The objective of this study was to screen out the effective shRNA which can inhibit the gene expression of tumour necrosis factor-alpha (TNF-alpha), to construct the recombinant plasmid and to determine its sequence so as to provide the new approach for searching gene therapy of TNF-alpha related diseases. The primary macrophages were added into 15% DMEM, then cells were adjusted as 2 x 10(7) cells/L and were inoculated in 6-well plate with 3 ml/well, and were cultured at 37 degrees C in a fully humidified atmosphere with 5% CO(2). Cells were stimulated with lipopolysaccharide (LPS) and the concentration of TNF-alpha in the supernatant at different time points was determined by enzyme-linked immunosorbent assay (ELISA). The 5 synthesized DNA sequences which can be transcripted into shRNA were transfected into cells with lipofectamine 2000, then the cells were stimulated with LPS for 24 hours. The concentration of TNF-alpha in the supernatant and the expression of TNF-alpha mRNA were determined by ELISA and reverse transcription polymerase chain reaction (RT-PCR) respectively. The most effective shRNA was inserted into plasmid, and the recombinant plasmid was identified by sequence analysis. The results showed that the concentration of TNF-alpha in the supernatant reached peak after the stimulation with LPS for 24 hours. In the RNA interference group, the shRNA 1 was the most effective one, which could inhibit the expression of TNF-alpha by 59.46% and the expression of TNF-alpha mRNA by 61.2%. The recombinant plasmid was cloned and the sequence of interest was obtained. In conclusion, the most effective shRNA targeting TNF-alpha was successfully screened out and the recombinant plasmid was constructed. The recombinant plasmid may be helpful to search new gene therapy for TNF-alpha related disease.
Animals ; Cells, Cultured ; Gene Expression ; Genetic Therapy ; Genetic Vectors ; Macrophages ; cytology ; Male ; Mice ; Mice, Inbred C57BL ; Plasmids ; RNA Interference ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Recombination, Genetic ; Transfection ; Tumor Necrosis Factor-alpha ; genetics

OBJECTIVETo investigate the placement of a long tube into the small intestine under fluoroscopic guidance and to evaluate its decompression effect on early postoperative small bowel obstruction (EPSBO).

METHODSFifty-four patients with EPSBO requiring decompression between April 2010 and July 2014 were enrolled in the study. Insertion of a long tube was guided by fluoroscopy. We first used the guide wire to pass the pylorus and then used the 10 Fr feeding tube as an exchangeable tube to put the superstiff wire into the duodenum. Finally the long tube could be passed over the guide wire through the pylorus into the intestine. The total procedure time, the radiation exposure time, and the incidence of complications were evaluated.

RESULTSThe long tubes passed into the jejunum on initial insertion for all patients, so the success rate of this technique was 100%. The long tube was inserted into ileum in 18 patients. The mean total procedure time was 34.4 ± 8.6 minutes, and the mean radiation exposure time 18.9 ± 6.8 minutes. A total of 47 patients (87%) experienced full recovery following long-tube decompression and without the need for surgical intervention.

CONCLUSIONSUsing the wire-exchange technique, it is easy to place a long tube into the small bowel under fluoroscopic guidance. This decompression method is safe and effective for management of EPSBO.

Adult ; Aged ; Decompression, Surgical ; methods ; Female ; Fluoroscopy ; Humans ; Intestinal Obstruction ; surgery ; Male ; Middle Aged ; Postoperative Complications ; surgery ; Retrospective Studies

Objective To investigate the prognostic value of maximum diameter in axial plane of primary tumor (MDAPPT) on MRI in nasopharyngeal carcinoma (NPC).Methods From 2005 to 2007,333 patients with newly diagnosed and biopsy-proven NPC without distant metastasis,who underwent MRI scans of the nasopharynx and neck,were included in our study.MDAPPT was measured on MRI.The univariate analysis with the log-rank test and multivariate analysis with the Cox proportional hazards model were used to analyze the relationship between MDAPPT and prognosis.Results The median values of MDAPPT in patients with T1,T2,T3,and T4 NPC were 21.2,30.0,38.0,and 52.3 mm,respectively.For all patients with a MDAPPT of ≤30 mm,＞ 30-50 mm,and ＞ 50 mm,the 5-year overall survival (OS) rates were 81.3％,70.1％,and 51.5％,respectively (P =0.000) ; the 5-year progression-free survival (PFS) rates were 81.3％,70.0％,and 48.9％,respectively (P =0.000) ;the 5-year distant metastasisfree survival (DMFS) rates were 85.5 ％,86.5 ％,and 67.2 ％,respectively (P =0.000) ; the 5-year local relapse-free survival (LRFS) rates were 97.7％,91.5％,and 83.3％,respectively (P =0.013).The multivariate analysis showed that MDAPPT was a prognostic factor for PFS and DMFS.For the T3-T4 patients with a MDAPPT of ≤50 mm and ＞50 mm,the 5-year OS rates were 69.4％ and 52.2％ (P =0.004),the 5-year PFS rates were 68.0％ and 49.6％ (P =0.001),and the 5-year DMFS rates were 84.0％ and 66.8％ (P=0.001).In the patients with a MDAPPT ≤30 mm,the 5-year LRFS rates for those with T1,T2,T3,and T4 NPC were 10 0 ％,9 5.8 ％,9 6.3 ％,and 10 0 ％,respectively (P =0.6 4 3).Conclusions MDAPPT is a prognostic factor for PFS and DMFS in NPC,and it is an important prognostic factor in patients with T3-T4 NPC.In the NPC patients with a small MDAPPT,local control rate varies little in different T stages.

Objective To evaluate the prognostic impact of MRI-detected prevertebral space involvement in nasopharyngeal carcinoma (NPC) treated with radiotherapy and chemotherapy.Methods A retrospective analysis was performed on the clinical data of 333 patients who had newly diagnosed biopsyproven NPC without distant metastasis from 2005 to 2007.All patients underwent MRI scans of the nasopharynx and neck and were treated with two-and three-dimensional radiotherapy without or without chemotherapy.The Kaplan-Meier method was used to calculate overall survival (OS),distant metastasis-free survival (DMFS),and locoregional relapse-free survival (LRFS),and the log-rank test was used for survival difference analysis;the Cox proportional hazards regression analysis was used to assess the prognostic value of prevertebral space involvement.Results The follow-up rate was 95.2％.Prevertebral space involvement was seen in 139(41.7％) of these patients.The patients with prevertebral space involvement had significantly higher T stage and clinical stage than those without prevertebral space involvement (x2 =90.41,P =0.000;x2 =54.03,P =0.000).The 5-year OS,DMFS,and LRFS for NPC patients with and without prevertebral space involvement were 58.8％ vs.77.5％ (x2 =11.95,P =0.000),77.8％ vs.85.0％(x2=2.56,P=0.110),and 88.3％ vs.91.8％ (x2=1.51,P=0.220),respectively.After adjusting for N stage,a significant difference was still seen between the two groups with regard to 5-year OS (x2 =9.93,P =0.002).The multivariate analysis showed that prevertebral space involvement was not the independent prognostic factor for OS,DMFS,and LRFS (x2 =0.43,P =0.512 ; x2 =0.08,P =0.783 ; x2 =0.00,P =0.971).Conclusions The frequency of prevertebral space involvement is very high in NPC.The OS for the patients with prevertebral space involvement is significantly lower than those without prevertebral space involvement.But prevertebral space involvement is not the independent prognostic factor in NPC patients.

Objective To explore the effect of Okam on airway inflammation in asthmatic mouse.Methods Thirty-two SPF grade Kunming Strain mice were randomly divided into positive control group,glucocorticoid inhalation group,Okam group and negative control group with 8 mice in each group.The mice were sensitized and repeatedly challenged with ovalbumin(OVA) to establish the models of chronic asthma.The glucocorticoid group were given Budesonide(200 ?g) and saline everyday by inhalation,the Okam group were given 50 mg/kg Okam by gavage,and the positive group had saline at the same time,the negative control group received saline at all stages.The inflammation of the lung tissue were scored underwent HE staining.Bronchoalveolar lavage fluid(BALF) cell count and differential were studied,and interferon-?(IFN-?),interleukin-4(IL-4) in BALF were determined by enzyme-linked immunosorbent assay(ELISA).Results There were no inflammatory cell infiltrate of bronchiole in the negative control group.Inflammatory infiltration of lung tissue were obvious in the positive control group.Inflammatory infiltration of lung tissue lightened obviously in the Budesonide and Okam groups.The total cell number,Eosinophils(EOS) and IL-4 level in BALF,and the score of the lung tissue in Okam group were all markedly lower than those in positive control group(t=5.942,7.089,7.078 Pa0.05),IFN-? lower(t=4.275 P

The objective of this study was to investigate the function and mechanism of hyperbaric oxygen (HBO) in antagonizing acute graft-versus-host disease (aGVHD) and improving the rate of survival. The lethally irradiated C57BL/6 recipients were injected with bone marrow and lymphocyte of spleen from BALB/c donors and were treated with HBO, cyclosporine A (CsA) and methotrexate (MTX). T lymphocytes and subsets, adhesion molecules and cytokines were detected by flow cytometry, ELISA and RT-PCR respectively. The results showed that the survival rate in HBO group was much higher than that in allogenetic bone marrow transplantation (allo-BMT) group and CsA + MTX group; the numbers of CD3(+), CD4(+), CD8(+), CD4(+)CD11a(+), CD4(+)CD18(+), CD8(+)CD11a(+), CD8(+)CD18(+) lymphocytes in spleen were decreased markedly by HBO and CsA + MTX (p < 0.05); the levels of IL-2 and TNFalpha mRNA and their serum concentrations in HBO group were much lower than those in allo-BMT group but were higher than those in CsA + MTX group; the levels of IL-4 and IL-10 mRNA in HBO group were much higher than those in allo-BMT group and CsA + MTX group. It is concluded that HBO has more remarkable advantage in improving the rate of survival than CsA + MTX, its mechanism of anti-aGVHD is tightly correlated with the transform of T cell and its subsets and the expression of adhesion molecules and cytokines.
Acute Disease ; Animals ; Bone Marrow Transplantation ; adverse effects ; Cytokines ; biosynthesis ; Female ; Graft vs Host Disease ; etiology ; therapy ; Hyperbaric Oxygenation ; Lymphocyte Transfusion ; adverse effects ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; T-Lymphocytes ; immunology ; Whole-Body Irradiation

The aim of this study was to explore the effect of hyperbaric oxygen (HBO) preconditioning on the rejection of skin allograft in mice and its molecular mechanism. BALB/c donor mice and C57BL/6 recipients received hyperbaric oxygen preconditioning once a day for 7 days. After skin transplantation, the recipients were treated with cyclosporine A (CsA) intraperitoneally. Immunofluorescent staining technique and flow cytometry were used to observe the influence HBO on percentage of spleen lymphocytes CD3+, CD4+, CD8+ and cell adhesion molecule LFA-1 (CD11a/CD18). The results showed that as compared with control, the numbers of CD3+, CD4+, CD8+, CD4+CD11a+, CD4+ CD18+, CD8+CD11a+, CD8+CD18+ lymphocytes of spleen decreased in HBO preconditioning groups and CsA group, and decreased markedly in HBO preconditioning combined with CsA group (p<0.05); the general state of recipient mice in HBO preconditioning combined with CsA group was better than that of recipient mice received HBO preconditioning or CsA only. It is concluded that the method of HBO preconditioning combined with traditional immunosuppressive agent CsA has remarkable advantage in inhibiting the rejection of skin graft. Its molecular protective mechanism is correlated with the expression of adhesive molecules on T cell subsets.
Animals ; Cell Adhesion ; Cell Adhesion Molecules ; pharmacology ; Cyclosporine ; pharmacology ; Female ; Graft Rejection ; prevention & control ; Graft Survival ; Hyperbaric Oxygenation ; Lymphocyte Count ; Lymphocytes ; cytology ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Skin Transplantation ; immunology ; Spleen ; cytology ; Transplantation Conditioning ; methods

OBJECTIVE: To study DNA quantification and STR typing of samples pre-treated with pyramidon. METHODS: The blood samples of ten unrelated individuals were anticoagulated in EDTA. The blood stains were made on the filter paper. The experimental groups were divided into six groups in accordance with the storage time, 30 min, 1 h, 3 h, 6 h, 12 h and 24h after pre-treated with pyramidon. DNA was extracted by three methods: magnetic bead-based extraction, QIAcube DNA purification method and Chelex-100 method. The quantification of DNA was made by fluorescent quantitative PCR. STR typing was detected by PCR-STR fluorescent technology. RESULTS: In the same DNA extraction method, the sample DNA decreased gradually with times after pre-treatment with pyramidon. In the same storage time, the DNA quantification in different extraction methods had significant differences. Sixteen loci DNA typing were detected in 90.56% of samples. CONCLUSION Pyramidon pre-treatment could cause DNA degradation, but effective STR typing can be achieved within 24 h. The magnetic bead-based extraction is the best method for STR profiling and DNA extraction.
Aminopyrine/pharmacology* ; Blood Stains ; DNA/isolation & purification* ; DNA Fingerprinting ; Forensic Medicine ; Humans ; Polymerase Chain Reaction ; Reproducibility of Results ; Specimen Handling

Coronary artery disease (CAD) is one of the leading causes of death. Safflower attracts great attention owing to its anti-ischemia/reperfusion injury effect. Ninety-three patients with CAD were included and randomized into safflower treatment group, PCI group and control group. Low-dose dobutamine stress echocardiography (DSE) was performed to measure end-systolic volume (ESV), end-diastolic volume (EDV), left ventricular ejection fraction (LVEF) and wall motion score index (WMSI) to determine the recovery of hibernating myocardium and cardiac function in all patients before treatment and after 3-month follow-up. The study was to investigate the effects of safflower on hibernating myocardial revascularization and cardiac function. It was found that LVEF was significantly improved, while the ESV and WMSI were significantly reduced after 2-week treatment in safflower and PCI treatment groups. No significant differences were found between safflower and PCI treatment groups in ESV, EDV, WMSI and LVEF after treatment Safflower injection effectively improved hibernating myocardial function.
Aged ; Carthamus tinctorius ; chemistry ; Coronary Artery Disease ; drug therapy ; physiopathology ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Heart ; drug effects ; physiopathology ; Humans ; Male ; Middle Aged ; Myocardial Revascularization ; Myocardial Stunning ; drug therapy ; physiopathology ; surgery ; Recovery of Function

OBJECTIVETo investigate the effects of the quinoline derivative PQ1 combined with cisplatin on the proliferation and gap junction communication of prostate cancer PC3 cells.

METHODSWe cultured in vitro prostate cancer PC3 cells and divided them into DMSO blank control, cisplatin control, and cisplatin (10 mg/ml) plus PQ1 (1, 2, 5, 10, and 15 μmol/L) groups. We measured the proliferation of the prostate cancer PC3 cells, determined the expressions of the connexin 43 (Cx43) mRNA and protein by RT-PCR and Western blot, and compared the indexes among different groups.

RESULTSCisplatin combined with PQl at 1 - 10 μmol/L significantly inhibited the proliferation of the PC3 cells and the inhibition rate rose in a concentration- and time-dependent manner, from (48.72 ± 0.98)% vs (50.33 ± 0.62)% at 0 μmol/L to (77.38 ± 1.12)% vs (83.50 ± 1.05)% at 15 μmol/L at 24 and 48 hours (P < 0.05). Compared with the cisplatin control, cisplatin combined with PQ1 at 1, 2, 5, 10, and 15 μmol/L increased the expression of Cx43 mRNA from 0.379 ± 0.113 to 0.669 ± 0.031, 0.831 ± 0. 127, 0.769 ± 0.100, 0.532 ± 0.086, and 0.475 ± 0.134, respectively (P < 0.05), and cisplatin combined with PQ1 at 1, 2, 5, and 10 μmol/L elevated that of Cx43 protein from 0.138 ± 0.146 to 0.263 ± 0.111, 0.306 ± 0.152, 0.415 ± 0.280, and 0.643 ± 0.310, respectively (P < 0.05).

CONCLUSIONThe quinoline derivative PQ1 can promote the gap junction communication of prostate cancer PC3 cells and enhance the killing effect of cisplatin on PC3 cells by upregulating the expressions of Cx43 mRNA and protein.

Aminoquinolines ; pharmacology ; Antineoplastic Combined Chemotherapy Protocols ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Connexin 43 ; genetics ; metabolism ; Dose-Response Relationship, Drug ; Gap Junctions ; drug effects ; physiology ; Humans ; Male ; Prostatic Neoplasms ; metabolism ; pathology ; physiopathology ; RNA, Messenger ; metabolism ; Time Factors