Killing effects of 5-Aza-CdR combined with EBNA1-DC vaccine-induced lymphocytes on nasopharyngeal carcinoma C666-1 cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨EB病毒核抗原1（EBNA1） mRNA修饰的DC（EBNA1-DC）诱导的淋巴细胞联合甲基化抑制剂5-Aza-CdR对鼻咽癌C666-1细胞的杀伤作用。方法：以构建的EBNA1-pCDNA3.1质粒为模板，体外转录获得EBNA1 mRNA，通过脂质体转染至健康人外周血来源DC，构建EBNA1-DC疫苗。流式细胞术检测转染后DC表型及5-Aza-CdR处理后的C666-1细胞凋亡情况。实时无标记动态细胞分析技术检测EBNA1-DC疫苗诱导的淋巴细胞联合5-Aza-CdR的特异性抗肿瘤活性。结果：转染EBNA1 mRNA后EBNA1-DC表面EBNA1阳性率为（59.3±5.85）%，HLA-DR的表达与未转染DC相比显著升高[（84.9±5.5）% vs （68.0±5.8）%，P=0.026]，CD80的表达也显著升高[（88.2±3.9）% vs （61.1±4.4）%，P=0.015]。低剂量5-Aza-CdR处理后的C666-1细胞凋亡情况与未处理的细胞相比无显著差异。经低浓度5-Aza-CdR预处理的C666-1细胞中IRF7基因表达与未处理的细胞相比显著升高（P=0.000 1）。与空载的DC相比，EBNA1-DC诱导的淋巴细胞对EBV阳性表达的C666-1细胞具有更强的特异性杀伤活性（P=0.049）；经低浓度5-Aza-CdR预处理的C666-1细胞对EBNA1-DC诱导的特异性免疫杀伤更敏感（P=0.019）。结论：5-Aza-CdR与EBNA1-DC疫苗联合可显著增强对C666-1细胞的特异性免疫杀伤，本研究为开拓以mRNA为基础的DC疫苗及其在临床综合治疗中的应用转化提供前期研究基础。

UHRF1/DNMT1-MZF1 axis-modulated intragenic site-specific CpGI methylation confers divergent expression and opposing functions of PRSS3 isoforms in lung cancer

As confusion mounts over RNA isoforms involved in phenotypic plasticity, aberrant CpG methylation-mediated disruption of alternative splicing is increasingly recognized as a driver of intratumor heterogeneity (ITH). Protease serine 3 (PRSS3), possessing four splice variants (PRSS3-SVs; PRSS3-V1-V4), is an indispensable trypsin that shows paradoxical effects on cancer development. Here, we found that PRSS3 transcripts and their isoforms were divergently expressed in lung cancer, exhibiting opposing functions and clinical outcomes, namely, oncogenic PRSS3-V1 and PRSS3-V2 versus tumor-suppressive PRSS3-V3, by targeting different downstream genes. We identified an intragenic CpG island (iCpGI) in PRSS3. Hypermethylation of iCpGI was mediated by UHRF1/DNMT1 complex interference with the binding of myeloid zinc finger 1 (MZF1) to regulate PRSS3 transcription. The garlic-derived compound diallyl trisulfide cooperated with 5-aza-2'-deoxycytidine to exert antitumor effects in lung adenocarcinoma cells through site-specific iCpGI demethylation specifically allowing MZF1 to upregulate PRSS3-V3 expression. Epigenetic silencing of PRSS3-V3 via iCpGI methylation (iCpGIm) in BALF and tumor tissues was associated with early clinical progression in patients with lung cancer but not in those with squamous cell carcinoma or inflammatory disease. Thus, UHRF1/DNMT1-MZF1 axis-modulated site-specific iCpGIm regulates divergent expression of PRSS3-SVs, conferring nongenetic functional ITH, with implications for early detection of lung cancer and targeted therapies.

CircularRNA CDR1as promotes osteogenic differentiation and angiogenesis related genes expression in mouse bone marrow mesenchymal stem cells / 口腔疾病防治

Objective@# To investigate the effects of over expression and low expression of antisense transcripts of circular RNA cerebellar degeneration associated protein 1 (CDR1as) in Balb/C mouse bone marrow mesenchymal stem cells (BMSCs) on factors related to osteogenesis and angiogenesis.@*Methods@#BMSCs were cultured and identified in vitro. The lentiviral (LV) vector containing the overexpressed and silenced circRNA CDR1as genes and the control lentivirus were respectively transfected into mouse BMSCs, and stable cell lines were screened. The cells were divided into the circRNACDR1as over expression group and the over expression control group, and the CircRNACDR1as low expression group and the low expression control group. The components were stained with Alizarin Red S and alkaline phosphatase after 14 and 21 days of osteoinduction; qRT-PCR was used to detect the target genes circRNA CDR1as, osteogenic differentiation markers alkaline phosphatase (ALP), runt- related transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN), osterix(Osx), collagen I (COL-1), and the mRNA expression levels of vascular endothelial grown factor (VEGF) and angiogenin-1 (Ang-1). @*Results@# The results of alizarin red staining and alkaline phosphatase staining showed that the extracellular matrix calcium precipitation and ALP staining area of the over expression experimental group was greater than its control group, and those of the low expression experimental group was less than its control group. As the number of days of osteogenic induction increased, the calcium precipitation and ALP staining in each group also increased. RT-PCR results showed that the mRNA expression levels of circRNA CDR1as, ALP, RUNX2, OCN, OPN, OSX, COL-1, VEGF and Ang-1 in the over expression experimental group BMSCs were significantly increased (P<0.001). In the low expression experimental group, the mRNA expression levels of circRNA CDR1as, ALP, RUNX2, OCN, OPN, OSX, COL-1, VEGF and Ang-1 in BMSCs were significantly reduced (P<0.001). @*Conclusion@# Over expression of the circRNA CDR1as gene promotes the osteogenic differentiation and angiogenesis of BMSCs. Low expression of the circRNA CDR1as gene inhibits the osteogenic differentiation and angiogenesis of BMSCs.

Affinity transfer of nanobodies to a CDR3 only single-domain antibody fragment NBL42 / 国际生物医学工程杂志

Objective:To evaluate the potential of a previously identified CDR3 only single-domain antibodies (sdAbs) fragment, NBL42, as a general framework for affinity transfer.Methods:The H3 loops of VHH-A4(A4), VHH-H5(H5), cAb-Lys3(L3) and B6H12 which bind with alliinase, PD-1, lysozyme and CD47, respectively, were grafted into the corresponding loop of NBL42. The genes of the reconstituted CDR3 only sdAbs were synthesized, expressed in E. coliand purified with Ni 2+ column affinity chromatography. The antigen binding and stability of the recombinant CDR3 only sdAbs were assayed by ELISA. Results:The recombinant NBL42-A4CDR3, NBL42-H5CDR3, NBL42-L3CDR3 and NBL42-B6H12CDR3 ran as a single peak at 15, 15, 28 and 16 kDa, respectively, in SDS-PAGE as expected molecular weight. Grafted sdAbs NBL42-A4CDR3 and NBL42-H5CDR3 expressed in a soluble form and specifically bind with alliinase and PD-1, respectively, but lost about 50% of their binding activity. In contrast, the grafted sdAbs NBL42-Lys3CDR3 and NBL42-B6H12CDR3 completely lost their antigen binding capacity. NBL42 sdAbs and grafted sdAbs NBL42-A4CDR3 and NBL42-H5CDR3 retain roughly half of their binding activity after 90 ℃ heat treatment, indicating high stability. The C88Y mutation in NBL42 and the Swiss Mode 3D model predicted that the C88Y residue in FR3 may play a key role in NBL42 stability and CDR3 affinity transfer.Conclusions:The structure of NBL42 has potential as a framework for CDR3 transplantation and affinity transfer.

Circular RNA CDR1as: A Novel Regulator of Malignant Tumors / 中国生物化学与分子生物学报

Circular RNA (circRNA) is a new type of non-coding RNA with closed circular structures that are widely distributed in various tissues Compared with traditional linear RNA, circRNA does not have 5′ and 3′ ends and will not be easily degraded by exonuclease It can stably exist in a variety of body fluids and is evolutionarily conserved It has become a key research object of clinical non-coding RNA Malignant tumors have the characteristics of late detection, rapid progression, and easy recurrence Currently, effective treatment methods are lacking, and their morbidity and mortality have been high Therefore, how to carry out early diagnosis, treatment intervention and prognosis evaluation is one of the research frontier of contemporary medical research CDR1as is the most widely studied circRNA It can regulate the expression of downstream genes through sponge microRNA (miRNA) or directly bind to RNA-binding proteins (RBPs) to activate related signaling pathways, thereby promoting or inhibiting tumor progression, and even affecting tumor chemotherapy sensitivity CDR1as mainly exists in the cytoplasm and can be released into the blood at the early stage of the disease Therefore, CDR1as may become a bi-omarker for early diagnosis of malignant tumors or an ideal target for therapeutic intervention Focusing on the characteristics and biological functions of circRNA, this article reviews the expression level, mechanism of action and related signaling pathways of CDR1as in the occurrence and development of malignant tumors At the same time, this article analyzes the current research status of CDR1as, preliminarily summarizes the problems it may face in clinical applications in the future, and puts forward ideas and suggestions on the future research direction of CDR1as

Research on the expression and regulation of ASPP2 and its methylationin human gastric carcinoma cell / 中华内分泌外科杂志

Objective To investigate the relationship between the expression of ASPP2 mRNA and the methylation of ASPP2 gene in gastric cancer cells,to observe the inhibitory effect of 5-Aza-CdR on the growth of gastric cancer cells,to observe the effect of demethylation on the expression of ASPP2 mRNA and the methylation of ASPP2 gene in gastric cancer cells,and to explore the molecular mechanism of gastric cancer.Methods Real-time PCR was used to detect the expression of ASPP2 mRNA in two gastric cancer cells and normal gastric epithelial cells.BSP was used to detect the methylation of ASPP2 gene in two gastric cancer cells and normal gastric epithelial cells.CCK-8 was used to detect the growth inhibition rate of gastric cancer cells treated with 5-Aza-CdR of different concentrations,then they were used to detect expression of ASPP2 mRNA and the methylation of ASPP2 gene in gastric cancer cells again after the demethylation.Results ① The expression of ASPP2 mRNA in MKN-45 cells was significantly lower than that in GES-1 cells(P＜0.01).The expression of ASPP2 mRNA in MGC-803 cells was significantly lower than that in GES-1 cells (P＜0.01).There was no significant difference in MGC-803 cells and MKN-45 cells(P＞0.05).② The methylation rate of ASPP2 in MKN-45 cells was significantly higher than that in GES-1 cells (P＜0.01).The methylation rate of ASPP2 in MGC-803 cells was not significantly different from that in GES-1 cells (P＞0.05).The methylation rate of ASPP2 in MKN-45 cells was significantly higher than that in MGC-803 cells (P＜0.01).③ At the same time,the growth inhibition rate of each 5-Aza-CdR concentration group increased as the drug concentration depended.4.The expression of ASPP2 mRNA in MKN-45 cells was significantly higher than that before treatment (P＜0.01),the expression of ASPP2 mRNA in MGC-803 cells was not significantly different from that before treatment(P＞0.05).The methylation rate of ASPP2 in MKN-45 cells was significantly lower than that before treatment (P＜0.01).The methylation rate of ASPP2 in MGC-803 cells was not significantly different from that before treatment (P＞0.05).Conclusion ① Abnormal hypermethylation of ASPP2 gene in MKN-45 cells may be a molecular mechanism of decreased ASPP2 mRNA expression.② 5-Aza-CdR can inhibit the growth of MKN-45 and MGC-803 cells,and it can enhance the expression of ASPP2 mRNA in MKN-45 cells.Reversal of methylation in the promoter region of ASPP2 gene is the possible mechanism.③ Abnormal hypermethylation of the promoter region of ASPP2 gene may lead to silencing of mRNA expression that may be associated with gastric cancer.

Cis-2-dodecenoic Acid Mediates Its Synergistic Effect with Triazoles by Interfering with Efflux Pumps in Fluconazole-resistant Candida albicans / 生物医学与环境科学（英文）

OBJECTIVE@#To evaluate the synergy of the Burkholderia signaling molecule cis-2-dodecenoic acid (BDSF) and fluconazole (FLU) or itraconazole (ITRA) against two azole-resistant C. albicans clinical isolates in vitro and in vivo.@*METHODS@#Minimum inhibitory concentrations (MICs) of antibiotics against two azole-resistant C. albicans were measured by the checkerboard technique, E-test, and time-kill assay. In vivo antifungal synergy testing was performed on mice. Analysis of the relative gene expression levels of the strains was conducted by quantitative reverse-transcription polymerase chain reaction (qRT-PCR).@*RESULTS@#BDSF showed highly synergistic effects in combination with FLU or ITRA with a fractional inhibitory concentration index of ⪕ 0.08. BDSF was not cytotoxic to normal human foreskin fibroblast cells at concentrations of up to 300 μg/mL. The qRT-PCR results showed that the combination of BDSF and FLU/ITRA significantly inhibits the expression of the efflux pump genes CDR1 and MDR1 via suppression of the transcription factors TAC1 and MRR1, respectively, when compared with FLU or ITRA alone. No dramatic difference in the mRNA expression levels of ERG1, ERG11, and UPC2 was found, which indicates that the drug combinations do not significantly interfere with UPC2-mediated ergosterol levels. In vivo experiments revealed that combination therapy can be an effective therapeutic approach to treat candidiasis.@*CONCLUSION@#The synergistic effects of BDSF and azoles may be useful as an alternative approach to control azole-resistant Candida infections.

Improvement in affinity and thermostability of a fully human antibody against interleukin-17A by yeast-display technology and CDR grafting

Monoclonal antibodies (mAbs) are widely used in many fields due to their high specificity and ability to recognize a broad range of antigens. IL-17A can induce a rapid inflammatory response both alone and synergistically with other proinflammatory cytokines. Accumulating evidence suggests that therapeutic intervention of IL-17A signaling offers an attractive treatment option for autoimmune diseases and cancer. Here, we present a combinatorial approach for optimizing the affinity and thermostability of a novel anti-hIL-17A antibody. From a large naïve phage-displayed library, we isolated the anti-IL-17A mAb 7H9 that can neutralize the effects of recombinant human IL-17A. However, the modest neutralization potency and poor thermostability limit its therapeutic applications. affinity optimization was then used to generate 8D3 by using yeast-displayed random mutagenesis libraries. This resulted in four key amino acid changes and provided an approximately 15-fold potency increase in a cell-based neutralization assay. Complementarity-determining regions (CDRs) of 8D3 were further grafted onto the stable framework of the huFv 4D5 to improve thermostability. The resulting hybrid antibody 9NT/S has superior stabilization and affinities beyond its original antibody. Human fibrosarcoma cell-based assays and analyses in mice indicated that the anti-IL-17A antibody 9NT/S efficiently inhibited the secretion of IL-17A-induced proinflammatory cytokines. Therefore, this lead anti-IL-17A mAb might be used as a potential best-in-class candidate for treating IL-17A related diseases.

Advances and challenges in therapeutic monoclonal antibodies drug development

The use of serum containing polyclonal antibodies from animals immunized with toxins marked the beginning of the application of antibody-based therapy in late nineteenth century. Advances in basic research led to the development of the hybridoma technology in 1975. Eleven years later, the first therapeutic monoclonal antibody (mAb) was approved, and since then, driven by technological advances, the development of mAbs has played a prominent role in the pharmaceutical industry. In this review, we present the developments to circumvent problems of safety and efficacy arising from the murine origin of the first mAbs and generate structures more similar to human antibodies. As of October 2017, there are 61 mAbs and 11 Fc-fusion proteins in clinical use. An overview of all mAbs currently approved is provided, showing the development of sophisticated mAbs formats that were engineered based on the challenges posed by therapeutic indications, including antibody-drug conjugates (ADC) and glycoengineered mAbs. In the field of immunotherapy, the use of immunomodulators, bispecific mAbs and CAR-T cells are highlighted. As an example of promising therapy to treat infectious diseases, we discuss the generation of neutralizing monoclonal-oligoclonal antibodies obtained from human B cells. Scientific and technological advances represent mAbs successful translation to the clinic

Demethylation effect of emodin combined with 5AzA-cdR on tumor suppressor gene p16, RASSF1A and ppENK in subcutaneous xenografts of pancreatic cancer nude mice model / 中华胰腺病杂志

Objective To investigate whether emodin combined with 5AzA-cdR can enhance the demethylation of tumor suppressor genes p16,RASSF1A and ppENK in nude mice with subcutaneously transplanted pancreatic cancer.Methods Pancreatic cancer cells Panc1 burdened subcutaneous xenograft nude mice model was established,which were randomly divided into control group,emodin group,5AzA-cdR group and emodin combined 5AzA-cdR group (combined group).The growth of transplanted tumors wasobserved in each group.Methylation specific PCR (MSP) was used to detect the methylation levels of p16,RASSF1A and ppENK in the xenograft tumor tissue among three groups.The mRNA and protein expression of three tumor suppressor genes were detected by FQ-PCR and Western blotting,respectively.Results The weight of xenografts in the control group,emodin group,5AzA-cdR group,and combination group were (0.28 ±0.01),(0.17 ± 0.01),(0.12 ± 0.02),(0.08 ± 0.01)g,respectively.The tumor volume was (517 ±0.02),(382 ± 0.01),(232 ± 0.03),(169 ± 0.01) mm3.The methylation levels of p16 were 1.00 ± 0.00,0.89 ± 0.02,0.63 ± 0.02,and 0.19 ± 0.01;the methylation levels of RASSF1A were 1.00 ± 0.00,0.88 ± 0.02,0.51 ± 0.01,and 0.32 ± 0.01;the methylation degree of ppENK was 1.00 ± 0.00,0.92 ± 0.02,0.77 ± 0.02 and 0.31 ± 0.01,respectively.The expression of p16 mRNA was 1.00 ± 0.00,1.71 ±0.02,2.67 ± 0.02,3.81 ± 0.01.The expression of RASSF1A mRNA was 1.00 ± 0.00,1.92 ±0.02,2.73 ± 0.03,3.77 ± 0.01.The expression of ppENK mRNA was 1.00 ± 0.00,1.69 ± 0.03,2.17 ± 0.02 and 4.28 ± 0.01.The expression of p16 protein was 1.00 ± 0.00,1.71 ± 0.02,2.67 ± 0.02,3.81 ± 0.01;the expression of RASSF1A protein was 1.00 ± 0.00,1.92 ± 0.02,2.73 ± 0.03.3.77 ± 0.01;ppENK protein expression levels were 1.00 ±0.00,1.69 ±0.03,2.17 ±0.02,4.28 ±0.01.The weight and volume of xenografts in the three treatment groups were significantly smaller than those in the control group.The methylation of three tumor suppressor genes was lower than that of the control group,and the expression of tumor suppressor mRNA and protein was all significantly higher than the control group,which the combination drug group was also significantly stronger than that in emodin group and 5AzA-cdR group,and the differences were statistically significant (P ＜ 0.05 or ＜ 0.01).Conclusions The combination of emodin and 5AzA-cdR can enhance the demethylation effect of 5A6A-cdR on the tumor suppressor genes p16,RASSF1A and ppENK in the tumor tissue of pancreatic cancer xenograft model.

Preparation of anti-hCG single domain antibody by antibody grafting technique using an antigen-binding peptide / 生物工程学报

We used the antibody grafting technology to prepare anti-hCG single-domain antibodies on the basis of antigen-binding peptide to simplify the single-domain antibody preparation process and improving the biochemical stability of peptide. By using a universal single-domain antibody backbone (cAbBCII10), CDR1 or CDR3 was replaced by the hCG-binding peptide, and the grafted antibody gene sequences were synthesized and cloned into the prokaryotic expression vector pET30a(+) in fusion with a C-terminal sfGFP gene, i.e. pET30a-(His6)-cAbBCII10-CDR1/hCGBP1-sfGFP and pET30a-(His6)-cAbBCII10-CDR3/hCGBP3-sfGFP. The recombinant plasmids were transformed into E. coli BL21(DE3), and the fusion proteins were induced by IPTG. Highly soluble recombinant fusion proteins were obtained and purified by Ni-NTA affinity column. SDS-PAGE confirmed the purified protein as the target protein. The antigen-antibody binding assay showed that both the CDR1 and CDR3 grafted antibodies have hCG-binding activities. While the titers of the two grafted antibodies were similar, the binding affinity of CDR3 grafted antibody was higher than that of CDR1 grafted protein (about 2-3 times). The grafted antibodies retained the relatively high biochemical stability of the single-domain antibody backbone and were relatively thermostable and alkaline tolerant. The obtained antibodies also had a relatively high antigen-binding specificity to hCG. This study provided a reliable experimental basis for further optimization of anti-hCG single domain antibody by antibody grafting technology using antigen-binding peptide.

Effect of CDR1/CDR2 or MDR1 genes overexpression on oxidative stress in Candida albicans / 中国人兽共患病学报

To investigate the effect of CDR1/CDR2 or MDR1 genes overexpression on oxidative stress in Candida albicans,we evaluated the effect of H2O2 on cell viability in C.albicans overexpressing genes CDR1 /CDR2 or MDR1 and their parent strains.After establishing an oxidative stress model with H2O2,we detected reactive oxygen species (ROS),mitochondrial membrane potential (Δψm) and the expression of oxidative stress response-related genes (CAP1 and GRP2) and ROS clearance related-genes (SOD2 and SOD5).The results showed that C.albicans growth were inhibited by 100％ after the treatment of 5 mmol/L H2O2.HeO2 caused more ROS accumulation and Δψm reduction in parent strains than in CDR1/CDR2 or MDR1 genes overexpressed strains (P＜0.05).Compared to parent strains,the up-regulated expression of CAP1 and GRP2 were relatively less in CDR1/CDR2 or MDR1 genes overexpressed strains,moreover,the down-regulated expression of SOD2 and SOD5 were also relatively less in CDR1/CDR2 or MDR1 genes overexpressed strains (P＜0.05).In conclusion,the overexpression of CDR1/CDR2 and MDR1 genes could reduce the oxidative stress response and enhance the adaptability of C.albicans to oxidative stress.

Bioinformatic analysis of antibody repertoire development in response to influenza vaccination / 中华微生物学和免疫学杂志

Objective To analyze the immunogenomic characteristics of antibody repertoire in re-sponse to influenza vaccine in order to provide a theoretical basis for further development of antibody. Meth-ods Based on a time-series immunoglobulin heavy chain ( IGH) repertoire sequencing dataset, we analyzed the immunogenomic characteristics of antibody repertoire in response to trivalent influenza vaccine ( TIV ) from three aspects which included the features in complementarity-determining region 3 ( CDR3 ) , antibody mutation and VDJ usage. Results The frequency of antibody mutation increased significantly upon vaccina-tion. Analysis of the CDR3 region indicated that polar and aromatic amino acids had a higher preference. The length of CDR3 region in naive B cells followed a normal distribution, while specific CDR3 sequences with 15 to 18 amino acids in length occupied a dominant position after vaccination. In addition, the VDJ us-age altered obviously and IGHV3-7-derived antibody had a significant response to the vaccine. Response in-tensity reached the peak on day 7 and gradually weakened over time. Conclusion Antibody repertoire evolves dynamically to express specific antibody upon vaccination and the characteristics of immune responses at sequence level could be used to evaluate their effectiveness.

Construction of Hospital Knowledge Service System Based on Clinical Data Center / 医学信息学杂志

Based on the new trend of transition from information service to knowledge service currently emerging in the field of information service,the paper presents the construction of a knowledge service system based on clinical data center.It introduces the architecture of the system,analyzes its features,describes its application effect and discusses the issues to be concerned for the further development of the knowledge service system in the future.

Preliminary Study on the Effect of 5-Aza-CdR on the Demethylation of RNF180 in Prostate Cancer Cell Line DU145 / 中国医科大学学报

Objective To investigate the mechanism and cause of the inactivation of tumor suppressor gene RNF180 in prostate cancer cell line by observing the effect of 5-Aza-CdR on the RNF180 gene in prostate cancer cell line DU145. Methods MTT method was adopted to study the effect of 5-Aza-CdR(0,1,2,5,10,15 and 20μmoI/L)on the proliferation of prostate cancer cells. Western blotting,real-time PCR,and methyla-tion specific PCR(MSP)were separately used to detect the expression of RNF180 in prostate cancer cells before and after the treatment of the most suitable drug concentration(5μmoI/L). Results In a certain range,the effect of 5-Aza-CdR on the proliferation of prostate cancer cell line DU145 was increased with the increase of drug concentration and the time of drug treatment(P<0.05). After the treatment of the most suitable drug concentration,the protein and mRNA expression of RNF180 in prostate cancer cells was significantly increased(P<0.05),but the methyla-tion of the promoter region was obviously decreased. Conclusion 5-Aza-CdR can reverse the methylation status of RNF180 gene in DU145 pros-tate cancer cell line,and relieve the silencing status of RNF180gene expression.

Characteristics of IgH-CDR3 repertoire of peripheral B cells in a patient with primary biliary cholangitis: a preliminary study using high-throughput sequencing / 中华肝脏病杂志

Objective@#To analyze the characteristics of immunoglobulin heavy chain complementarity-determining region (IgH-CDR3) repertoire of peripheral B cells in a patient with primary biliary cholangitis (PBC) and to investigate the diversity of the immune system.@*Methods@#Arm-PCR was used to amplify the IgH-CDR3 region of circulating B cells isolated from a PBC patient, and high-throughput sequencing was used to analyze the amplified product. The characteristics of immune repertoire were analyzed by bioinformatics.@*Results@#In total, 329219 sequence reads were generated from the sample, with 325540 total CDR3 sequences and 72774 distinct CDR3 sequences, and the D50 of IGH-CDR3 was 7.7. The dominant CDR3 length of the sample was 45 nt (9.6%); the N addition with the highest frequency ranged from 13 to 14 nt (5.25%); the J trimming with the highest frequency was 0 nt (12.7%); the three most frequent V alleles were V4-59 (9.5%), V3-23 (8.1%), and V1-69 (6.4%).@*Conclusion@#The diversity of IgH-CDR3 repertoire is relatively low in this patient with PBC, with several B-cell clonal expansions. The specificity needs to be further verified after increasing the sample size.

Research on the tiered homogeneity medical work platform based on medical image and cloud computing / 中国医学装备

Objective:In the medical cooperation, in order to realize the homogeneity of the medical, teaching, research, management, avoid to the waste of resources and the low benefit of patients with traditional cooperative mode. So combining the Internet and cloud computing technology to realize the reconstruction of the traditional mode of cooperation.Methods: Because of the need for equivalent cooperation, Beijing Friendship Hospital set up The Tiered Medical Work Platform Based on Medical image and Cloud Computing which between Beijing Friendship Hospital and Beijing Pinggu Hospital. The Work Platform was able to provides applications such as remote reading, remote diagnosis, remote consultation, remote difficult case discussion, remote teaching, training and so on.Results: The Work Platform solved the problem of wasting of human resource cost in our hospital, and improved the allocation of medical resources, improved the level of medical service of Beijing Pinggu Hospital, made people get benefits.Conclusion: Telemedicine technology can realize the reconstruction of traditional medical cooperation mode, establish standardized information sharing platform, increase the efficiency of medical resources allocation, improve the ability of primary diagnosis and treatment services, provide assist in forming the pattern of Tiered Medical.

Study of DNA demethylation acting on DNA methyltransferase gene and microRNA in B-cell acute lymphocytic leukemia cell line / 白血病·淋巴瘤

Objective To explore the function of 5-Aza-CdR in B-cell acute lymphocytic leukemia cell line NALM-6 and its influence on the expression of microRNA (miRNA) in the cells. Methods NALM-6 was treated with different concentrations of 5-Aza-CdR. Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) test, and DNA methyltransferase (DNMT) mRNA expression level was detected by reverse transcription PCR (RT-PCR). The expression changes of miRNA were detected by miScript miRNA PCR Array chip in cells after methylation. Results NALM-6 cell growth was inhibited by different concentrations of 5-Aza-CdR processing time, reaching to the maximum inhibitory rate was (74.163 ±0.381) %. 5-Aza-CdR affected concentrations was inversely proportional with expression level of DNMT mRNA. After 1 000 μmol/L of 5-Aza-CdR was dealed with cell 72 h, the relative expression of DNMT-1 was reduced to 0.453 ±0.021, DNMT-3L was 0.003±0.001, DNMT-3B was 0.395±0.019. MiScript miRNA PCR array sieved out 3 miRNA (miR-184, miR-23a-3p, miR-34a-5p) associated with DNA methylation. Conclusions 5-Aza-CdR down regulates the expression of DNMT gene in NALM-6 cells, and inhibits the proliferation of cells. MiR-184, miR-23a-3p and miR-34a-5p are related to DNA methylation in the occurrence and development of B-cell acute lymphocytic leukemia.

Cellular senescence is regulated by p21Waf1/Cip1 methylation / 重庆医学

Objective To explore the effect of p21Waf1/Cip1 methylation changes on the process of cellular senescence .Methods Bisulfite sequencing was used to analyze the methylation changes of p21Waf1/Cip1 in the process of cellular senescence;p21Waf1/Cip1 ex‐pression was detected by RT‐PCR and Western‐blot ;Middle‐aged 2BS cells was treated by 5‐aza‐CdR and cellular senescence was detected by MTT and SA‐β‐Gal staining .Results Bisulfite sequencing analysis of p21Waf1/Cip1 promoter showed that CpGs were methylated by 1 .25% in the young 2BS cells ,by 27 .27% in the middle‐aged 2BS cells ,while only by 0 .64% in the senescent cells . The expression of p21Waf1/Cip1 was low in the young(28 PD) 2BS cells ,it increased first(35 PD) but decreased later in the middle‐aged(42 PD) cells .In the senescent 2BS cells ,p21Waf1/Cip1 expression was further increased .5‐aza‐CdR treatment resulted in de‐creased growth rate but increasedβ‐Gal staining of middle‐aged 2BS cells .Conclusion The process of cellular senescence is regula‐ted by the status of p21Waf1/Cip1 methylation ,and p21Waf1/Cip1 demethylation accelerates cellular senescence .

Analysis of BCR CDR3 repertoire of peripheral blood with HBsAb titer higher than 10 000 mU/ml / 中国免疫学杂志

Objective:To acquire potential HBsAb sequences,we have analyzed the BCR CDR3 repertoire of the peripheral blood with HBsAb titer higher than 10 000 mU/ml,which could provide a data basis for follow-up study.Methods:Genomic DNA of pe-ripheral blood mononuclear cells was extracted from samples with HBsAb titer higher than 10 000 mU/ml.We have adopted Illumina Solexa high-throughput sequencing technology of the Adaptive Biotechnologies ImmunoSEQ platform to acquire sequence data.IMGT/High V-QUEST was used to preliminary analyze our sequence data,including usage of IGHV,IGHJ and IGHD gene subgroups,IGHV-J matching,distribution of CDR3 amino acid (AA) length and usage of total CDR3 AA.And these sequences were compared with the HBsAb sequences from NCBI database.Results:Experimental samples have highly selected gene subgroups IGHV3,IGHV4,IGHJ4, IGHJ6,IGHD3,IGHD6,and IGHV3-J4 pairing,IGHV3-J6 pairing.The AA length distributions of CDR3 region were normal distribution with the length of 14/15 AA as the midline.In the regard to amino acid usage in CDR3 region, each sample prior used Alanine, Tyrosine,Glycine,Alanine,Aspartic acid and Serine.The amino acid usages of 107,108,109,113,114 positions were diversified but 105,106,115,116,117 positions taking conservative amino acids usages.We have found 48 unique sequences that have same IGHV, IGHJ and CDR3 AA length with the HBsAb sequences from NCBI database.Conclusion:There were almost the same characteristics of BCR CDR3 repertoire of the peripheral blood with HBsAb titer higher than 10 000 mU/ml.The 48 unique sequences provided a solid data basis for the follow-up study.