Objective To construct the eukaryotic expression plasmids of human Fas and TNFR1 gene(pcDNA3.0-hFas and pcDNA3.0-hTNFR1)and microRNA(miRNA)expression plasmid of hFas and hTNFR1 named p-hFasmiRNA and p-hTNFR1miRNA,and to investigate their inhibitory effects in vitro.Methods The eukaryotic expression plasmids of human Fas and TNFR1 gene were constructed(pcDNA3.0-hFas and pcDNA3.0-hTNFR1)and have been shown successfully to express hFas and hTNFR1 protein.miRNA expression plasmids of hFas and hTNFR1 named p-hFasmiRNA and p-hTNFR1miRNA complimentary to the sequence responsible for hFas and hTNFR1 respective were constructed,meanwhile irrelevant miRNA plasmid was used as a control.By respective co-transfection of p-hFasmiRNA and pcDNA3.0-hFas,p-hTNFR1 miRNA and pcDNA3.0-hTNFR1 expression construct into 293T cells,the inhibition of hFas and hTNFR1 expression was analyzed by real-time PCR and Western blot.Results The experiments showed the significant inhibitory effect of p-hFasmiRNA on hFas and p-hTNFR1miRNA on hTNFR1 expression at 48 h post-transfection both at RNA level and protein level as well in 293T cell lines with the inhibitory efficiency being as high as 87% for hFas and 80% for hTNFR1,respectively.Conclusion The p-hFasmiRNA and p-hTNFR1miRNA were constructed successfully,and it was verified that they could specifically inhibit the hFas and hTNFR1 expression at the cellular level.

Previous study on TNFRl-mediated hepatocyte apoptosis has been implicated in the development of fulminant viral hepatitis.To interfere with the potentially effective target,plasmid named p-mTNFR1shRNA complimentary to the sequence responsible for mTNFR1 was also constructed and further confirmed by sequence analysis.To investigate the effect of mTNFR1shRNA plasmid on mTNFR1 expression in vivo and the disease progress in MHV-3 induced fulminant hepatitis mice model.By hydrodynamic injection of mTNFRlshRNA plasmid,the survival rate of mice,hepatic pathological change were examined and compared between mice with/without mTNFR1 shRNA plasmid intervention.The expression of mTNFR1 was detected by Real-time PCR,immunohistochemistry staining.The mTNFR1 shRNA plasmid significantly reduced mTNFR1 expression in vivo,markedly ameliorates inflammatory infiltration,prolonged the survival time period and elevated the survival rate from 0 up to 13.3% in Balb/cJ mice with MHV-3 induced fulminant hepatitis.This study was designed to explore the opportunity of RNA interference technique in inhibiting TNFR1 expression,which has been reported to be involved in the development of a variety of diseases including fulminant viral hepatitis and severe chronic hepatitis B.

ObjectiveTo investigate the role of active protein C (APC) in lipopolysaccharide (LPS) induced microglia activation.MethodsMicroglia from one day old Sprague-Dawley newborn rat was collected, purified and identified by primary culture and immunofluorescence staining, and then was randomly divided into four groups including LPS group (1.0μg/ml LPS plus 10μl phosphate buffered saline 12 h later), LPS+ APC group (1.0μg/ml LPS plus 0.1μg/ml APC 12 h later), APC group (10μl phosphate buffered saline plus 0.1μg/ml APC 12 h later) and control group (10μl phosphate buffered saline at each time point). The morphology of micaroglia in all groups was observed under microscope, and the expression of tumor necrosis factor-α (TNF-α) and protease-activated receptor-1 (PAR-1) were determined by immunofluorescence staining. One-way analysis of variance and LSD test were applied for statistical analysis.ResultsPrimary culture microglia was successful and the purity was no less than 99%. In LPS group, the microglia morphology was activated, and the expression of TNF-α was increased significantly than the control group (2.11±0.35 vs 1.38±0.28, LSD test,P=0.002). In LPS+APC group, the microglia morphological change was reversed, and the expression of TNF-α had no significant difference with the control group (1.35±0.36 vs 1.38±0.28, LSD test,P>0.05). The expression of PAR-1 in LPS+APC group was higher comparing with that in the control group (4.60±0.84 vs 2.64±0.41, LSD test,P=0.008) and the LPS group (2.44±0.86, LSD test,P=0.002). The expression of PAR-1 in APC group and LPS group had no obvious difference with control group (2.62±0.69, 2.44±0.86 vs 2.64±0.41, LSD test, bothP>0.05).ConclusionsBy increasing the level of PAR-1 in microglia, active protein C could inhibit the activation of miciroglia and the expression of TNF-α induced by lipopolysaccharide, therefore, protecting the brain tissues from inflammation-induced damage.

Objective To study the NR5A1 gene mutation in patients with idiopathic hypogonadotropic hypogonadism(IHH), and to find the new mutation point.Methods Sixty-one IHH patients and 100 normal control subjects were collected and genomic DNA was extracted from blood samples.These patients were with normal karyotype and no abnormality was discovered in magnetic resonance imaging (MRI) scan of the pituitary.Other endocrine diseases were also excluded.The 2-7 exons and splice-sites of NR5A1 gene were amplified with polymerase chain reaction.DNA of the coding sequence and splice-sites of NR5A 1 were sequenced by double deoxidizing terminal end sequencing method in 61 IHH and 100 normal control subjects.The results of sequencing were compared with their corresponding sequence data.61 IHH kindreds were investigated and the clinical data of these patients were collected.Finally, the phenotype and genotype positive cases were analyzed.Results Six patients carried NR5A1 gene mutational sites in 61 cases of IHH.Analysis of sequencing results from 100 age and ethnicity matched control subjects did not show any of these novel changes.Conclusions One mutation in NR5A1 gene may affect protein structure and function, which should be considered in male IHH patients with normal karyotype and without insufficiency of adrenal function.

In previous study, glutaric acid (GA) induced apoptosis of primary striatal neuron in vitro. In order to investigate the neurotoxic effects of GA on neonatal rat corpus striatum and the possible mechanism, 34 male pups were randomly assigned to NS group, low dose GA (LGA, 5 μmol GA/g body weight) group and high dose GA (HGA, 10 μmol GA/g body weight) group. These pups were subcutaneously administered with three injections from postnatal day 3 to 22 at 7:30 am, 15:00 pm and 22:30 pm and killed 12 h after the last injection. Microscopic pathology in corpus striatum was evaluated by HE staining. The apoptotic cells were identified by TUNEL staining. The transcript levels of caspase-3, 8, 9, Bax, Bcl-2 were detected by using real-time PCR and the protein levels of procaspase-3 and the active fraction were evaluated by Western blotting. In LGA and HGA groups, ventricle collapse, cortical atrophy by a macroscope and interstitial edema, vacuolations, widened perivascular space of bilateral striatum by a microscope were observed. TUNEL assay revealed that the apoptotic cells were increased in LGA and HGA groups. The transcript of caspase-3 was up-regulated to 2.5 fold, accompanied by the up-regulation of caspase-9, Bax and down-regulation of Bcl-2. The protein levels of procaspase-3 and the active fraction were up-regulated in LGA and HGA groups. The rat model for GA I showed mitochondrial apoptotic pathway may be involved in the GA-induced striatal lesion. Further studies should be taken to investigate the underlying mechanisms.

Objective To investigate correlation between aspirin resistance(AR) and inflammatory factors. Methods One hundred and ten patients with coronary heart disease took aspirin 0.1 mg/d for 14 days.It was detected platelet aggregation function induced with adenosine disphosphate (ADP) and arachidonic acid (AA), and investigated correlation between AR and inflammatory factors. Interleukin-1? (IL-1?),interleukin-6 (IL-6) and high sensitive C-reaction protein (hs-CRP) levels. Results IL-6 level of patients with AR was significantly higher than that of aspirin sensitive (AS) patients. The other two index were not different between the two groups. Conclusion IL-6 levels could be used as predictor.

This study was designed to explore the RNA interference technique in inhibition of the expression of the mouse fibrinogen like protein 2 (mfgl2), which has been reported to be involved in the development a variety of diseases including fulminant viral hepatitis. A plasmid named p-mfgl2shRNA,complementary to the sequence of mfgl2 was constructed, while another short hairpin RNA (shRNA)which was a mutated form of the mfgl2shRNA sequences was used as a control. A plasmid named pEGFP-mfgl2 expressing the mfgl2-EGFP fusion protein was also constructed for the screening of the effect of p-mfgl2shRNA on mfgl2 expression. By cotransfection of p-mfgl2shRNA and pEGFP-mfgl2 or pcDNA3.1-mfgl2 expression construct into CHO cells or HeLa cells, the inhibition of mfgl2 expression by mfgl2shRNA was analyzed by direct observation through fluorescent microscopy, FACS, RT-PCR and immunohistochemistry staining. The experiments showed the significant inhibitory effect of p-mfgl2shRNA on mfgl2 expression at 48h post-transfection in both CHO and Hela cell lines with the inhibitory efficiency as high as 80.1%. The study demonstrated that the construct of p-mfgl2shRNA successfully interfered with the mfgl2 expression in vitro.

SARS coronavirus (SARS-CoV) is the etiologic agent of severe acute respiratory syndrome. The aim of this study was to construct Sars-CoV membrane (M), nucleocapsid (N) and spike 2 (S2) gene eukaryotic expression plasmids, and identify their expression in vitro. Gene fragments encoding N protein, M protein and S2 protein of SARS-CoV were amplified by PCR using cDNA obtained from lung samples of SARS patients as template, and subcloned into pcDNA3.1 vector to form eukaryotic expression plasmids. SARS-CoV protein eukaryotic expression plasmids were transfected respectively into CHO cells. Immunohistochemistry was employed to detect the expression of the structural proteins of SARS-CoV in transfected cells. SARS-CoV protein eukaryotic expression plasmids were successfully constructed by identification with digestion of restriction enzymes and sequencing. M, N and S2 proteins of SARS-CoV were detected in the cytoplasm of transfected CHO cells. It was concluded that these recombinant eukaryotic expression plasmids were constructed successfully, and SARS-CoV encoding proteins could activate transcription and expression of hfgl2 gene.

Multidrug-resistant (MDR) bacterial infection is a common complication of severe acute pancreatitis (SAP).This study aimed to explore the association between human leukocyte antigen-antigen D-related (HLA-DR) expression and multidrug-resistant infection in patients with SAP.A total of 24 SAP patients who were admitted to Nanjing Drum Tower Hospital between May 2015 and December 2016 were enrolled in the study.The percentages of CD4+,CD8+,natural killer (NK),and HLA-DR (CD14+) cells and the CD4+/CD8+ cell ratio on days 1,7,14,and 28 after admission were determined by flow cytometry.Eighteen patients presented with the symptoms of infection.Among them,55.6％ patients (10/18) developed MDR infection.The most common causative MDR organisms were Enterobacter cloacae and Acinetobacter baumannii.The CD4+/CD8+ cell ratio and the percentage of NK cells were similar between patients with non-MDR and patients with MDR infections.In patients without infection,the HLA-DR percentage was maintained at a high level throughout the 28 days.Compared to the patients without any infection,the HLA-DR percentage in patients with non-MDR infection was reduced on day 1 but increased and reached similar levels on day 28.In patients with MDR infection,the HLA-DR percentage remained below normal levels at all-time points.It was concluded that persistent down-regulation of HLA-DR expression is associated with MDR bacterial infection in patients with SAP.

Liver failure is a complex process involving many factors. The molecular therapy targeting the cytokines and receptors associated with liver failure has become the research hotspots in recent years. Immunotherapy for liver failure includes promoting the regeneration of hepatocytes and immunoregulation by thymosin, and the combination of immunomodulatory therapy with traditional therapy is widely applied in clinical treatment. Research on the animal model of liver failure suggested that importing ARC protein or hepatocyte nuclear factor 4 protected hepatocytes and promoted the recovery of their function. Block therapy against liver failure-related molecules (fibrinogen-like protein 2, apoptosis-related molecules, transforming growth factor β, osteopontin, nuclear factor-κB, and Toll-like receptors, etc.) successfully cured the animal model of liver failure. The research on molecular therapy offers a new way for treatment of liver failure, but it is still in the stage of laboratory research at present and lacks clinical data.