Objective To investigate the clinical efficacy and improvement of patients' quality of life in tension-free vaginal tape(TVT)and tension-free vaginal tape obturator(TVT-O)for surgical treatment of severe female stress urinary incontinence.Methods This study was a randomized,singleblinded,controlled trial.Patients were randomized by a computer-generated randomization schedule with allocation to either TVT or TVT-O procedure.TVT procedure was performed in 35 cases and TVT-O in 34 cases.None had received surgery for urinary incontinence or was in pregnancy.Transvaginal hysterectomy and prolapse reparation were done simultaneously in some of the patients.All patients were requested to complete the Urinary Distress Inventory(UDI-6)and Incontinence Impact Questionnaire(ⅡQ-7)as part of their pre-and postoperative assessment.Results All patients were evaluable and the mean follow-up was 14.5 months.The mean operative time was(18±5)min in the TVT-O group,Significantly shorter than in the TVT group(27±5)min(P＜0.01).The two groups did not differ significantly in perioperative blood loss,postoperative complications(including tape erosion,pain in thigh or behind pubis),postvoid residual volume,hospital stays or expenses(all P＞ 0.05).Sixty patients were successfully treated for stress urinary incontinence(88.6%and 85.3%for TVT and TVT-O groups,respectively).There were significant improvements in postoperative scores for both the ⅡQ-7 and the UDI-6(P＜0.01),except in subscale measuring symptoms of voiding dysfunction(P＞0.05).Conclusions Both techniques appear to be equally effective in the surgical treatment of severe stress urinary incontinence in a short term review.Significant improvements could also be seen in patients' quality of life.However.TVT-O has a shorter operative time.No evidence of increasing risk of urethral obstruction after the operation could be found.Long term followups axe necessary to evaluate outcomes of different types of surgery for stress urinary incontinence.

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

Objective To explore the correlation between quantitative parameters of blood perfusion with contrast-enhanced ultrasound (CEUS) and microvessel density (MVD),microvessel area (MVA) in papillary thyroid carcinoma (PTC).And to investigate the value of CEUS in evaluating the angiogenesis in PTC before operation.Methods Totally 69 cases of patients with papillary thyroid carcinoma were selected from April 2014 to October 2016 in the Affiliated Hospital of Qingdao University.The CEUS characteristics of 69 patients with papillary thyroid carcinoma confirmed by pathology were retrospectively analyzed.The patients were divided into three groups according to maximum diameter of lesions (＜ 1 cm group,1-2 cm group and ＞ 2.0 cm group),and two groups according to pathologic reports (neck lymph node metastatic and nonmetastatic groups).The blood perfusion parameters between or among different groups were evaluated by ttest or one-way ANOVA.Immunohistochemical staining were performed to evaluate the MVD,MVA in the surgical specimens,and the correlation of quantitative parameters with MVD,MVA were assessed by Spearman.Results (1) Peak Intensity (Peak),area under the curve (AUC),MVD and MVA of thyroid carcinoma were lower than the surrounding normal thyroid tissue (14.95 ± 4.96 vs 22.67±6.11,970.01±263.20 vs 1798.35±563.67,118.91±31.32 vs 206.27±39.58,8.58±-2.68 vs 18.47±3.13),and the differences were statistically significant (t=-8.700,-11.061,-14.377 and-20.532,all P ＜ 0.05).(2)With the increase of the lesion's maximum diameter,Peak,AUC,MVD and MVA increased,and the differences were statistically significant (t=0.000,0.000,0.000,0.000;t=0.027,0.044,0.033,0.000;t=0.027,0.044,0.033,0.000,all P ＜ 0.05).(3) Papillary thyroid carcinoma with lymphatic involvement had significantly higher values of Peak,AUC,MVD and MVA than those without lymphatic involvement (16.86±4.36 vs 13.80±3.55,1128.16±290.85 vs 874.39±192.27,114.12±30.69 vs 103.67±22.19,10.30 ± 2.44 vs 7.54 ± 2.29),and the differences were statistically significant (t=3.177,4.366,6.336 and 4.742,all P ＜ 0.05).(4) A positive correlation existed between the Peak,AUC and MVD,and the differences were statistically significant (r=0.506,0.478,all P ＜0.05).Peak,AUC and MVA showed positive correlation,and the differences were statistically significant (r=0.648,0.653,all P ＜ 0.05).TP,MTT and MVD,MVA showed no correlations (all P ＞ 0.05).Conclusions The values of Peak and AUC calculated from CEUS were correlated to MVD and MVA.CEUS may be used to evaluated the angiogenesis of PTC before operation.And CEUS is helpful for prediction of prognosis of PTC.

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

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

Through stating the current situation of the doctor -patient relationship , and combining with its characteristics in emergency ICU (EICU), to analyze the application basements and skills of progressive doctor -patient communication in EICU , in order to explore the present stage to build a harmonious doctor -patient rela-tionship , a feasible way to safeguard the rights and interests of both doctors to provide the reference .

The aim of this study was to analyze the promoter methylation patterns of inhibitory killer cell immunoglobulin-like receptor (KIR) which gene expression and the effect of demethylation treatment were studied, and to explore the possible regulation mechanism of inhibitory kir gene expression. The promoter methylation levels of kir2DL1 and kir2DL2/kir2DL3 in NK-92MI cell line were detected by bisulfite sequencing technique. Then NK-92MI cells were treated with 5-azacytidine to induce the demethylation of CpG islands. The levels of gene expression of kir were determined by RT-PCR. The results demonstrated that the methylation frequencies of CpG dinucleotides surrounding the promoter regions of kir2DL1 and kir2DL2/kir2DL3 genes were 25% to 88% and 5% to 80% respectively. DNA-demethylating treatment with 5-azacytidine resulted in re-expression of kir2DL1 gene and increased expressions of kir2DL1, kir2DL2 and kir2DL3 genes in NK-92MI cells. In conclusion, the promoter DNA methylation participates in the regulation of kir gene expression in NK-92MI cells.
Cell Line ; DNA Methylation ; Gene Expression ; Humans ; Killer Cells, Natural ; metabolism ; Receptors, KIR2DL1 ; genetics ; metabolism ; Receptors, KIR2DL2 ; genetics ; metabolism ; Receptors, KIR2DL3 ; genetics ; metabolism

OBJECTIVETo investigate the mechanisms of apoptosis induced in human hepatoma cell line SMMC7721 by plasmid pVHN constructed with Newcastle disease virus (NDV) HN gene.

METHODSTwenty-four h after transfection with liposome-plasmid pVHN complexes in vitro, the mortality rate of SMMC7721 cells was determined by MTT staining and flow cytometry (FCM) with PI staining. The alteration of mitochondrial trans-membrane potential of the cells was detected by FCM with rhodamine 123 staining. Cell genomic DNA was detected by agarose electrophoresis. The activation of caspase-3 was assayed by its substrate color reaction.

RESULTSSignificant apoptosis was induced by transfection with plasmid pVHN into the cells for 24 h and the mortality rate was 50.0% (the mortality rate of control group was 5.2%). Genomic DNA was fragmented and mitochondrial trans-membrane potential was decreased, but caspase-3 activity increased.

CONCLUSIONSignificant apoptosis in SMMC7721 cells can be induced by NDV HN gene. Apoptosis may be resulted from the decrease of mitochondrial trans-membrane potential and activation of Caspase-3.

Apoptosis ; physiology ; Cancer Vaccines ; immunology ; Carcinoma, Hepatocellular ; pathology ; Caspase 3 ; metabolism ; Cell Line, Tumor ; HN Protein ; genetics ; Humans ; Liver Neoplasms ; pathology ; Newcastle disease virus ; genetics ; Transfection ; Vaccines, DNA ; immunology

BACKGROUNDOne of the reasons for poor neuroregeneration after central nervous system injury is the presence of inhibitory factors such as Nogo. Here, we tested the inhibition of Nogo by RNA interference both in vitro and in vivo, using recombinant adenovirus-mediated transfection of short hairpin RNAs, to explore a new method of treatment for spinal cord injury.

METHODSWe designed and cloned two Nogo-specific short hairpin RNAs and an unrelated short hairpin RNA, packaged the clones into adenovirus, and amplified the recombinant virus in 293 cells. We then tested the inhibition of Nogo expression both in vitro in adenovirus-transfected oligodendrocytes and in vivo in spinal cord tissue from adenovirus-transfected spinal cord injury model rats. We tested Nogo expression at the mRNA level by reverse-transcription PCR and at the protein level by Western blotting and immunohistochemistry.

RESULTSIn vitro, the two specific Nogo short hairpin RNAs decreased Nogo mRNA expression by 51% and 49%, respectively, compared with Nogo expression in cells transfected with the unrelated control small hairpin RNA (P < 0.005). Similarly, Nogo protein expression decreased by 50% and 48%, respectively (P < 0.005). In vivo, in spinal cord injury model rats, the two specific Nogo short hairpin RNAs decreased Nogo mRNA expression by 45% and 40%, respectively, compared with Nogo expression in spinal cord injury model rats transfected with the unrelated control short hairpin RNA (P < 0.005). The Nogo protein level was similarly decreased.

CONCLUSIONSWe were successful in specifically downregulating Nogo at the mRNA and protein levels by adenovirus-mediated delivery of short hairpin RNAs, both in vitro and in vivo. This confirms the effectiveness of RNA interference for the inhibition of Nogo gene expression and the efficiency of using adenovirus for delivery. Thus gene therapy may be an effective treatment for spinal cord injury.

Adenoviridae ; genetics ; Animals ; Blotting, Western ; Humans ; Immunohistochemistry ; Myelin Proteins ; genetics ; metabolism ; Nogo Proteins ; RNA Interference ; RNA, Small Interfering ; genetics ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; therapy

OBJECTIVETo develop a method for quantifying gene expressions in the mouse single oocyte and preimplantation embryo.

METHODSWe quantified the message RNA (mRNA) expression of the TSC2 gene in the single oocyte and preimplantation embryo by capillary electrophoresis using the exogenic mutation TSC2 gene as the reference and amplification by competition polymerase chain reaction (PCR).

RESULTSWe successfully established the method for quantifying the mRNA expression of the TSC2 gene, with good linear relations between the mRNA level of the TSC2 gene and the dilution degree of the reference gene (r = -0.987).

CONCLUSIONThe level of the mouse TSC2 gene expression can be effectively quantified by competition PCR and capillary electrophoresis, which has provided a molecular base for evaluating the quality of human oocytes and preimplantation embryos.

Animals ; Blastocyst ; metabolism ; Female ; Gene Expression ; Male ; Mice ; Mice, Inbred ICR ; Oocytes ; metabolism ; Polymerase Chain Reaction ; methods ; RNA, Messenger ; genetics ; Tumor Suppressor Proteins ; genetics