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Objective To establish the A549 cell line with stable expression of HIF1α by using lentiviral vector system.Methods Primers were designed and synthesized with human HIF1α gene coding sequence by the National Center of Biotechnogical Information(NCBI) as the template.HIF1α was amplified by PCR.The HIF1α fragment recycled by enzyme digestion was recombined with prepared lentiviral vector HBLV-RFP-Puro.The recombinant plasmid was identified by PCR and gene sequencing.The recombinant plasmid and the auxiliary plasmid were co-transfect into 293T cell.After filtration and concentration of packaged virus,the viral titer was detected by using the dilution counting method.The prepared lentivirus was infected A549 cells.The drug screening was adopted to stabilize the transfected cell line.The transfection effect was detected and observed by fluorescence microscope and Western blotting.Results The HIF1α fragment amplified by PCR was successfully verified and the recombinant plasmid was successfully constructed by PCR and gene sequencing identification.High-titer LV-HIF1α was obtained by successful package.After LV-HIF1α infecting A549 cells,the cells showed the red fluorescence by fluorescence microscope.The expression level of HIF1α in the LV-HIF1α group was significant higher than that in the control group by Western blot.Conclusion The 549 cell line with HIF1α stable expression mediated by lentivirus is constructed successfully.
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Objective to construct the high titers rat Hes1 adenovirus expression vector (Ad-Hes1).Methods With the rat cDNA as a template,the Hes1 fragment was amplified by PCR,which constructed pShuttle-CMV-Hes1 shuttle plasmid by directly clone.Based on pShuttle-CMV-Hes1,pAdeno-Hes1 virus plasmid was constructed,pAdeno-Hes1 was transfected into 293 cells to package Ad-Hes1,virus titers were determined by modified TCID50.Hes1 was detected by Western blot after Ad-Hes1 infected with H9c2 myocardial cells.Results pShuttle-CMV-Hes1 shuttle plasmid and pAdeno-Hes1 plasmid were constructed successfully,with a general titer of 1.6 × 1011 PFU,Ad-Hes1 can be expressed in H9c2 myocardial cells,and its MOI value was 30.Conclusion Ad-Hes1 is successfully constructedand packaged,thus provide basis for further research on the protection effect of Hes1 on myocardium.
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BACKGROUND: Gentamicin bead chain is an effective drug delivery system for treatment of osteomyelitis, but it cannot be degraded, need to be removed by second operation, and can breed pathogens. As a result, biodegradable drug delivery systems become a hotspot. Nano-hydroxyapatite/poly(β-hydroxybutyrate-co-β-hydroxyvalerate)-polyethylene glycol-gentamicin (nano-HA/PHBV-PEG-GM-DDS) is considered to be a good choice for the current predicament. OBJECTIVE: To evaluate the acute or chronic toxic reactions of the whole body and local tissues, intracutaneous stimulation, cytotoxicity and hemolytic reactions after bone remodeling and implantation of nano-HA/PHBV-PEG-GM-DDS, thus providing a new kind of material for treating osteomyelitis. METHODS: Plastic nano-HA/PHBV-PEG-GM-DDS was prepared using plastic fibrin glue as microsphere scaffold and nano-HA as the core carrier of GM that was coated with PHBV and PEG. The acute, subacute/chronic toxicity, implantation, hemolysis, cytotoxicity and intracutaneous stimulation tests were performed according to the evaluated criteria of medical implanted materials as wel as biological and animal trials recommended in GB/T16886.1-1997. RESULTS AND CONCLUSION: The plastic nano-HA/PHBV-PEG-GM-DDS was nontoxic and caused no apparent changes in liver and kidney function and serum biochemical indexes. Pathological examination showed that the implanted material was covered with tissues, and inflammation changes accorded with the general regularity of inflammatory outcomes. After implantation, the nano-HA/PHBV-PEG-GM-DDS was biodegraded and replaced by osseous tissues. The hemolytic rate of the material extract to the composite diffusion solution was 1.2%, which was below the standard criteria (5%). Human bone marrow cells cultured in vitro with the plastic nano-HA/PHBV-PEG-GM-DDS grew normally with good morphology. There was no stimulation reaction according to the criteria after the diffusion solution was subcutaneously injected into the back of the animal. These findings indicate that the plastic nano-HA/PHBV-PEG-GM-DDS for treating osteomyelitis possesses excel ent biocompatibility and security.
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<p><b>OBJECTIVE</b>To construct rat N1ICD lentiviral over-expression vector (LV-N1ICD) and N1ICD lentivirus interference vector (LV-N1ICD-shRNA.</p><p><b>METHODS</b>With the rat cDNA as a template, the N1ICD fragment was amplified by PCR to construct pGC-FU-N1ICD-3Flag shuttle plasmid by directly clone. Four pairs of N1ICD-shRNA oligonucleotide sequences were syn- thesized to construct the GVC112-N1ICD-shRNA interference plasmid. pGC-FU-N1ICD-3Flag and GVC112 -N1ICD-shRNA plasmids were co-transfected into 293T cells to screen for the best interference plasmid in the 4 GVC112-N1ICD-shRNA plasmids by detecting Flag expression. pGC-FU-N1ICD-3Flag or GVC112-N1ICD-shRNA plasmid along with with pHelper 1.0 and pHelper 2.0 plasmids were co-transfect into 293T cells to package LV-N1ICD and LV-N1ICD-shRNA, and the virus titer was determined by real-time PCR and drug screening method, respectively. H9c2 cells infected with LV-N1ICD and LV- N1ICD-shRNA respectively were assessed for cell viability using CCK-8 assay.</p><p><b>RESULTS</b>pGC-FU-N1ICD-3Flag and GVC112- N1ICD-shRNA plasmid were verified by PCR, gene sequencing and Western blotting. Co-transfection of the plasmids with pHelper 1.0, and pHelper 2.0 plasmids into 293T cells obtained high-titer LV-N1ICD and LV-N1ICD-shRNA. LV-N1ICD was capable of promoting the cell viability and LV-N1ICD-shRNA produced an opposite effect.</p><p><b>CONCLUSION</b>The vectors LV-N1ICD and LV-N1ICD-shRNA have been successfully constructed and packaged, which have the biological functions of Notch1 signaling.</p>
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Animals , Rats , Genetic Vectors , Lentivirus , Plasmids , RNA, Small Interfering , Real-Time Polymerase Chain Reaction , TransfectionABSTRACT
BACKGROUND:Ligamentum flavum hypertrophy is one of the most important factors of lumbar spinal stenosis, but the molecular mechanism is stil not very clear. OBJECTIVE:To explore the role of basic fibroblast growth factor, connective tissue growth factor and transforming growth factor β1 in hypertrophy of the lumbar ligamentum flavum. METHODS: The ligamentum flavum samples were divided into three groups according to different diseases: control group (acquired from the patients with lumbar spinal canal tumor,n=6), lumbar disc herniation (LDH) group (acquired from the patients with LDH,n=6) and lumbar spinal stenosis (LSS) group (acquired from the patients with LSS,n=6). Then the mRNA expressions of basic fibroblast growth factor, connective tissue growth factor, transforming growth factor β1 and colagen I, III, V of the ligamentum flavum were detected using real-time quantitative RT-PCR method. The roles of basic fibroblast growth factor, connective tissue growth factor and transforming growth factor β1 were explored. RESULTS AND CONCLUSION:The expression of basic fibroblast growth factor mRNA in the LSS group was significantly higher than that in the LDH and control groups (bothP 0.05); the expression of transforming growth factor β1 mRNA was significantly higher in the LSS group than in the LDH and control groups (bothP 0.05). This study indicate that both basic fibroblast growth factor and transforming growth factor β1 play important roles in the formation process of the lumbar ligamentum flavum hypertrophy, and the main type of the colagen in the hypertrophied ligamentum flavum is colagen I.
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Ras is best known for its ability to regulate cell growth, proliferation and differentiation. Mutations in Ras are associated with the abnormal cell proliferation which can result in incidence of all human cancers. Extracellular signal-regulated kinase (ERK) is a downstream effector of Ras and plays important roles in prognosis of tumors. Recently, evidence has gradually accumulated to demonstrate that there are other effectors between Ras and ERK, these proteins interact each other and constitute the thorough Ras/Raf/MEK/ERK signaling pathway. The pathway has profound effects on incidence of esophageal carcinoma and clinical applications of some chemotherapeutic drugs targeting the pathway. Further understanding of the relevant molecular mechanisms of Ras/Raf/MEK/ERK signaling pathway can be helpful for the development of efficient targeting therapeutic approaches which contribute to the treatment of esophageal cancer. In this article, roles of Ras/Raf/MEK/ERK signaling pathway in esophageal carcinoma as well as pharmacological targeting point in the pathway are reviewed.
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Objective To promote comprehension of pulmonary sequestration by investigating the diagnosis and surgical treatment methods of this disease.Methods A retrospective analysis of the clinical records of 27 patients with pulmonary sequestration was made,15 male patients and 12 female patients,with the age ranging from 4 to 56 years old.The patients were examined by chest radiography,computerized tomography (CT),computerized tomography angiography (CTA),magnetic resonance imaging (MRI) before the surgery.Eleven patients were confirmed preoperatively as pulmonary sequestration by the contrast enhanced CT and 64slice spiral CT examinations.Sequestrectomy was performed on patients with extralobar sequest ration (ELS) and lobectomy was performed on patients with intralobar sequestration (ILS).There were 21 cases of left lower lobectomy,5 cases of right lowerlobectomy,and 1 case of right sequestrectomy.Results All recovered uneventfully.No significant complications occurred in the perioperative period.All patients were cured and discharged from hospital.No relapse was reported during follow up period.Conclusion Diagnosis mainly depended on the chest radiography,CT,MRI and CTA.Surgical operation should be the best choice of treatment.
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Objective To determine the effectivity of 5-azacytidine(5-Aza) inducing the cardiomyogenic SCs were isolated and purified from Sprague-Dawley (SD) rats. Some BMSCs were induced with 5-Aza,group Ⅰ was not induced with 5-Aza and the third generation of cells were used for the next experiment; group Ⅱ were cultured and induced with 5-Aza( 10 μmol/L) at the third day of the culture for 24 h; group Ⅲ were induced with 5-Aza( 10 μmol/L) for 24 h when the third generation of cell were fused; group Ⅳ were induced with 5-Aza( 10 μmol/L) for 24h at the third day of primary cuhure,which were induced with 5-Aza( 10 μmol/L) for 24 h before the third gener-phosis was found in the BMSCs among groups. CD34 was negatively expressed and CD29 and CD44 were positively gle 5-Aza inducing group(group Ⅲ ) was much higher than that in no 5-Aza inducing group( group Ⅰ ) [ MHC : (6. 82±2.05 ) % vs (3.61±1.14) % ], cTnI: (5.63±1.86) % vs (2.76±0.82) %, P < 0.05 ], but was significantly lower than the percentage in the double 5-Aza inducing group ( group Ⅳ ) [ MHC : ( 12.18±3.16) % vs ( 6.82±2. 05)% ] ,cTni:(9.93±2.79)% vs(5.63±1.86)% ,P<0.05]. Conclusions 5-Aza could promote the cardio-myogenic differentiation of BMSCs in a "Cardiac-like" Milieu. 5-Aza double induing is better than the single indu-cing.
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Three cases of terminal stage heart disease received orthotopic heart transplantation at the Department of Cardiothoracic Surgery, the First Hospital of Nanchang University between August 2001 and December 2003. These three cases were all female and died of brain death. Body weight difference between donor and recipient was less than 20%. All three cases underwent superior and inferior vena cave osculation and received immunosuppressive therapy of cyclosporine A, prednisone, and mycophenolate postoperatively. They were successfully discarded. Heart function recovered to grade Ⅰ-Ⅱ(NYHA). No infection or serious rejection was found during the surgery. These results indicate that good donor heart preservation, consummate perioperative processing, and proper immunosuppressiva therapy are the key measures of successful heart transplantation.
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0.05). Conclusions One mechanism of SF pretreatment cardioprotective effect is mediated by bradykinin. The combined use of SF and CP doesn′t result in significant improvement, and thenefore is not advocated.
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Aim To study the protective effects of pharmacological preconditioning induced by sodiumferulate(SF)on isolated rat heart and its machanisms.Methods Isolated SD rat hearts were divided randomly into 5 groups : Control group (hearts were perfused by oxygenated perfusate for 100 minutes); Ischemia/Reperfusion(I/R) group (hearts suffered from 40 min global ischemia/30 min reperfusion after oxygenated perfusate for 30 minutes); Ischemia preconditioning group(hearts were preconditioned by 3 periods of 5-minute global ischemia/5-minute reperfusion before it suffered from I/R); SF group (previously hearts were perfused with oxygenated perfusate administered with 1.69 mmol?L-1 SF then subjected to I/R); Glibenclamide group (previously hearts were perfused with oxygenated perfusate administered with 1.69 mmol?L-1SF and 30 ?mol?L-1 Glibenclamide before it suffered from I/R). Results Compared with I/R group, 1.69 mmol?L-1 SF precondition improved significantly heart function, reduced the incidence and severity of ventricular arrhythmias, alleviated calcium overload in myocardial cell; furthered activities of Na+,K-ATPase and Ca2+-ATPase in myocardium. These effects were attenuated by 30 ?mol?L-1 Glibenclamide.Conclusions SF precondition can improve heart function and resist arrhythmia and Ca2+-overload. The cardioprotective effects of SF precondition maybe related with the opening of ATP-sensitive potassium (KATP) channels.