Objective To investigate the levels of soluble tyrosine kinase-1(sFlt-1)and chemokine C-C ligand 3(CCL3)in serum of patients with coronary heart disease(CHD)and their correlation with the severity of the disease.Methods A total of 230 elderly CHD patients admitted to the De-partment of Cardiovascular Medicine of Xinxiang Central Hospital from November 2020 to No-vember 2022 were collected as the study subjects(CHD group),and according to their Gensini score,they were divided into mild(n=89),moderate(n=95),and severe(n=46)CHD sub-groups.Another 230 healthy individuals who taking physical examination during the same period served as the control group.ELISA was applied to measure serum levels of sFlt-1 and CCL3.ROC curve was plotted to analyze the diagnostic values of serum sFlt-1 and CCL3 levels for CHD.Pear-son correlation analysis was employed to analyze the relationship between serum sFlt-1 and CCL3 levels and the CHD severity.Results The serum levels of sFlt-1 and CCL3 were obviously higher in the CHD group than the control group(121.71±29.80 ng/L vs 98.70±17.57 ng/L,18.22± 5.41 ng/L vs 13.68±3.89 ng/L,P＜0.01).ROC curve analysis showed that the AUC value of the two indicators combined together was significantly greater than that of them alone in diagnosis of CHD(0.886 vs 0.791,0.775,P＜0.01).The serum levels of sFlt-1 and CCL3 were increased along with the severity of the disease and Gensini score when the levels and the score were compared among the mild,moderate and severe subgroups(P＜0.05).Pearson correlation analysis indicated that the serum levels of sFlt-1 and CCL3 were positively correlated with the Gensini score(r=0.420,r=0.479,P＜0.01).Conclusion The levels of serum sFlt-1 and CCL3 are obviously ele-vated in CHD patients,and closely associated with the severity of coronary lesions.

Objective:To investigate the expression profile of IL-36 family members in patients with coronary atherosclerotic heart disease (CAHD) and to assess the regulatory effects of exogenous IL-36 on CD8 + T cell function in CAHD patients. Methods:Twenty controls and 82 CAHD patients including 31 with stable angina pectoris (SAP), 27 with unstable angina pectoris (UAP) and 24 with acute myocardial infarction (AMI) were enrolled in this study. Anti-coagulant peripheral blood samples were collected. Plasma and peripheral blood mononuclear cells were isolated. The levels of IL-36α, IL-36β, IL-36γ and IL-36 receptor antagonist (IL-36RA) in plasma were measured by ELISA. CD8 + T cells were enriched. The expression of IL-36 receptor subunits at mRNA level was semi-quantified by real time PCR. Flow cytometry was used to detect the expression of programmed death-1 (PD-1), cytotoxic T lymphocytes associated protein-4(CTLA-4) and lymphocyte-activation gene-3 (LAG-3) in CD8 + T cells. Levels of periforin, granzyme B, granulysin, IFN-γ and TNF-α in the culture supernatants of CD8 + T cells were measured by ELISA. Purified CD8 + T cells from controls and AMI patients were stimulated with recombinant human IL-36RA. Changes in the expression of immune checkpoint molecules and the secretion of cytotoxic molecules and cytokines after IL-36RA stimulation were analyzed. One-way analysis of variance or paired t-test was used for statistical analysis. Results:There were no significant differences in plasma IL-36α, IL-36β or IL-36γ level between the control, SAP, UAP and AMI groups ( P>0.05). Plasma IL-36RA level was significantly down-regulated in the AMI group as compared with that in the control, SAP and UAP groups[(1 159.57±297.83) pg/ml vs (1 773.47±754.29) pg/ml, (1 600.12±740.48) pg/ml and (1 578.72±720.42) pg/ml; P<0.05]. The expression of IL-1 receptor 6 (IL-1R6) and IL-1 receptor accessory protein (IL-1RAcP) at mRNA level, the expression of PD-1 and CTLA-4, and the secretion of IFN-γ and TNF-α by CD8 + T cells showed no significant differences between the four groups ( P>0.05). Periforin, granzyme B and granulysin levels secreted by CD8 + T cells of the AMI group were significantly higherthan those of the control, SAP and UAP groups ( P<0.05). In the control group, recombinant human IL-36RA stimulation did not affect the expression of immune checkpoint molecule or the secretion of cytotoxic molecules and cytokines by CD8 + T cells ( P>0.05). In the AMI group, the percentage of PD-1 + CD8 + T cells increased after recombinant human IL-36RA stimulation ( P=0.033), but no significant change in the percentage of CTLA-4 + CD8 + T cells was observed ( P=0.288). Moreover, recombinant human IL-36RA stimulation suppressed the CD8 + T cells of AMI patients to secrete periforin, granzyme B and granulysin ( P<0.05), but not affect the secretion of IFN-γ and TNF-α ( P>0.05). Conclusions:The reduced IL-36RA level in AMI patients might induce the enhancement of CD8 + T cell activity by promoting CD8 + T cells to secrete cytotoxic molecules, which was involved in the immunopathogenesis of AMI.

OBJECTIVES: The study aimed at identifying salivary microbiota in caries-free Chinese preschool children using high-throughput sequencing. METHODS: Saliva samples were obtained from 35 caries-free preschool children (18 boys and 17 girls) with primary dentition, and 16S ribosomal DNA (rDNA) V3-V4 hypervariable regions of the microorganisms were analyzed using Illumina MiSeq. RESULTS: At 97% similarity level, all of these reads were clustered into 334 operational taxonomic units (OTUs). Among these, five phyla (Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Candidate division TM7) and 13 genera ( CONCLUSIONS Our results revealed the diversity and composition of salivary microbiota in caries-free preschool children, with little difference between male and female subjects. Identity of the core microbiome, coupled with prediction of gene function, deepens our understanding of oral microbiota in caries-free populations and provides basic information for associating salivary microecology and oral health.

Objective To investigate the mutations of epigenetic regulation factor ASXL1 gene in myelodysplastic syndrome(MDS).Methods Mutation analysis of ASXL1 gene in 53 de novo MDS patients and 20 healthy persons was performed by using polymerase chain reaction(PCR)followed by sequence analysis at DNA level.The clinical and laboratory characteristics were compared in MDS patients with ASXL1 gene mutation and ASXL1 wild type.ASXL1 mutation in mRNA level was detected by using reverse transcription PCR(RT-PCR)followed by sequence analysis.Results ASXL1 gene mutations were observed in 9 cases(16.9%)of 53 MDS patients.6 mutation types were detected,including 4 frameshift mutations types(2 cases with p.Glu635ArgfsX15,3 cases with p.Gly646TrpfsX12,1 case with p.Ala640GlyfsX14 and 1 case with p.Gly790TrpfsX10)and 2 nonsense mutation types(1 case with p.Gln1063X and 1 case with p.Gln695X).All the mutations were heterozygous,and p.Gly790TrpfsX10 and p.Gln695X were new mutation types.In addition,a single nucletide polymorphism(SNP)p.Gly652Ser was also detected in 4 cases with MDS.5 cases of p.G652S SNP and 1 case of p.Leu1173Leu SNP were detected in 20 healthy people.Frameshift mutation(p.Gly646TrpfsX12)could be detected at mRNA level by using RT-PCR.Differences were not observed in red blood cell counts,white blood cell counts,platelet counts,hemoglobin levels,reticulocyte,neutrophil granulocyte,the peripheral blood lymphocytes percentage,T-cell subsets in the peripheral blood,the proportion of primitive cell in the marrow and MDS types between the patients with ASXL1 gene mutation and ASXL1 wild type patients(P >0.05).Conclusion There is a high frequency of ASXL1 gene mutation in MDS patients,which can be detected at mRNA level.

Objective: To investigate the effect of biology and mTOR pathway activity of down-regulated TSC2 gene expression on U937 leukemia cells. Methods: Gene expression was down-regulated by lentivirus induced RNA interference on TSC2 high expressed U937 cell line; the proliferation, apoptosis and differentiation were detected by CCK-8 assay, colony formation assay and flow cytometry; the gene expression level and protein kinase activity were detected by qRT-PCR and Western blot. Results: Down-regulated expression of TSC2 gene promoted U937 cell proliferation and colony formation ability (P<0.05) . The proportion in G₀/G₁ phase of TSC2 down-regulated U937 cell was much lower than that of the control cells [ (52.53±3.75) % vs (75.10±4.33) %, t=6.829, P=0.002], the S phase [ (22.43±1.00) % vs (15.47±1.20) %, t=-5.581, P=0.019] and G₂/M phase [ (25.03±4.34) % vs (14.33±0.91) %, t=-5.413, P=0.013] was remarkably higher than that of the control cells (P<0.05) . There were no statistically significant differences in cell apoptosis and differentiation (P>0.05) . Down-regulation of TSC2 led to the increased activity of mTOR, 4EBP1 and S6K1, but did not influence the activity of AKT. The expressions of proliferation related cyclinD1, c-myc and PTEN were also up-regulated after TSC2 silenced, but the expressions of P27KIP and BCL-XL were not changed. Conclusion Downregulation of TSC2 could promote the proliferation of U937 cells through up-regulation of mTOR activity.

Objective To explore the value of the plasma miR-193a-5p level on diagnosis and monitoring the response to treatment in acute myeloid leukemia (AML). Methods Peripheral blood samples were obtained from AML patients enrolled in hematology department of the Second Hospital of Shanxi Medical University from July 2015 to December 2015, including 30 de novo AML patients, 9 patients in complete remission (CR) and 6 patients in relapse. Peripheral blood samples from 15 healthy people were randomly choosed as the health control group. Plasma miR-193a-5p expression levels were detected by using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Results The plasma miR-193a-5p relative expression level of AML patients group [0.465 6 (0.103 1-5.000 2)] was obviously lower than that of health control group [0.766 1 (0.052 1-3.134 4)] (U= 122, P= 0.018 7). The plasma miR-193a-5p relative expression levels of de novo group and relapse AML group were significantly lower than those of CR group and health control group (P<0.05), and there was no significant difference between the CR group and health control group (U= 56, P= 0.511 9). No significant correlation was found between the plasma miR-193a-5p level and age, gender, blast percentage of the bone marrow, peripheral blood leukocyte count, platelet count, CD34+cells'percentage and so on. Conclusion The decreased plasma miR-193a-5p expression level can be served as a new and noninvasive biomarker for the evaluation of diagnosis and treatment in AML.

OBJECTIVETo identify the MPL L391-V392ins12 spliceosome and analyze its frequencies in patients with myeloproliferative neoplasms (MPN).

METHODSMPL aberrant spliceosome was identified through reverse transcription polymerase chain reaction (RT-PCR)combined with cloning sequencing. The mutation of this spliceosome in 248 MPN patients and 200 normal people was determined by allele-specific polymerase chain reaction (AS-PCR).

RESULTSA novel aberrant spliceosome of MPL gene (MPL L391-V392ins12)was identified, i.e. 36 bp intron was retained between exon7 and exon8, and there were 12 amino acids (EGLKLLPADIPV)inserted. MPL L391-V392ins12 mutation was detected in 19 (7.66%)of the 248 patients with MPN, including 1 (1.92%) of 52 patients with PV, 14 (9.66%) of 145 with ET, and 4 (7.84%) of 51 with PMF. And the mutation was not detected in the group of 200 normal people.

CONCLUSIONMPL L391-V392ins12 spliceosome is an aberrant spliceosome present in the MPN. It can be detected in PV, ET and PMF, and more frequently in ET and PMF. This mutation may play an important role in the process of MPN.

Humans ; Mutation ; Myeloproliferative Disorders ; genetics ; Neoplasms ; genetics ; Polymerase Chain Reaction ; Receptors, Thrombopoietin ; genetics ; Spliceosomes

Objective To explore the clinical significance of dyslipidemia in patients with systemic lupus erythematosus (SLE).Methods By independent-samples t test,serum lipid level was compared between 326 SLE patients and 300 healthy controls.The total cholesterol (TC),triglyceride (TG),lowdensity lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) levels were partially compared in subgroups of SLE patients.The correlation of serum TC,TG,LDL-C and HDL-C with clinical manifestations and laboratory findings in SLE was analyzed by Pearson or Spearman correlation analysis.Results ①The serum levels of TC [(3.8±1.5) mmol/L],TG [(2.1±1.6) mmol/L] and LDL-C [(2.1±0.9) mmol/L] were significantly higher in SLE group than those of the control group [(3.4±0.6),(0.8±0.4),(1.9± 0.5) mmol/L],and the serum level of HDL-C [(1.2±0.9) mmol/L] was significantly lower in SLE group than that of the control group [(2.0±0.5) mmolFL] (t=4.953,P=0.000; t=14.569,P=0.000; t=3.204,P=0.001; t=-14.335,P=0.000].② The serum levels of TC [(4.0± 1.7) mmol/L],TG [(2.5± 1.7) mmol/L] and LDL-C [(2.2±1.0) mmol/L] were significantly higher in LN group than those of the non-LN group [(3.6±1.0),(1.6± 1.0),(1.9±0.7) mmol/L; t=2.646,P=0.009; t=6.292,P=0.000; t=3.261,P=0.001].③ The serum level of TG [(2.2±1.6) vs (1.8±1.4) mmol/L] was significantly higher in SLE patients with hypocomplementemia than that of the normal ones (t =2.098,P=0.038).The serum level of HDL-C [(1.1 ±0.4) vs (1.6± 1.7) mmol/L] was significantly lower in SLE patients with hypocomplementemia than that of the normal ones (t=-2.375,P=0.020).④ The serum level of TG [(2.3±1.7) vs (2.0±1.4) mmol/L] was significantly higher in anti-dsDNA antibody positive patients than that of negative ones (t=1.989,P=0.048).The serum level of HDL-C [(1.5± 0.4) vs (1.4±1.2) mmol/L] was significantly lower in anti-dsDNA antibody positive patients than that of negative ones (t=-2.979,P=0.003).⑤ The lipid level was correlated with the clinical manifestations and laboratory findings in SLE patients.Conclusion Dyslipidemia exists in patients with SLE and has close correlation with LN,hypocomplementemia and positive anti-dsDNA antibody.

Objective To investigate the therapeutic effect of tumor necrosis factor (TNF)-αsiRNA on typeⅡcolla-gen induced arthritis (CIA) in rats. Methods The expression vectors of siRNA against TNF-αgene were constructed suc-cessfully and were injected by tail veil into CIA rats. Twenty-four CIA rats were randomly divided into 4 groups including model group, empty vector group, TNF-α-siRNA1 group and TNF-α-siRNA2 group. CIA rats were injected with the same dose of phosphate buffered sodium (PBS) and pGFP-V-RS vector respectively in model group and empty vector group, while TNF-α-siRNA1 group and TNF-α-siRNA2 group were injected with TNF-α-siRNA1 eukaryotic expression vector and TNF-α-siRNA2 eukaryotic expression vector respectively. Another 6 rats, which were not established CIA model, were in-jected with PBS (blank control group). The serum expression levels of IL-1 were detected by ELISA on day 1, 5, 9 and 13 af-ter injection. The expression level of TNF-αmRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) on day 13. Results The expression level of IL-1 was significantly higher on day 1, 5, 9 and 13 in model group than that of blank group (P＜0.05). The expression levels of IL-1 were significantly lower on day 1, 5 and 9 in TNF-α-siRNA1 group and TNF-α-siRNA2 group than that of model group and blank group (P ＜ 0.05). The expression level of TNF-αmRNA was significantly higher on day 13 in model group than that of blank group (P＜0.05). The expression levels of TNF-αmRNA were significantly lower in TNF-α-siRNA1 group and TNF-α-siRNA2 group than those of model group and emp-ty vector group (P＜0.05). Conclusion TNF-αspecific siRNA can suppress the levels of TNF-αmRNA and IL-1, which provides experimental basis for gene therapy of rheumatoid arthritis.