Objective To construct the replication-incompetent lentivirus expressing rat TLR4 shRNA,and to observe its inhibitory effect on TLR4 expression in alveolar macrophage and on lipopolysaccharide (LPS)-induced release of IL-1?,IL-6.Methods We designed and constructed four shRNA expressing plasmids with silencing effect,then they were co-transfected into HEK293T cells with the TLR4 eukaryotic vector constructed previously;the best shRNA was selected to be packed into lentivirus;and the titer was determined.The packed lentvirus was used to infect the alveolar macrophage(NR8383) in presence of LPS,and the LPS-induced IL1?,IL-6 expression was examined by ELISA.Results The best shRNA was successfully screened out and was correctly inserted into the lentivirus.The titer of the recombinant lentivirus was 2.0 ? 106 TU/ml.The LPS-induced expression of IL-1?,IL-6 in the alveolar macrophages was greatly reduced after virus infection (P

Th17 cells play an important role in the pathogenesis of various immune-mediated diseases,including psoriasis,rheumatoid arthritis,multiple sclerosis,inflammatory bowel disease,asthma,and so on.The discovery of Th17 cells has offered scientists a new insight into the etiology and treatment of a broad spectrum of diseases.This article presents an overview of the differentiation,cytokine expression and trafficking of Th17 cells.

Objective To summarize the experience of heart-lung transplantation.Methods Four patients with Eisenmenger’s syndrome underwent heart-lung homoplastic transplantation. All patients were complicated with severe pulmonary hypertension in New York Heart Association ( NYHA ) functional class IV. Cannulation for cardiopulmonary bypass consisted of a cannula in the high ascending aorta and separate vena caval cannulas. The heart-lung graft was moved into the chest, beginning with passage of the lung before the phrenic nerve pedicle. The bronchus was trimmed, leaving two cartilaginous rings proximal to the orifice of the upper lobe. The tracheal anastomosis was performed with a continuous 4-0 polypropylene suture, with the posterior portion continuously and anterior interrupted. The lungs were then ventilated (

The most important cause of late mortality after lung transplantation is obliterative bronchiolitis ( OB) . It is clear that a good animal model is indispensable to further unravel and clarify the pathogenesis of bronchiolitis obliterans syndrome ( BOS) .Many animal models have been developed to study BOS, however, so far, none of these models truly mimics the human condition.In recent years mouse models of orthotopic lung transplantation have been established, which provide potential possibilities for further studies of OB/BOS after lung transplantation.The aim of this article was to review the pros and cons of those animal models, and discuss the possible approaches to establish animal models of chronic rejec-tion after lung transplantation.

Objective To construct eukaryotic expressed plasmid vector pLenti6/V5- DEST-Cx43 in human for further study on bioinformative communication between myocardial cell, embryonic development of heart and differentiation of myocardial cell. Methods Cx43 cDNA fragments were obtained from plasmid p18-T by restriction enzyme BamH1、Xho1, after the agarose gel electrophoresis was performed, the Cx43 cDNA was retrieved. pENTR11 were extracted by enzyme BamH1、Xho1, then connected with Cx43 cDNA by T4 DNA ligase. LR reaction was performed and Cx43 cDNA were cloned into Lentiviral vector pLenti6/V5-DEST. Results Agarose electrophoresis and sequent examination, identified that Cx43 cDNA was cloned into Lentiviral vector pLenti6/V5-DEST. Conclusion The present experiment demonstrate a successful cloning of human Cx43 cDNA into the Lentiviral vector pLenti6/V5-DEST.

Objective To explore the electrophysiological properties and the disparity of visual cortical neurons in developing rats. Methods Whole cell patch clamp recording and intracellular labeling of brain slices were performed on rats at postnatal 14 and 28 days. The electrophysiological data were analyzed according to the cellular input resistance and resting membrane potential. Results When the input impedance of visual cortical neurons(IR) was lower, increased peak value of postsynaptic currents(PSCs), prolonged rise and decay time were found. At postnatal 14th day(before eyes opened), the intermediate cells and mature cells were 59.2% and 14.9% respectively, but at postnatal 28th day(14 days after eyes opened), the mature cells and immature cells were 62.5% and 12.5% respectively. Conclusion During the period of postnatal development, the visual cortical neurons become mature gradually, but maturation is not completely correlated with age.

Objective By using cardiac ischemia reperfusion model, the muscarinic receptor path way of cardiac signal conduction in both young and aged myocardium was studied. The aim was to study the pathogenesis of ischemia reperfusion injury in aged myocardium. Methods 20 dogs (15～20 kg) were randomly divided into two groups: group A (young, 3～4 years old, n=10), group B (old, 7～8 years old, n=10). All dogs underwent 60 minutes of global ischemia and 30 minutes of reperfusion under cardiopulmonary bypass (CPB). A tunnel was inserted through the left ventricular apex, to measure the LVEDP、+dp/dt、-dp/dt and ECG. . Myocardial specimens were collected at the beginning of reperfusion, 30 min after reperfusion and 60 min after reperfusion. Myocardium was prepared or studied of density of muscarinic receptor, G protein level, cGMP, guanylate cyclase activity, value of NO, protein level of eNOS, mRNA level of iNOS and eNOS. Results (1) Group B had higher LVEDP but lower +dp/dt max and -dp/dt max than group A in each stage. After CPB, the LVEDP showed no difference between pre and post surgery in two groups, while +dp/dt max and -dp/dt max were decreased in both groups, more significantly in group B. (2) At 30 min and 60 min after reperfusion, the density of M receptor was significantly increased in group B than that in group A. (3) There was no significant difference in Gs pre and post surgery in group A. Compared with group B, the level of Gi decreased significantly after the reperfusion in group A. There were decreasing in Gs and Gi after reperfusion in group B, more greater in the former. The ratio of Gi/Gs in group B was much higher than that in group A. (4) Compared with group A, the mRNA level of eNOS and protein level of eNOS decreased more significantly in group B during each stage of reperfusion. After ischemia reperfusion, the mRNA level of iNOS, NO, cGMP and guanylate cyclase activity were obviously increased in group B than that in group A.Conclusion (1) After the cardiac surgery in elder model, the M receptor path way increased significantly, which is negatively correlated with cardiac function, hence promote the ischemia reperfusion injury in elder myocardium. (2) In the ischemia reperfusion injury in elder myocardium, higher vagotonic does more harm to the old myocardium through NO and NO cGMP pathway.

Objective:To establish a rapid method to detect drug-resistance genotypes of extended spectrum-β-Lactamases (ESBLs) produced by gram negative bacillus using the real -time fluorescence quantitative PCR. Methods: According to clinical common genotypes of ESBLs, SHV, TEM.CTX-M.OXA and their homology, 9 pairs of specific primers were designed including SHV, TEM, CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, OXA-1, OXA-2 and OXA-10. To extract DNA template by boiling assay, and then establish and grade up SYBR GREEN I real-time fluorescence quantitative PCR reaction system, finally definite real-time fluorescence quantitative PCR method. Its precision and range of linearity were tested. With established assay 51 multi- drug resistant ESBLs- E. coli K. pneumoniae were detected and compared with improved three dimensional extract tests. Results: Except OXA-2, 8 genotypes SHV, TEM, CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, OXA-1 and OXA-10 were amplified by quantitative PCR from 39 ESBLs+ and 51 multi-drug resistant ESBLs-E. coli K. pneumoniae and confirmed by sequence testing. The range of linearity was 3×10~3-3×10~8 copies/mL, r =-0.994 7. Repetitive experiments showed that the average coefficient of variation between -runs was 9.6%. Comparing with three dimensional extract test, there was no significant difference (χ2 = 1.125,P> 0.05). Conclusion: Testing drug-resistance genotypes of ESBLs with SYBR GREEN I real-time fluorescence quantitative PCR is a rapid,specific and sensitive method, which is capable of inspecting genotypes of ESBLs from clinical strains.

Objective This study evaluated the effects of combined procedure of surgical treatment and cell transplantation on dilated cardiomyopathy. Methods Eight patients (5 men and 3 women) with moderate to severe mitral regurgitation from end-stage dilated cardiomyopathy underwent surgery. Four patients were in functional class (FC) Ⅳ, six were in FC Ⅲ. There age ranged from 15 to 56 years. The preoperative ejection fraction (EF) ranged from 0.15 to 0.32 (mean 0.26 ± 0.08). Mitral valve replacement was performed in 5 patients and mitral valve repair in 3. Auto-bone marrow monenuclear cells were harvested, isolated, washed, and resuspended for direct injection after surgical procedure. Results All patients survived and were discharged from the hospital. After a mean follow-up period of 18 months ( 12 - 42 months). Echocardiography showed postoperative ejection fraction and wall movement velocity increased after 6 months. Radionuclide ventriculography showed myocardial perfusion improved significantly. Conclusion Combined procedure of surgical treatment and cell transplantation led to significant improvement in cardiac function in patients with dilated cardiomyopathy.

BACKGROUND:Side population(SP)cells,with varied contents,are widely distributed in adult tissues,embryos,and certain tumor cells.Haematological tumor is one of the main pathological conditions,which endangers human life.Thus,it is important to recognize SP cells in haematological tumor cell lines.OBJECTIVE:To identify whether hematologic tumor cell lines contain SP cells,and to explore a stable detection and separation methods.METHODS:Cells with the characteristics of stem cells being capable of fluorescent dye Hoechst33342 were isolated by flow cytometry;whether there were SP cells in logarithmic growth period of NB4,Raji,K562/ADM and K562 or not were detected.After sorting K562 subpopulations,the purity of sorted cells was detected.RESULTS AND CONCLUSION:After Hoechst33342 staining,flow cytometry results showed that the SP cells appeared in the haematological tumor NB4,Raji,K562/ADM and K562 cell lines,which accounted for 0.8%,2.7%,1.3% and 2.7%,respectively.These cells could be blocked by Verapamil.The purity was greater after a second detection of SP and Non SP cells in K562 cells.