cancer patients by chi-square statistics.The correction of recovery cells with the spiked cells were assayed by linear correlation.Results The recovery rate was ranging from 68％ to 82％ by spiking varying numbers of A549 lung cancer cells into 2ml blood samples of healthy volunteers.Regression analysis of number of recovered vs.spiked A549 cells yielded a regression equation of Y =0.6419X + 8.8875.The number of CTCs detected has signification correlate with the cells spiked (R2 =0.9916,P ＜ 0.05),Eighteen of the 42 patients (43％) were found have CTCs in peripheral blood.The detection rate of lung cancer cells was 0 at stage Ⅰ,the detection rate of lung cancer cells was 11.1％ at stage Ⅱ,the detection rate of lung cancer cells was 62.5％ at stage Ⅲ and the detection rate of lung cancer cells was 70％ at stage Ⅳ.The positive rate of CTCs has no signification correlate with ages and gender of patients and tumor size (P ＞ 0.05),has signification with the clinical stage (P ＜ 0.05).None of the peripheral blood samples of the 20 healthy subjects analyzed was found to have CTCs.Conclusions This novel immunomagnetic separation technology is a sensitive and specific method,which provides a new tool allowing for feasible and specific detection of CTCs in lung cancer patients.The level of CTCs increases with the clinical stage and tumor size increased,which has important value to discover the early stage micrometastasis and redefine the clinical stage.But further multicenter and large sample clinical research are needed to confirm its clinical value.
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Objective To set up a real-time quantitative PCR approach for detection and quantification for bcr-abl transcripts in CML patients,and detect minimal residual disease (MRD) in CML by real-time quantitative PCR (RQ-PCR)and evaluate the significance of MRD detection.Methods The ber-abl.fusion gene expression in 80 patients with CML was analyzed by RQ-PCR. The patients were divided into three groups according to the different treatment, allogeneic hematopoietic stem cell transplantation group,imatinib group and hydroxyurea group. The change of bcr-abl fusion gene was monitored in CML patients before and after treatment.Results The average of RQ-PCR detection on newly diagnosed patients with CML in chronic phase was 6847.67 copies / 104 cells,the accelerated phase was 306 176.08 copies / 104 cells,and the average results were 944.33, 2.37, 0.29, 0 copies / 104 cells after allogeneic hematopoietic stem cell transplantation one month,6 months,12 months or 24 months respectively.The average of RQ-PCR detection after use imatinib mesylate 3 months was 3720.23 copies / 104 cells and not be detected after one year. The average was 7290.11 and 3143.24 copies / 104 cells after hydroxyurea treatment 0 and 9 months respectively.The difference in first two groups was not significant (t=1.74,P=0.17), but the difference between the third group and the first two groups was significant (t=3.74,P=0.01.t=2.97,P=0.02). The upregulation of bcr-abl transcript levels could be detected when disease progression. The transcripts level in accelerated phase was significantly higher than that in chronic phase. Conclusion RQ-PCR can be used to detect the MRD,monitor the treatment outcome,predict disease recurrence and give early intervention.
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Objective To investigate the correlation between the expression level of BLM and its clinical significance in leukemia.Methods 125 bone marrow specimens of inpatients and outpatients with leukemia were collected in Anhui provincial hospital from January 2011 to December 2011.125 leukemia patients were diagnosed and classified into acute leukemia (AL,n =66) and chronic myelogenous leukemia (CML,n =59) by Morphologic and Immunologic criteria,5 non-tumor individuals were included as control group.The BLM mRNA expressions were by reverse transcription-polymerase chain reaction(RT-PCR).The specimens were devided into groups according to the age,gender,leukemia type,peripheral blood leukocyte counts,hepatomegalia and(or) splenomegaly,fusion gene,chromosome karyotype,whether first visit and transplantation.The expression of BLM gene in each group and the correlation with above factors were retrospectively analysed.The statistical methods such as chi-square test,single factor variance analysis,t test and Pearson correlation test were mainly used.Results BLM mRNA was detected in leukemia.In bone marrow cells,the BLM gene expression was positive in 71 patients and negative in 54 patients.But none of 5 non-tumor bone marrow cells expressed BLM gene.The difference of BLM expression between patients and controls was statistically significant in two groups,i.e.peripheral blood leukocyte counts and fusion gene (x2 =14.730,22.399 ; P ＜ 0.05),but there is no statistical significant differences in other groups.The expressions of BLM mRNA in leukemia patients who had been treated with chemotherapy were lower than those newly diagnosed (0.1788 ± 0.1091 vs 0.3276 ± 0.2016 ; P ＜ 0.05).Moreover,BLM mRNA level in post-bone marrow transplant patients was lower than those not treated (0.1271 ± 0.1009 vs 0.2902 ±0.2034 ; P ＜ 0.05).Pearson correlation analysis showed that higher BLM mRNA expression positively correlated with fusion gene (r =0.357,P ＜ 0.01) and chromosome abnormality (r =0.279,P ＜ 0.05).Conclusion The BLM mRNA expression level of measurement can be used as judgment for leukemia patients disease severity and the index of prognosis,testing the level may provide a basis for clinical and curative effect judgment.
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Objective To establish a real-time fluorescence quantitative PCR method for detection of the different expression level of FPGS in methotrexate enantiomer-resistant A549 cell lines,and observe FPGS mRNA expression in patients with leukemia.Methods A real-time fluorescence quantitative PCR method for detection FPGS mRNA was established using SYBR Green Ⅰ as fluorescence and β-actin as reference.The method was evaluated by Ct,correlation coefficient,slope,repeatability curve,melting curve and amplification efficiency curve.The expression levels of FPGS gene in methotrexate enantiomer-resistant A549 cell lines and methotrexate resistant leukemia cells in bone marrow were detected by the method.Results The standard curves had a high linear relationship between cycle threshold and template concentration.The correlation coefficients of FPGS and β-actin were 0.996 8 and 0.998 7,and the slopes were -3.595 and -3.740,respectively.The inter-coefficient of variation was from 1.27% to 2.95%.The intra-coefficient of variation was 3.82%.The method was characterized with specific melting curve and similar amplification efficiency(slope was 0.021 7).The relative contents of FPGS mRNA were(3.51 ±0.66),(0.16 ±0.01) and(1.00 ±0.31) in L-(+)-MTX/A549 cells(L),D-(-)-MTX/A549 cells(D)and A549 parent cells,and there was statistically difference among the three groups(F = 64.45 ,P＜ 0.01)Statistical difference was observed between L and D(q =9.29,P＜0.01).After treated with MTX,the expression level of FPGS mRNA was(0.35 ± 0.04) in methotrexate resistant leukemia patients,compared with(1.00 ± 0.44) before treatment.Statistical difference was observed(t = 8.83 ,P＜ 0.01).Conclusions The real-time fluorescence quantitative PCR is suitable for the quantification of FPGS.The expression levels of FPGS in methotrexate resistant leukemia cells in bone marrow and drug resistant cells are different.Two enantiomer forms of methotrexate may play different roles in drug resistance mechanisms.
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Objective To investigate the influence of electroacupuncture on the expression of neuropeptides in fibroeartilage callus tissue after femoral fracture. Methods Forty male Wistar rats were randomly divided into a fracture group and an electroacupuncture group. Femoral fracture models were established in both groups, and the rats in the electroacupuncture group were given electroacupuncture. Rats were sacrificed on the 4th, 7th, 14th and 28th days after surgery. The calluses were stained immunohistochemically to detect the expression of calcitonin gene-related peptide (CGRP) and substance P (SP). Results CGRP and SP levels increased rapidly after eleetroacu-puncture, with neuropeptides expressed strongly in callus cell tissue. The optical density (OD) in the electroacu-puncture group was significantly higher than that in the fracture group ( P≤0. 05 ). Conclusion Electroaeupune-ture can significantly increase the expression of neuropeptides in rats after femoral fracture. Electroaeupuneture can promote fracture healing through regulating neuropeptides.
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Objective To observe the effect of treating recurrent oral ulcer with integrated therapy of traditional Chinese and western medicine. Methods 60 patients of recurrent oral ulcer were randomly divided into treatment group and control group. Patients in the control group were treated with Levamisole (take orally 45rag for each time, and once daily,and hold on for 5 days after take it for successive two days) and vitamin C (100mg daily). On the basis of treatment for the control group, patients in the treatment group were also treated with Huanglian Jiedu Decoction taken orally and Shuanghuang powder applied externally. Results The total effective rate was 97.2% and 83.3% respectively in the treatment group and the control group. Conclusion The therapeutic effect of treating recurrent oral ulcer with integrated therapy of traditional Chinese and western medicine is exceUent.