Comparison of methods for isolating exosomes derived from laryngocarcinoma Hep-2 cells / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To provide a reference about choosing the methods of isolating exosomes derived from tumor cells including laryngocarcinima Hep-2 cells by comparing advantages and defects of two methods of isolation and extraction exosomes.@*METHOD@#Previously, laryngocarcinoma Hep-2 cells were cultivated massively, then the cells were processed with hot shock in 42 degrees C for 1 h. Sucrose density gradient centrifugation ultrafiltration (method 1): cells culture supernatant 90 ml was gathered, the supernatant was clarified through a 3/0.8 μm small filter to remove impurities and fragments which in larger diameter. Then the filtering fluid was concentrated and purified through sucrose density gradient centrifugation and ultrafiltration, the concentrated fluid was obtained. Exosome Isolation Kit (method 2): cells culture supernatant 4 ml was gathered, the solutions of the kit were added into the supernatant in proper sequence, then filtered by the special column, the concentrated fluid was obtained. Both products are observed by high resolution transmission electron microscopy.@*RESULT@#Both methods could isolate and extract exosomes feasibly. In single high power view of transmission electron microscopy, exosomes of method 1 disperse better, but lower density, and more impurity in background, exosomes of method 2 arrange closer, higher density, and less impurity.@*CONCLUSION@#Exosome isolation Kit require less supernatant, cost less time, process procedure briefly, harvest higher yield. It may become a new option of isolating exosomes derived from Laryngocarcinoma Hep-2 cells.

Expressions of tumor angiogenesis related factors CD105 and Galectin-3 in laryngeal squamous cell carcinoma and biological assessment / 中国耳鼻咽喉头颈外科

[ABSTRACT]OBJECTIVETo investigate the expressions of Endoglin (CD105) and Galectin-3 protein in laryngeal squamous cell carcinoma (LSCC) as well as the relationship between their expressions and the clinicopathological factors of LSCC.METHODSThe expressions of CD105 and Galectin-3 protein were detected in 76 samples of LSCC and 25 normal laryngeal tissues (NLT) by immunohistochemical staining (S-P).RESULTS 1.The mean of Microvessel density (MVD) value marked by CD105 in LSCC was 10.33±2.29, which was significantly higher than that in NLT (1.20±1.04) (P<0.05). The expression of MVD marked by CD105 (CD105-MVD) was correlated with histological grading, T stage, clinical stage, lymph node metastasis, recurrence and prognosis in LSCC (P<0.05). 2.The positive expression rate of Galectin-3 protein in LSCC was 86.84%, which was significantly higher than that in NLT (36%)(P<0.05). The expression of Galectin-3 was correlated with T stage,clinical stage, lymph node metastasis, recurrence and prognosis in LSCC (P<0.05). 3.There was a positive correlation between CD105 and Galectin-3 protein. 4.Survival analysis indicated that the expressions of CD105 and Galectin-3, histological grading, lymph node metastasis,T stage and recurrence were independent factors for tumor prognosis in LSCC (P<0.05). CONCLUSIONThe expressions of CD105 and Galectin-3 protein have a positive correlation in LSCC. They may play important roles in the tumorigenesis, malignant progression and poor prognosis of LSCC. Combined detection of them may be great value in diagnosis and predicting prognosis of LSCC.

Mechanisms of pulmonary embolism and/or deep vein thrombosis secondary to chronic obstructive pulmonary disease exacerbation in elders / 中华全科医师杂志

Objective To explore the inflammatory mechanisms of pulmonary embolism ( PTE ) and/or deep venous thrombosis ( DVT ) in elders secondary to chronic obstructive pulmonary disease ( COPD) exacerbation.Methods A total of 26 elders with acute exacerbation of high-risk COPD secondary PTE and/or DVT and 26 patients with low-risk COPD during stable phase diagnosed during the period of January 2008 to December 2012 were enrolled.The relevant parameters of routine blood examination , blood viscosity, D-dimer, fibrinogen ( FIB), arterial blood gas, blood cytokine, erythrocyte sedimentation rate ( ESR ) and C-reactive protein ( CRP ) were retrospectively analyzed.Results The major nonspecific symptoms were cough, sputum and dyspnea.The mean of neutrophile percentage (N%), D-dimer, FIB, interleukin-6 (IL-6), tumor necrosis factor (TNF), C-reactive protein (CRP), low and high shear blood viscosity in blood samples of patients with acute exacerbation of high-risk COPD secondary PTE and ( or ) DVT were higher than those of the control group ( t =3.339, 2.700, 2.207, 2.431, 2.257, 2.143, 2.223, 2.797, all P<0.05).However arterial partial pressure of oxygen ( PaO2 ) was lower than that of lower-risk COPD patients (t=4.312, P<0.05).IL-6 in blood of patients with acute exacerbation of high-risk COPD secondary PTE and ( or) DVT was positively correlated with low-shear blood viscosity , D-dimer and FIB (r=0.437, 0.624, 0.429, all P<0.05).TNF in blood of patients with acute exacerbation of high-risk COPD secondary PTE and ( or ) DVT was positively correlated to FIB , low and high cut blood viscosity ( r =0.624, 0.519, 0.513, all P <0.05 ).Plasma CRP in blood of patients with acute exacerbation of high-risk COPD secondary PTE and/or DVT was positively correlated with D-dimer, FIB, IL-6 and TNF ( r=0.478, 0.541, 0.533, 0.491, all P<0.05).Conclusions Inflammation may exist in elders with acute exacerbation of high-risk COPD secondary thrombotic disease.IL-6 and TNF may promote thrombosis secondary to acute exacerbation of COPD disease.Early screening and/or prophylactic anticoagulation are necessary for prevention.

Melanoma antigen A3 sourced from laryngeal cancer was expressed in mouse melanoma cell model / 中国免疫学杂志

Objective:To construct tumor cell model by determination of the pIRES2-EGFP/MAGE-A3 eukaryotic expression plasmid expressing steadily in mouse melanoma B16 cells.Methods:The pIRES2-EGFP/MAGE-A3 eukaryotic expression plasmid being constructed from the melanoma-associated antigen A3 genes sourcing laryngocarcinoma in advance was translated into the mouse melanoma B16 cells under the mediation of lipofectamine,and the positive clones were detected with G418.The expression of enhanced green fluorescent protein( EGFP) and MAGE-A3 mRNA in positive clones were detected by fluorescence microscopy and fluorescence quantitative PCR ( qRT-PCR ) assay, respectively.Results:The pIRES2-EGFP/MAGE-A3 eukaryotic expression plasmid has been transfected into B16 cells successfully, the green fluorescence of fusion protein expression was found, and MAGE-A3 mRNA transcription in B16 cells expressions were detected in positive clones.Conclusion:The pIRES2-EGFP/MAGE-A3 eukaryotic expression plasmid has been transfected effectively and expressed stably by liposome method in the B16 cells.The expression of MAGE-A3 tumor cell model has been successfully established,which provide data for the study of laryngocarcinoma immunotherapy.

Construction of the superantigen SEA transfected laryngocarcinoma cells / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To construct an eukaryotic expression vectors containing superantigen staphylococcal enterotoxin A (SEA) gene, and to identify its expression in laryngeal squamous carcinoma cells.@*METHOD@#SEA full-length gene fragment was obtained from ATCC13565 genome of the staphylococcus, referencing standard strains producing SEA. Coding sequence of SEA was artificially synthetized. Than, SEA gene fragments was subcloned into eukaryotic expression vector pIRES-EGFP. The recombinant plasmid pSEA-IRES-EGFP was constructed and was transfected to laryngocarcinoma Hep-2 cells. Resistant clones were screened by G418. The expression of SEA in laryngocarcinoma cells was identified with ELISA and RT-PCR method.@*RESULT@#The subclone of artificially synthetized SEA gene was subclone to eukaryotic expression vector pires-EGFP. Flanking sequence confirmed that SEA sequence was fully identical to the coding sequence of standard staphylococcus strains ATCC13565 in Genbank. After recombinant plasmid transfected to laryngocarcinoma cells, the resistant clones was obtained after screening for two weeks. The clones were selected. The specific gene fragment was obtained by RT-PCR amplification. ELISA assay confirmed that the content of SEA protein in supernatant fluid of cell culture had reached about Pg level.@*CONCLUSION@#The recombinant eukaryotic expression vector containing superantigen SEA gene is successfully constructed, and is capable of effective expression and continued secretion of SEA protein in laryngochrcinoma Hep-2 cells after recombinant plasmid transfected to laryngocarcinoma cells.

Establish and evaluate allergic rhiultis model in guinea pigs with Toluene-2,4-diisocyanate / 中国医师杂志

Objective To understand the changes of allergic rhinitis from immunology,pathophysiology and morphology method were applied.Methods Toluene 2,4-diisocyanate was dissolved with florence oil to final concentration 10%,this solution was dropped into the nasal vestibules of animals to induce sensitization process.8 guinea pigs were prepared as allergic rhinitis with 10% Toluene 2,4-diisocyanate,and other 8 animals were used as blank controh.After model successfully established,the blood were obtained to determine RBC-C3b receptor rosette rate and RBC-imuuunocomplex rosette rate.The nasal mucosas were obtained from 2 groups,distribution and changes of substance P in nasal mucosa were observed with the application of immunohistochemical staining.The content of blood histamine was determined with ELASA method.The pathological changes of nasal mucosas were observed with the application of light microscope and transmission electron microscope.Results The behavior scores of the modeling animals were significantly higher than that of controls(P＜0.01).The value of RBC-C3b reeeptor rosette rate and RBC-imuuunoeomplex rosette rate of the modeling animals were significantly decreased than that of the controls(P＜0.05).Substance P presented in the normal nasal mucosal epithelial cells,epithelium cells of blood vessels,glandular cells and its duets,the staining density and the positive staining cells in the same aero in modeling group significantly increased,compared with controls.The counts of substance P-positive cells of control group were less than those of modeling group(P＜0.05).The content of blood histamine of the modeling animals were significantly increased compared with the controls(P＜0.01).The structure of false multiple coat cilium columnar epithelial cells in nasal mucosa of control group were successive,intact,and distinct.There were normal mucosal epithelium,lamina propria and submucosa.But modeling group showed that mucosal epithalamiums were damaged and shed,goblet cell proliferated,squamous metaphase,and epithelial necrosis happened,serous glands in lamina propria vigorously proliferated,blood vessels expanded,tissue edema formed,plenty of inflammatory cell such as eosinophil and mast cells were more in number and infiltrated.The structure of epithelial eells,cilia and mierotubule of nasal mucosa of control group were regular and distinct,there were abundant cellular organs in cytoplasm.Eosinophil cells were intact.But iu model group,mucosal epithalamium,cilia and its microtubule,mierovillus,goblet cell were damaged,the cell bulk and nucleus changed,mast cells and eosinophil cells changed,blood vessels expanded,serous glands vigorously proliferated.This morphological change was roughly identical to clinical manifestation of allergic rhinitis.Conclusion Toluene-2,4-diisocyanate can be used to establish allergic rhinitis model in guinea pigs,and some ehanges of the allergic rhinitis in model guinea pig were similar with clinic observation.

Measurement of components of the phospholipid of the surfactant in irrigating fluid from the nasopharynx of patients with chronic sinusitis / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To investigate the effect of chronic sinusitis on components of the phospholipid of nasopharyngeal surfactant, and to study biochemical component of phospholipid of surface active substance.@*METHOD@#The concentrations of surfactant in nasopharyngeal irrigating fluid were implemented in normal controls and patients with chronic sinusitis. Components of phospholipid such as Phosphatidylserine, Phosphatidylethanolamine, Phosphatidylcholine and Sphingophospholipid were measured by the high-performance liquid chromatograph.@*RESULT@#Results showed as follows (1) There was surfactant in nasopharynx. 4 compositions of phospholipid could be measured. (2) Compared with controls, Phosphatidylserine signficantly decreased in patients with chronic sinusitis (P < 0.05). (3) Only Phosphatidylserine signficantly decreased between sinusitis III stages and controls (P < 0.05). The rests had no signficant difference between chronic sinusitis' stages and controls, and among stages. But as the chronic sinusitis' stages proceeded, proportion of Phosphatidylserine may decreased.@*CONCLUSION@#(1) There is surfactant in nasopharynx, nasopharyngeal surfactant is made of Phosphatidylserine, Phosphatidylethanolamine, Phosphatidylcholine and Sphingophospholipid. The proportion of Phosphatidylcholine shows most, and determines biochemical effect of nasopharyngeal surfactant. (2) chronic sinusitis may cause decrease of some components of nasopharyngeal surfactant. (3) As the chronic sinusitis' stages proceed, the proportion of some phospholipids progressively decrease. Which, above assessed, may cause the change of surfactant in eustachian tube, and cause dysfunction of middle ear and eustachian tube.