We present a minimal invasive technique for the treatment of broncho-pleural fistula (BPF) after pulmonary lobectomy with lung cancer. 2 cases of BPF were found at the 4th and 7th day after pulmonary lobectomy respectively. They were reoperated on by VATS, direct resuture of stump and consolidation with acrylic or fibrin glue. Both cases were cured. Authors consider mentioned procedure might be a feasible therapeutic approach for early broncho-pleural fistula.

Objective To study the clinical application of video-assisted thoracoscopic surgery (VATS). Methods Clinical records of 78 cases of VATS from July 1998 to December 2002 were retrospectively analyzed. There were 40 cases of bullectomy for spontaneous pneumothorax, 12 cases of surgical exploration for chest trauma, 9 cases of wedge resection for solitary pulmonary nodules, 6 cases of pleural biopsy combined with pleurodesis, 4 cases of resection for mediastinal tumor, 4 cases of pulmonary lobectomy, 2 cases of broncho-pleural fistula following lobectomy and 1 case of resection for esophageal leiomyoma. Results A conversion to open surgery was required in no cases, but a supplementary mini incision was needed in 5 cases because of the adhesion of pleural cupula. The intrathoracic drain was all removed within 48 postoperative hours with exception of 3 middle-old aged patients with spontaneous pneumothorax in whom the continuous air leakage was observed and the drain was removed on 7, 8 and 13 postoperative days, respectively. The delayed healing of drain site was seen in 5 cases. The incidence of operative complications was 10 3% (8/78). Conclusions Broad prospects exists in the clinical application of VATS, but the costs of the disposable is subject to reduce. The application of suture or knotting under thoracoscope may save medical costs.

Objective To establish the cell model of restenosis in vitro and investigate the effects of fenofibrate on the prevention and treatment of the restenosis.Methods Human umbilical vein endothelial cells(HUVECs) were cultured in vitro.The study was designated to 5 groups:(1) control group,(2) LPC group,(3) low-concentration fenofibrate(10 ?mol/L) group,(4) midium-concentration fenofibrate(50 ?mol/L) group,and(5) high-concentration fenofibrate(100 ?mol/L) group.Proliferation and apoptosis of HUVECs were assessed by MTT assay,flow cytometry(FCM) and fluorescence microscopy respectively.Results Compared with the control group,LPC could inhibit the growth and induce apoptosis of HUVECs as well as decrease NO production in HUVECs.Fenofibrate could increase the proliferation and decrease the apoptosis of HUVECs,and also enhance NO production of HUVECs.Conclusion Fenofibrate could improve proliferation and reduce apoptosis and enhance NO production of HUVECs through lysophosphatidylcholine,which may be an important route for fenofibrate to prevent and treat the restenosis after percutaneous coronary intervention.

Objective To compare the effect of insulin pump continuous subcutaneous insulin (CSII) and multiple subcutaneous insulin (short-acting insulin before meals + glargine,MSII) for the short duration of type 2 diabetes mellitus (T2DM) patients.Methods Fifty-two newly diagnosed T2DM patients were randomly divided into CSII(n =29) and MSII(n =23) group.Patients in CSII group were given insulin pump continuous subcutaneous plus metformin.And patients in MSII group were given insulin by multiple subcutaneous insulin injection plus metformin.The treating periods was 2 weeks and followed up one month.Results The periods from point of insulin injection to blood glucose back to normal level in CSII group was (4.70 ±2.01) d,shorter than that in MSII group(6.90 ± 1.50) d,and the difference was significant(t =2.056,P ＜0.05).The levels of C peptide in two hours postprandial before and after treatment in CSII group were (4.24 ± 0.25) ng/L,(6.29 ± 0.56) ng/L,and (3.20 ±0.11) ng/L and (7.33 ±0.41) ng/L respectively in MSII group.The levels of C peptide in two hours postprandial after treatment were higher than that of before treatment in two groups (t =2.018,2.436 respectively,P ＜0.05),but there were no significant differences between two groups(t =0.985,P ＞ 0.05).Nineteen cases (65.5％) in CSII group were off insulin treatment in one month,and 9 cases (39.1％) in MSII group.There were significant differences in two groups(x2 =5.11,P ＜0.05).Conclusion The two treatment plans can make the improvement in terms of glucose control.However,CSII plan showed more effective than MSII.

To screen potential human soluble protein tyrosine phosphatase 1B (PTP1B) inhibitors, a high-throughput screening (HTS) model in 384-well microplate with total volume of 50 microL was established. Recombinant PTP1B was cloned and expressed in E. coli. with its specific substrate 4-nitrophenyl phosphate disodium salt hexahydrate (PNPP). The HTS model was based on enzyme reaction rate with enhanced sensitivity and specificity (Z' = 0.78). A total of 24,240 samples were screened, among them 80 samples with inhibition greater than 70% were selected for further rescreening. Finally, six compounds with high inhibitory activity were identified, whose IC50 values were 21.58, 18.39, 15.37, 11.92, 37.27, and 36.61 microg x mL(-1), separately. The results indicated that the method was stable, sensitive, reproducible and also suitable for high-throughput screening.

ObjectiveTo study the effect of bone mesenchymal stem cells (BMSC) transplantation into traumatic brain injury(TBI) rats by the external carotid artery on neurological function and learning and memory.MethodsTen adult SD rats were randomly divided into TBI group ( n =5 ) and BMSC transplantation group ( n=5).Feeney free falling method was used to establish TBI models.The experimental rats were administrated with BMSC via external carotid artery (ECA),while TBI rats were injected with sterile liquid medium of equal volume via right ECA.Neurological function were evaluated according to the modified neurological severity score (NSS) at 1,3,7,15 days.Morris water maze test was used to observe the animal capabilities of place navigation and space exploration at 15 days,then animals were sacrificed.Survival and migration of implanted BMSC in brains under fluorescence microscope. ResultAfter traumatic brain,varying degrees convulsions,paralysis,loss of balance function in rats were found.Compared with TBI group,BMSC transplantation decreased significantly NSS (P ＜0.01 ).BMSC transplantation significantly decreased on escape latency ( ( 20.48 ± 2.29 ) s ) than the TBI group ( ( 85.93 ± 47.48 ) s) (P ＜ 0.01 ).Moreover,BMSC group in the target quadrant dwell time ( ( 28.62 ± 1.72) ％ )and distance ( (29.05 ± 3.08 )％ ) as well as the number of passing the platform (8.00 ± 2.45 ) were significantly higher than the TBI group ( ( 19.37 ± 2.81 ) ％,(21.78 ± 3.06) ％,(2.00 ± 1.87) respectively,P ＜ 0.01 ).Transplanted BMSC could survive and migrate around injury brain through Hochest mark immunofluorescence.ConclusionBMSC can survive and migrate around injury brain by transplantation of external carotid artery,which results in a significant neurological function improvement and learning and memory increase in rats with traumatic brain injury.

Objective To determine the effect of ghrelin on protecting the human umbilical vein endothelial cells (HUVEC) from injury by angiotesin Ⅱ(AngⅡ) in vitro. Methods (1)HUVEC was incubated for 24 h with AngⅡ whose final concentration in the medium varied from 10-9 to 10-6mol/L or pretreated with 10-9to 10-6mol/L ghrelin for 2 h before incubation for 24 h with AngⅡwhose final concentration in the medium was 10-6mol/L. HUVECs were harvested to measure the cell vitality and cell apoptosis. The cell vitality was determined by MTT and cell apoptosis rates were measured by Annexin V-FITC apoptosis detection kit. (2)HUVECs were incubated for 3,6,12,or 24 h with 10-9,10-8,10-7,or 10-6mol/L AngⅡ, respectively. Before HUVECs were incubated with 10-6 mol/L AngⅡfor 24 h, ghrelin (10-9,10-8,10-7, and 10-6 mol/L) was used to pretreat the cells for 2 h. Growth hormone secregogue receptor 1a blocker [D-Lys3]GHRP-6 was added to the cells which were incubated for 24 h with 10-6mol/L AngⅡ and pretreated with 10-6 mol/L ghrelin for 2 h. Cell reactive oxygen species were measured by dichlorofluorescin (DCF) fluorescence probe method. (3)HUVECs were incubated for 24 h with 10-9,10-8,10-7, or 10-6mol/L AngⅡ and ghrelin, respectively,and then were incubated with 10-6mol/L of AngⅡ for 3,6,12,or 24 h. Furthermore,HUVECs were pretreated with 10-9,10-8,10-7, or 10-6mol/L ghrelin for 30 min,1 h,or 2 h, and then were incubated with the inhibitor of mitogen-activated protein kinase /extracellular regulated kinase (MAPK/ERK1/2),PD98059, the inhibitor of phosphoinositide-3-kinase/serine threonine kinase( PI3K/Akt)wortmannin, and [D-Lys3]GHRP-6 for 24 h. NO production was compared among groups. HUVECs were pretreated with ghrelin,PD98059, wortmannin, and [D-Lys3]GHRP-6 for 2 h and co-cultured with 10-6mol/L AngⅡfor 24 h,or pretreated with ghrelin plus PD98059, wortmannin, and [D-Lys3]GHRP-6 before incubation with AngⅡfor 24 h. NO was measured in the endothelial cell supernatants by Griess method. (4)HUVECs were cultivated with blank or AngⅡwith or without pretreatment with ghrelin or both ghrelin and wortmannin. The protein expression of eNOS and phospho-protein expression of Akt were measured by Western blot analysis. Results AngⅡinjuried the endothelial cell vitality,increased the cell apoptosis rates in HUVECs, and decreased NO production in HUVEC supernatants,whereas ghrelin protected HUVECs from AngⅡ injury. Ghrelin decreased the reactive oxygen species in HUVECs induced by AngⅡ. The effect could be attenuated by [D-Lys3]GHRP-6 pretreatment; PD98059 alleviated AngⅡ inhibition of NO production in HUVEC supernatants. Wortmannin and [D-Lys3]GHRP-6 could abolish protection of ghrelin from reducing NO production in HUVEC supernatants. AngⅡreduced the expression of eNOS,but ghrelin increased eNOS expression. Wortmannin could cancel this effect of ghrelin. Ghrelin increased p-Akt expression and reached the peak in 10 and 20 min. Conclusion Ghrelin may protect HUVECs from AngⅡinduced injury, which is related to decreasing oxidative stress, increasing the protein expression of eNOS, and activating PI3K/Akt signal pathway through GHSR1a receptor.

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.
Cell Line, Tumor ; Exosomes ; ultrastructure ; Humans ; Laryngeal Neoplasms ; pathology ; Microscopy, Electron, Transmission

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.

[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.