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Background The main pathological feature of silicosis is pulmonary fibrosis. Multiple miRNAs regulate the development of silicosis. Objective Using a fibroblast cell line, to explore the effect of miR-18a on the expression of extracellular matrix-related genes, and verify the mechanism. Methods The fibroblast cell line NIH-3T3 cells were transfected with miR-18a mimics or neurogenic locus notch homolog protein 2 (Notch2) small interfering RNA (siRNA). The mRNA expression changes of Acta2, Col1a1, and Notch2 were detected by real-time quantitative reverse transcription PCR (qRT-PCR), α-smooth muscle actin (α-SMA) and Notch2 were also detected at the protein level by Western blotting. To verify whether miR-18a could directly act on the complementary sequences of the Notch2 gene, human embryonic kidney HEK293T cells and the psiCHECKTM-2 vector were used. Results The results of qRT-PCR showed that in NIH-3T3 cells, the over-expression of miR-18a mimics for 36 h inhibited the mRNA expression of Col1a1 and Acta2 (P<0.05). The results of Western blotting showed that the protein expression abundance of α-SMA was decreased at 48 h of miR-18a mimics over-expression. The qRT-PCR results showed that the over-expression of miR-18a for 36 h had no significant effect on Notch2 gene expression, but the Western blotting results showed that the over-expression of miR-18a mimics inhibited the expression of Notch2 at the protein level. The results of the dual luciferase reporter vector assay showed that in HEK293T cells, both over-expressed miR-18a mimics and inhibitors for 24 h demonstrated that Notch2 is a direct target gene of miR-18a. When Notch2 was inhibited for 36 h, the qRT-PCR results showed that Acta2 and Col1a1 were down-regulated (P < 0.05), and the results of Western blotting showed that α-SMA protein was also inhibited. Conclusion The findings indicate that miR-18a could inhibit the expression of extracellular matrix-related genes of NIH-3T3 cells by directly acting on the 3’UTR of target gene Notch2.
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Background Although transforming growth factor-β (TGF-β)/Smad signaling pathway is important in regulating the occurrence and development of pulmonary fibrosis, the pathogenesis of pulmonary fibrosis remains elusive. Objective To explore the functions of genes associated with TGF-β/Smad signaling pathway in the progression of pulmonary fibrosis. Methods A NIH-3T3 fibroblast model induced by TGF-β1 was established. The experiment samples were divided into a control group and a TGF-β1 treatment group. The control group was exposed to normal saline, while the TGF-β1 treatment group was exposed to 10 ng·mL−1 TGF-β1 for 12 h. The RNAs of the two groups were extracted, sequenced, and analyzed by bioinformatics methods to identify seven key genes in TGF-β pathway, including Dcn, Smad3, Smad7, Fbn1, Thbs1, TGF-β1, and TGF-β3. The gene expression levels of five markers [Collagen1α1, Collagen1α2, α-smooth muscle actin (α-SMA), TGF-β1, and TGF-β3] and the seven key genes were detected by quantitative real-time PCR (qRT-PCR). The proteins of the two groups were extracted. The important marker protein expression levels of Smad3, the phosphorylation of Smad3 (P-Smad3), and α-SMA were detected by Western blotting. At the same time, 30 healthy SPF-grade C57BL/6 mice were randomly divided into three groups, with 10 mice in each group: a control group, a SiO2 inhalation exposure group for 28 d (10 mice), and a SiO2 inhalation exposure group for 56 d (10 mice). The mice in the two treatment groups were exposed to a natural SiO2 environment for 4 h per day with a 10-min pause for breathing fresh air at 2 h intervals. The lung tissues of the mice were taken after execution. The changes of pulmonary fibrosis were detected by Masson staining, and mRNAs and proteins were extracted to detect the expression of the above key genes and proteins. Results The expression levels of the five marker genes Collagen1α1, Collagen1α2, α-SMA, TGF-β1, and TGF-β3 were significantly increased in the TGF-β1-induced NIH-3T3 fibroblasts than those in the control group (P < 0.01); the expression levels of P-Smad3 and α-SMA proteins increased significantly (P < 0.01); the expression results of the seven key genes screened in the TGF pathway were that Dcn and Smad3 were obviously down-regulated (P < 0.01), and Smad7, Fbn1, Thbs1, TGF-β1, and TGF-β3 were obviously up-regulated (P < 0.01). The changes in gene expression levels of the transcriptome sequencing showed the same trend. The results of Masson staining showed that the content of collagen fibers in the lung tissues also increased in the SiO2 inhalation exposure groups over time. In the mouse experiment, five marker genes were obviously up-regulated compared with the control group (P < 0.01); no obvious change was found in the expression of Smad3 protein, and the expression levels of P-Smad3 and α-SMA were obviously higher in the SiO2 exposure groups than those in the control group (P < 0.01); the expression levels of Dcn and Smad3 showed a down-regulated trend, while the expression levels of Smad7, Fbn1, Thbs1, TGF-β1, and TGF-β3 showed an up-regulated trend with the increase of SiO2 inhalation exposure days (P < 0.01). The expression levels of the above five marker genes, three important marker proteins, and seven key genes were consistent with the expression trends of TGF-β1-induced NIH-3T3 fibroblasts. Conclusion The expression levels of pulmonary fibrosis-related marker genes and proteins change significantly in TGF-β1-induced fibroblast cells, and the lung tissues of mice under natural SiO2 inhalation exposure has obvious fibrosis characteristics. Seven genes (Dcn, Smad3, Smad7, Fbn1, Thbs1, TGF-β1, and TGF-β3) may be involved in the regulation of pulmonary fibrosis by the TGF-β/Smad signaling pathway.
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Objective@#To understand the viral etiology of a clustered case of human infection outbreak in the middle school of Huai’an city.@*Methods@#Nasopharyngeal swab samples from patients were collected and rapidly detected by Real-time RT-PCR and the target virus isolated in cells. Furthermore, HA1 segments of target virus were amplified by RT-PCR and sequenced. The genetic and phylogenetic analysis based on HA1 genes was computed.@*Results@#Influenza A(H1N1)pdm09 viral nucleic acid in 11 nasopharyngeal swab samples from patients in the outbreak were positive. Compared to the vaccine strains A/California/07/2009, the Huai’an isolates, nucleotide identity was 97.7%-98.1%, and amino acid identity was 96.6%-97.4%. Phylogenetic analysis of HA1 segment sequences indicated that the Huai’an strains from the outbreak were related closely to the viruses isolated in the year of 2014. Sequence analysis indicated that the Huai’an isolates had no amino acid substitution in the receptor binding sites and glycosylation sites, while in the Ca1 of antigenic determinant of HA1 the Huai’an isolates had an amino acid substitution of S for T at 220.@*Conclusions@#The pathogen of the clustered case of human infection was Influenza A(H1N1)pdm09 virus. Though the Huai’an isolates had one animo acid substitution in the Ca1 of antigenic determinant, the antigenicity characteristic remained unchanged.
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Objective To compare the accuracy of nucleic acid detection and antibody detection for diagnosing human immunode‐ficiency virus(HIV) infection .Methods Retrospectively analysed data of nucleic acid detection and antibody detection from 124 ca‐ses of patients diagnosed with HIV infection from 2005 to 2014 .The positive rates of the two methods were compared respectively in patients with early‐stage of HIV infection(76 cases) and patients with intermediate and advanced stage of HIV infection (48 ca‐ses) .Results In patients with early‐stage of HIV infectionn ,the positive rate of nucleic acid detection (94 .74% ) was higher than that of antibody detection (84 .21% );while in patients with intermediate and advanced stage of HIV infection ,the positive rate of antibody detection(97 .92% ) was higher than that of nucleic acid detection (81 .25% );both had statistically significant difference (P<0 .05) .Conclusion On the early stage of HIV infection ,the accuracy of nucleic acid detection is higher than that of antibody detection ;while on the intermediate and advanced stage of HIV infection ,antibody detection shows better accuracy .
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ObjectiveTo investigate the incidence and possible related factors for fatty liver among workers engaged in light manual labor in Tianjin, China. MethodsAmong 800 healthy workers engaged in light manual labor randomly selected from a company in Tianjin, a total of 602 healthy workers completed physical examination and data collection. The content of fat and liver stiffness were determined by controlled attenuation parameter (CAP) using FibroScan, and the disease history, body mass, height, blood pressure, and living habits in all subjects were investigated. The possible related factors for different degrees of fatty liver were analyzed. Between-group comparison of continuous data was performed using the Kruskal-Wallis H test, and comparison of categorical data was performed using χ2 test with row×column tables. Pairwise comparison was performed using χ2 segmentation method, and the agreement between CAP and color ultrasound was analyzed using the Kappa coefficient. ResultsAmong the 602 subjects, there were 180 (29.9%), 195 (32.3%), and 227 (37.7%) subjects with severe fatty liver, moderate fatty liver, and no fatty liver, respectively. There were significant differences in age, body mass index, waist-hip ratio, smoking, drinking, history of diabetes, and family history between the above three groups according to CAP (P＜005 for all). With higher sensitivity, CAP achieved a significantly higher detection rate of fatty liver than traditional color ultrasound (623% vs 44.0%, P=0.000). The agreement between two methods was poor (Kappa value ＜0.4). ConclusionAmong the so-called “healthy people” engaged in light manual labor, the incidence of fatty liver has exceeded 60%, which is associated with body mass index, waist-hip ratio, bad living habits, history of diabetes, and family history of fatty liver. CAP can noninvasively detect fatty liver with higher sensitivity and less time; however, its clinical significance needs further studies.
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Objective To establish a rapid method of real-time fluorescence RT-PCR to quantify Soul virus.Methods The pro-fessional software was adopted to design the primer and the TaqMan-BHQ probe.With artificially synthesized L gene segment as the template of Soul virus,the real-time RT-PCR for detecting Soul virus was researched.Results The Ct value of templates had a good linear relationship with the log value of the template diluted concentration.The standard curve was Y =-3.607X +41.84, r2 =0.998,the PCR amplification efficiency was 108.1%,its lowest detection limit was 53.2 copies/μL.Conclusion Applying the real-time fluorescence RT-PCR by the TaqMan-BHQ probe for detecting nucleic acid of Seoul virus has the characteristics of short time-consuming and high sensitivity.