Objective:Through differential miRNA expression profiles and bioinformatics in the peripheral blood of patients with Keshan disease (KD) and healthy control, to explore the possible pathogenesis of KD.Methods:Ten patients with chronic KD (KD group) were selected in the severe disease area of KD in Wulian County, and 10 healthy subjects (control group) were selected in non-KD area of Dongchangfu District, Shandong Province. Blood sample of elbow vein was collected and plasma was separated. RNA-seq technology was used to construct the differential expression profiles of miRNA in KD and control groups. Target mRNAs were screened using Starbase, miRTarBase, miRDB and TargetScan. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted to investigate the possible pathogenesis of KD.Results:Compared the control group and KD group, 132 differentially expressed miRNAs were screened out, including 90 upregulated and 42 downregulated miRNAs. Through Starbase, miRTarBase, miRDB and TargetScan, 53 miRNAs were obtained, 737 targeted mRNAs were obtained. GO analysis showed that the differential genes were mainly involved in the biological processes of Ras protein signal transduction, transmembrane transport, cell cycle regulation, cell adhesion, etc. KEGG pathway analysis showed that the differential genes were mainly involved in viral infection, endocytosis, adhesion spot and actin regulation.Conclusion:In this study, RNA-seq technology is used to obtain differential miRNA expression profiles of KD patients and healthy control, and target pathogenic genes and signaling pathways that may be related to KD are screened out.

Objective By constructing the differential expression profile of lncRNA/mRNA in peripheral blood plasma of patients with Keshan disease (KSD) and dilated cardiomyopathy (DCM),to explore the commonality and characteristics of the two diseases in molecular mechanism.Methods Ten patients with chronic KSD were selected in the severe disease area of KSD in Shandong Province,and 10 cases of DCM and 10 healthy subjects (control group) were selected in non-KSD area.Blood of elbow vein was collected and plasma was separated.RNA-seq technology was used to construct the differential lncRNA/mRNA expression profile between KSD and control group,DCM and control group,and co-expression and specific expression of partial genes in KSD and DCM were analyzed through Wien analysis.The lncRNA-mRNA co-expression network maps of specific part of KSD,specific part of DCM and common part of the two diseases were constructed,and Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were applied to distinguish the biological function of the two diseases.Results Compared with control group,102 dysregulated mRNAs and 22 dysregulated lncRNAs showed the same trend in KSD and DCM.And 3 606 mRNAs and 451 lncRNAs were only differentially expressed in KSD group,217 mRNAs and 137 lncRNAs were only differentially expressed in DCM group.The differentially expressed lncRNA/mRNA shared between the KSD and DCM groups were mainly about viral transcription,immuno-inflammatory response,oxidative stress signaling pathways.The KSD specific lncRNA/mRNA mainly participated in cell membrane damage and viral myocarditis.The DCM specific lncRNA/mRNA mainly regulated mitochondrial structure and oxidative phosphorylation related enzymes.Conclusion The differentially expressed lncRNA/mRNA shared in KSD and DCM groups are mainly involved in viral transcription,oxidative stress signaling pathways;KSD specific lncRNA/mRNA are mainly related to cell membrane damage and viral myocarditis;DCM specific lncRNA/mRNA mainly regulate mitochondrial structure.

Objective: To explore the influence and mechanism of time effect of the controlled micromovement on fracture healing. Methods: Forty-eight rabbit models of femoral fracture were prepared and fixed with unilateral two-bar external fixator. They were randomly divided into four groups: continuing immobilization group, instant micromovement group, 1-week micromovement group and 2-week micromovement group. Postoperative radiographs were taken at 1, 2, 3 and 5 weeks to observe callus growth. The maximum load, deflection and rigidity of callus at fracture end were measured 5 weeks after operation. At 1, 2 and 3 weeks after operation, the histological morphology of callus was observed, and the expression and distribution of osteocalcin (oc) in callus were detected. Results: At 5 weeks after operation, the X-ray scores of fracture line in 1-week micromovement group and 2-week micromovement group were 10.384±0.744 mm, 10.412±0.482 mm, significantly higher than those in continuing immobilization group (7.518±0.536). The anteroposterior diameter and the exterior and interior diameter of the external callus in 1-week micromovement group and 2-week micromovement group were 14.3±3.2 mm, 14.0±2.8 mm and 14.6±2.1 mm, 15.2±3.1 mm, which were smaller than those in the continuing immobilization group 15.3±2.3 mm and 16.7±1.9 mm, but there was no significant difference. The bone mineral density value and proportion rate in the fracture site were 0.446±0.020 g/cm², 0.416±0.021 g/cm² and 1.171%±0.056%, 1.143%±0.040% in 1-week micromovement group and 2-week micromovement group, which were significantly higher than those in continuing immobilization group which were 0.376±0.022 g/cm² and 0.912%±0.051%. The maximum load of callus in 1-week micromovement group and 2-week micromovement group was 415.6±27.2 N, 400.3±28.5 N, which was significantly higher than that in continuing immobilization group 329.2±18.4 N and instant micromovement group 272.8±22.7 N. There was no difference of the deflection of callus between groups. The rigidity of callus in 1-week micromovement group was 590.4±24.2 N/mm, which was significantly higher than that in other groups; the rigidity of callus in the 2-week micromovement group was 540.6±22.8 N/mm, which was significantly higher than those in the instant micromovement group and the continuing immobilization group (152.4±21.7 N/mm, 174.8±20.6 N/mm). Conclusion Micromovement begins from one or two weeks can significantly raise external callus formation and vagueness level of fracture line, accelerating bridging callus formation, and can significantly raise bone mineral density and rigidity of callus. It also accelerates the maturity, hypertrophy and mineralization of chondrocyte, resulting in the stimulation of the fracture healing through endochondral ossification; it seemingly can improve the amount and density of osteoclasts in callus to stimulate the maturity and mineralization of chondrocyte. The strengthening coupling of osteoblasts and osteoclasts can promote the transformation from soft callus to hard callus and the remolding of hard callus.

The problem of Keshan disease (KD) is confused with dilated cardiomyopathy (DCM) is still not solved.KD and DCM have different gene expression profiles,namely,different Micro RNAs (miRNAs) and Long noncoding RNAs (lncRNAs).There may be characteristic miRNAs and lncRNAs in KD and DCM.Systemically study differential gene expression profiles and regulation of noncoding RNAs gene expression and elucidate the different molecular pathogenesis in gene expression and gene expression regulation will provide theoretic basis in identification,prevention and treatment of KD and DCM.

Objective To observe the electrocardiogram (ECG) of urban residents in non-endemic areas of Keshan disease in Ji'nan City,Shandong Province,and to provide scientific basis for health evaluation and prevention and control of Keshan disease.Methods From July 2014 to June 2015,ECGs of residents undertaken healthy examination in Shandong Ciming Physical Examination Center were collected,retrospective analysis was used to analyze residents' heart health.Statistical analysis was conducted according to sex and age groups,and the diagnosis of ECG was based on Concise Pediatric Electrocardiogram and Clinical Electrocardiography.Results Totally 31 753 ECGs were recorded,including 18 664 males accounting for 58.8％ and 13 089 females accounting for 41.2％,who were aged 6-78 years old,including 304 people 6-20 years old,18 861 people 21-40 years old,10 451 people 41-60 years old,and 2 137 people 61-78 years old.Totally 27 699 normal ECG (87.2％) and 4 054 abnormal ECG (12.8％) were detected,there were 2 212 males and 1 842 females in the abnormal ECG,the detection rates were 11.9％ (2 212/18 664) and 14.1％ (1 842/13 089) for males and females,respectively.The detection rate of female was significantly higher than that of male (x2 =34.082,P ＜ 0.01).The detection rate of ST-T change in abnormal ECG was the highest,which was 316.5/10 000 (1 005/31 753),men and women were 273.3/10 000 (510/18 664) and 378.2/10 000 (495/13 089),respectively.The detection rate of arrhythmia was 4.8％ (1 523/31 753),including 1 023 males and 500 females,and their detection rate of arrhythmia was 548.1/10 000 (1 023/18 664) and 382.0/10 000 (500/13 089),respectively.In arrhythmia,degree Ⅰ atrioventricular block (155.3/10 000,493/31 753),complete rightbundle branch block (81.6/10 000,259/31 753),ventricular premature beats (79.0/10 000,251/31 753),and atrial premature beats (48.2/10 000,153/31 753) had higher detection rates.Conclusion The detection rate of women of urban residents in Ji'nan is higher than that of men,the most common abnormal ECG is ST-T change,and the detection rate of arrhythmia is low.

Objective To investigate the differences in gene expression profiles of peripheral blood from patients with Keshan disease (KD) and the apoptosis mechanism in KD,to obtain diagnostic markers and establish diagnostic centroids plot for KD.Methods RNA was isolated from ten patients with KD diagnosed according to the clinical criteria for KD in China and ten health controls.The expression profiles were evaluated by Agilent 4 ×44K Whole Human Genome density oligonucleotide microarray analysis.The data were extracted by Agilent Feature Extraction Software t test,Pathway studio analysis and prediction analysis for microarray (PAM) were used to identify differently expressed genes,gene pathways,diagnostic markers and establish diagnostic centroids plot.Results Totally 1 570 up-regulated genes and 1 498 down-regulated genes were identified.Thirty-eight enrichment pathways were also identified,and the highest ranked by Pathway studio analysis was related to apoptosis.Six genes involved in apoptosis pathway were up-regulated in KD included ataxia telangiectasia mutated (ATM),cAMP-dependent protein kinase,protein kinase A (PKA),baculoviral IAP repeat-containing 2 (BIRC2),NLR family,apoptosis inhibitory protein (NAIP),BCL2-1ike 11 (Bim),BCL2-related protein A1 (BCL2A1) and down-regulated were 7 which included caspase 8 (CASP8),BCL2 binding component 3 (BBC3),BCL2--associated athanogene (BAG1),BCL2-associated X protein (BAX),BCL2-1ike 1 (BCL2L1),BCL2-related ovarian killer (BOK),and caspase 6 (CASP6).Forty-two diagnostic markers were obtained through PAM analysis.Conclusions Apoptosis related to genes and pathways might play an important role in the pathogenesis of KD.Forty-two markers could be used as molecular markers for the diagnosis of KD,which is important to the diagnosis of KD.

Objective To do screening acute lymphoblastic leukemia patients scFv antibody single chain variable region to cre-ate conditions for the expression and obtain further specificity of antibody fragments.Methods In this study,patients with newly diagnosed acute lymphoblastic leukemia serum as coating antigen using phage display technology,screening phage an-tibody specificity from the semi-synthetic human phage antibody libraries,the first to target the immune antigen-coated tab-let,phage library was added,so that with the target antigen-specific binding phage antibody was immobilized on plates immu-nization,could not be specifically bound phages were rinsed.The eluted specific binding phage,E.coli infection.Could get the specific antibody gene containing phagemid.Results After three “adsorption-elution-amplification”screening process,got stronger leukemia patient antigen-specific phage antibody variable region fragment and identification.Conclusion Got better strain affinity antibody fragments,to create the conditions for the next fragment expression,identification and clinical appli-cation.

Objective To investigate the clinical diagnostic value and pathogenesis of serum protein identification in Keshan disease (KD).Methods A total of 65 chronic KD patients were selected as the patient group in KD endemic areas,while 29 cases of dilated cardiomyopathy (the DCM group),62 healthy cases from KD endemic areas (control 1 group) and 28 healthy cases from non-endemic areas (control 2 group) were selected as controls.Liquid chip time of flight mass spectrometry (ClinProtTM MALDI-TOF-MS) was used to determine the expression of proteins/peptide peaks.ClinProTools 2.2 software was used to analyze the protein profiles to determine differentially expressed proteins/peptide peaks.The Genetic Algorithm (GA),QuickClassifer Algorithm (QC) and Supervised Neural Network Algorithm (SNN) methods were used to screen marker proteins.Matrix-assisted laser desorption/ionization time-of-flight Mass Spectrometry technique (MALDI-TOF/TOF) was also used as a secondary mass spectrometry to identify differentially expressed peptides.Results Between the KD and control 1 groups,34 differentially expressed proteins/peptides and 5 marker proteins were identified,while 52 differentially expressed proteins/peptides and 5 marker proteins were identified between the KD and control 2 groups,and there were 67 differentially expressed proteins/peptides and 5 marker proteins between the KD and DCM groups.During secondary mass spectrometry,two peptides for mass-to-charge ratio (m/z) 2 079 and 1 465 were obtained,peptide of matching β-globin showed low expression while peptide of matching fibrinogen showed high expression in the KD patients.Conclusions Serum marker proteins can be used as biomarkers for diagnosis and differentiation of KD.β-globin and fibrinogen play an important role in the development of KD myocardial injury.

Objective To provide more valuable information for diagnosis of dilated cardiomyopathy (DCM),we detected differentially expressed proteins in serum from patients with DCM and healthy people and protein biomarkers were selected.Methods During the period from march 2011 to may,a total of 29 samples of resident patients with DCM from Qilu Hospital of Shandong University,Jinan Central Hospital and Jinan First Peoples Hospital and 30 local healthy people in Jinan were selected as DCM and control groups,respectively.Serum samples from these patients with DCM and controls were detected by ClinProt MALDII-TOF-MS.ClinProTools 2.2 software was used to get mass spectrometric data.The ClinPrott discrimination model was established to screen out differentially expressed proteins as potential biomarkers.Results Via comparing proteins/polypeptides peaks of DCM patients and healthy controls,57 of all 73 peaks were found to be significantly different between the two groups.Compared with the control group,35 peaks were up-expressed while the other 22 peaks were downexpressed.Five peaks were screened out as protein biomarkers.They were mass-to-charge ratio (m/z):4 247.95,4 209.37,1 058.69,1 074.78,and 2 364.71.The sensitivity and specificity of ClinPrott discrimination model was 99.14％ and 99.16％,respectively.Conclusion Patients with DCM have expressed serum proteins differently and we have found five protein markers which might have some value for diagnosis of DCM.

BACKGROUNDKeshan disease (KD) is an endemic cardiomyopathy in China. The etiology of KD is still under debate and there is no effective approach to preventing and curing this disease. Young women of child-bearing age are the most frequent victims in rural areas. The aim of this study was to determine the differences between molecular pathogenic mechanisms in male and female KD sufferers.

METHODSWe extracted RNA from the peripheral blood mononuclear cells of KD patients (12 women and 4 men) and controls (12 women and 4 men). Then the isolated RNA was amplified, labeled and hybridized to Agilent human 4×44k whole genome microarrays. Gene expression was examined using oligonucleotide microarray analysis. A quantitative polymerase chain reaction assay was also performed to validate our microarray results.

RESULTSAmong the genes differentially expressed in female KD patients we identified: HLA-DOA, HLA-DRA, and HLA-DQA1 associated with spontaneous autoimmunity; BMP5 and BMP7, involved in cardiomyocyte differentiation defect; and ADAMTS 8, CCL23, and TNFSF15, implicated in anti-angiogenic activities. These genes are involved in the canonical pathways and networks recognized for the female KD sufferers and might be related to the pathogenic mechanism of KD.

CONCLUSIONOur results might help to explain the higher susceptibility of women to this disease.

ADAM Proteins ; genetics ; ADAMTS Proteins ; Adult ; Autoimmunity ; genetics ; physiology ; Bone Morphogenetic Protein 5 ; genetics ; Bone Morphogenetic Protein 7 ; genetics ; Cardiomyopathies ; genetics ; pathology ; Cell Differentiation ; genetics ; physiology ; Chemokines, CC ; genetics ; Enterovirus Infections ; genetics ; pathology ; Female ; Gene Expression Profiling ; HLA-D Antigens ; genetics ; HLA-DQ alpha-Chains ; genetics ; HLA-DR alpha-Chains ; genetics ; Humans ; Male ; Middle Aged ; Myocytes, Cardiac ; cytology ; metabolism ; Oligonucleotide Array Sequence Analysis ; Sex Factors ; Tumor Necrosis Factor Ligand Superfamily Member 15 ; genetics