OBJECTIVE: To investigate the optimizing conditions in isolation of the side population in laryngeal carcinoma cell line Hep-2. METHOD: Single-cell suspension cells were detached from the culture flask with trypsin EDTA, at a concentration of 1 x 10(6) cells/ml. (1) The trail Samples were incubated with Hoechst33342 at a concentration of 5 microg/ml, 9 microg/ml, 10 microg/ml, 11 microg/ml for 90 minutes. (2) They were incubated with Hoechst for 50, 70, 90, 110, 130 min in water bath individually. (3) The single-cell suspension were incubated Hoechst in water bath and in thermostat each. (4) The two different density of cells were harvested, which were 100% and 70%, and then di gest into single-cell suspension. Once incubation finished, suspended in phosphate buffered saline (PBS), then test SP% by flow cytometry. Among all groups,Verapamil hydrochloride was added to the control samples, incubated at 37 degrees C for 30 minutes, the other condition were keep the same with their trial groups. RESULT: (1) The percentage of Hoechst-negative cells in trial group was (39.96 +/- 0.24)%, (26.23 +/- 0.39)%. (18.79 +/- 0.02)%, (19.01 +/- 0.14)% at the concentration of 5 microg/ml, 9 microg/ml, 10 microg/ml, 11 microg/ml respectively, when the PI-positive cells were (30.45 +/- 0.63)%, (49.9 +/- 0.42)%, (50.12 +/- 0.68)%, (64.16 +/- 0.39)% separately. (2) Varying the duration of staining incubation showed that there was a typical FACS pattern and SP% was constant when the incubation was at least 90 min. (3) Compare to water bath, SP% was more than in thermostat, the SP% was (18.67 +/- 0.45)%, (22.6 +/- 0.50)% respectively; (4) Cell density is also responsible for SP%. The low density the cell is, the less in SP%. SPSS13.0 was used in statistical analysis, the groups were compared using t-Test. P < 0.05 was considered statistically significant. CONCLUSION The optimum concentration and duration of incubation of Hoechst33342 in isolation of the side population cells in laryngeal carcinoma cell line Hep-2 is 10 microg/ml and 90 min. Incubated in water bath is better than in thermostat. The best staining cell density is around 80%-90%.
Cell Count ; Cell Line, Tumor ; Flow Cytometry ; Humans ; Laryngeal Neoplasms ; pathology ; Neoplastic Stem Cells ; cytology ; Side-Population Cells ; cytology

Objective To explore a novel surgical treatment for chronic suppurative otitis media (CSOM) and evaluate its treatment effect.Methods All 97 patients with chronic suppurative otitis media were chosen to be treated using this new surgical method.The skin of the external auditory canal was maintained intact.Open radical mastoidectomy was used to complete clean-up lesions ; the fascia of pedicled temporalis myofascia (PTM) was used to repair the tympanic membrane.The pedicled temporalis fascia,pedicled postauricular periosteal flap and intact skin of the external auditory canal were used in reconstruction of the posterior wall of external auditory canal.Pure tone audiometry (PTA) was performed before and after surgery,recording the air conduction and bone conduction thresholds at 0.5 kHz,1.0 kHz,2.0 kHz,4.0 kHz.The average of the patient's air and bone conduction hearing thresholds was recorded at the 4 frequencies.External auditory canal gauze was removed 3 weeks after surgery.All subjects were followed up for over 2 years.Comparison of hearing thresholds (PTA) was made ① Before and 4 weeks after surgery.② Before and 2 years after surgery.Hearing function comparison include air conduction (AC),bone conduction (BC) and air-bone gap (ABG) analysis.SPSS 16.0 was used in statistical analysis.Pre-and postoperated AC,BC and ABG were compared using T-test.P ＜ 0.05 was considered statistically significant.Results The healing rate of post-operated tympanic membrane was 95.88％ (93/97).Ninty-six ears had 2-year follow-up,and 1 patient was lost in follow-up.There were 2 patients presented with eardrum perforation during the follow-up,and the 2-year healing rate of tympanic membrane perforation was also 93.85％ (92/97).In 96 ears with 2-year followed-up,the average of pre-AC was (52.10 ±3.96) dB,the average of post-AC was (35.67 ±2.52) dB; the average of preABG was (36.6 ± 5.2) dB,and the average of post-ABG (± SD) was (12.14 ± 6.20) dB.Statistical analysis showed significant difference between preoperative and postoperative AC or ABG values (P ＜ 0.05).Conclusion The present surgical procedure broke through the existing conventional mastoidectomy of making a surgical incision in the posterior wall of the external auditory canal.This procedure cleared the lesion completely and preserved the physiological function of the external auditory canal.The acoustic systems and state of gasification to the mastoid tymnpanum were reconstructed,rehabilitated and maintained.The healing rate of hearing and tympanic membrane perforation was improved.

AIM:To observed the variation regularity of corneal endothelial cells in patients with different diabetes duration after phacoemulsification, and investigate the effects of diabetes and its disease duration on corneal endothelial cells.
METHODS: Ninety-seven ( 135 eyes ) cataract patients with diabetes were selected randomly and divided into GroupⅠ( which diabetes duration ≥10a) and GroupII(which diabetes duration <10a) according to their disease duration. Additionally 62 (89 eyes) age-related cataract patients were randomly selected as the control group. The corneal endothelial cell density ( CD ) , proportion of hexagonal cell and coefficient of variation ( CV ) in the three group patients were measured respectively before phacoemulsification and after surgery. And the measurement results were statistically analyzed.
RESULTS:The corneal endothelial CD and proportion of hexagonal cell in the three group were decreased after surgery compared with preoperative. But the CV of corneal endothelial cells was increased on the 1 st wk and in 1st mo after surgery compared with the preoperative. The difference was statistically significant (P<0. 05). The corneal endothelial CD and proportion of hexagonal cell in the two diabetic groups were lower than the control group after surgery. However, the CV of corneal endothelial cells was higher than the control group. The difference was statistically significant ( P < 0.05 ) . There was no significant difference in the corneal endothelial CD, proportion of hexagonal cell and CV between the two diabetic groups before phacoemulsification (P>0. 05). The proportion of hexagonal cell in Group Ⅰ was lower than which in GroupIIafter surgery. While the CV was higher than which in Group II. The difference was statistically significant (P<0. 05).
CONCLUSION: Phacoemulsification has some damage on the corneal endothelial. Since the impact of diabetes on the morphology and function of corneal endothelial cell was related to the diabetic duration. So phacoemulsification has more obvious damage on the corneal endothelial in diabetic patients. And the diabetic duration was longer, the damage on the corneal endothelial in phacoemulsification was more easily.

OBJECTIVE: To explore the possibility mechanism of non-side population cells (NSP) of Hep-2 be induced into stem-like cancer cells by chemotherapy drug--cisplatin. METHOD: Hep-2 cell lines were sorted by fluorescence-actived cell sorting. The acquired NSP cells in trail group were co-cultured with cisplatin for more than 48 hours,while the control group with normal saline(NS). Then identified the percentage of the side population (SP) cells by flow cytometer. The β-catenin, notch-1 mRNA in trial and control group were detected using quantitative realtime PCR, and the β-catenin, notch-1 protein in two groups were compared by Western blot. RESULT: The percentage of side population cells in two groups were (17.16 ± 0.18)%, (10.05 ± 1.20)%, respectively. There was significant difference between two groups (t = 5.844, P < 0.01). The expression of β-catenin, notch-1 was higher in trail group by qRT-PCR; the protein levels of β- catenin, notch-1 was found to inceased in the trail group by Western blot (t = 5.155, P = 0.031; t = 5.977, P = 0.004). Statistical analysis showed significant difference between two groups (P < 0.05). CONCLUSION NSP cells can be differentiated into stem-like cancer cells after being treating with cisplatin. The supposed mechanism is maybe through wnt/β-catenin, notch signaling transduction pathway abnormalities.
Antineoplastic Agents ; pharmacology ; Cell Differentiation ; Cell Line, Tumor ; Cell Separation ; Cisplatin ; pharmacology ; Flow Cytometry ; Humans ; Laryngeal Neoplasms ; pathology ; Neoplastic Stem Cells ; drug effects ; RNA, Messenger ; Signal Transduction ; beta Catenin

Objective: To search for bioactive compounds from marine microbes. Methods: Pyricularia oryzae was used to screen microbes. Compounds extracted with EtOAc from the fermentation broth of F8712 strain(an active microbe screened out) were separated. The chemical structures of the separated compounds were identified by HNMR, CNMR and ESI-MS techniques. Results: Six cyclic dipeptides, cyclo(Ala-Leu), cyclo(Ala-Ile), cyclo(Ile-Pro), cyclo(Val-Pro), cyclo(Leu-Pro) and cyclo (Leu-Val), were obtained. Conclusion: None of the 6 cyclodipeptides has any anti-Pyricularia oryzae activities. Compound V has potent inhibitory effects on Vibrio anguillarm with a minimum inhibitory concentration of 0.07 μg/ml.

Objective: The aim of the study was to establish a high speed counter-current chromatography (HSCCC) method for the isolation and purification of alpinetin and cardamomin from Alpinia katsumadai. Methods: Two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (5 : 5 : 7 : 3) was used. The flow rate of the mobile phase was 2.0 mL/min, the revolution speed was 800 r/min, the separation temperature was controlled at 25 °C, the reservation ratio of the stationary phase was 50%, and the detection wavelength was 300 nm. Results: Alpinetin (17.2 mg) and cardamomin (25.1 mg) could be obtained from 100 mg of the crude extract in one-step separation by the method. The purities of them were 98.1% and 99.2%, respectively, as determined by HPLC and their chemical structures were identified by 1H-NMR and 13C-NMR. Conclusion: The traditional method, column elution, could not eliminate irreversible adsorption, while the HSCCC method used for the isolation and purification of alpinetin and cardamomin from A. katsumadai has many advantages, such as facility, high efficiency, and high recovery as well.

Adult ; Aged ; Aged, 80 and over ; Antifungal Agents ; therapeutic use ; Female ; Hematologic Neoplasms ; drug therapy ; microbiology ; Humans ; Male ; Middle Aged ; Mycoses ; complications ; drug therapy ; Pyrimidines ; therapeutic use ; Treatment Outcome ; Triazoles ; therapeutic use ; Voriconazole ; Young Adult

Cyclohexanones ; poisoning ; Ethylene Dichlorides ; poisoning ; Humans ; Infant ; Male

Adult ; Asthma ; metabolism ; physiopathology ; Basic-Leucine Zipper Transcription Factors ; genetics ; physiology ; Fanconi Anemia Complementation Group Proteins ; genetics ; physiology ; Female ; Glutamate-Cysteine Ligase ; metabolism ; Humans ; Inflammation ; metabolism ; pathology ; Male ; NF-E2-Related Factor 2 ; genetics ; physiology ; RNA, Messenger ; genetics ; metabolism ; Sputum ; cytology

ObjectiveTo compare the diagnostic efficiency of 18F- FDG coincidence SPECT/CT and 99Tcm- MDP whole body bone scan (WBBS) in detecting malignancy.MethodsA total of 71 cases (male 45,female 26,mean age 59.2 ± 15.4 years) with clinically confirmed malignancy underwent both 99TcmMDP WBBS and 18F-FDG coincidence imaging within three weeks.The sensitivity,specificity,accuracy,positive and negative prediction value of these two imaging methods in detecting bone metastases were compared based on the results from pathology or clinical follow-up.x2test was used for data analysis.ResultsA total of 350 lesions (including primary,second malignancy and benign disease) in 71 patients were eval-uated.81.7％ (286/350) malignant lesions were identified by either 99Tcm-MDP WBBS (209/350,59.7％ ) or 18F-FDG coincidence imaging ( 141/350,40.3％ ) (x2 =25.65,P ＜ 0.01 ).The imaging findings of osteoblastic,osteolytic,mixed types of bone metastases by99Tcm-MDP WBBS and 18F-FDG coincidence imaging were significantly different (x2 =20.78,2.89 and 9.94,all P ＜ 0.05 ).The sensitivity,specificity,accuracy,false-positive,false-negative,positive and negative predictive values for detecting bone metastases by 18 F-FDG coincidence study and 99Tcm-MDP WBBS were as follows:11.72％ ( 15/128),91.67％(22/24),24.34％ (37/152),8.33％ (2/24),88.28％ (113/128),88.24％ (15/17),16.30％ (22/135) ; and 53.91％ (69/128),75.00％ ( 18/24),57.24％ (87/152),25.00％ (6/24),46.09％ (59/128),92.00％ (69/75),23.38％ ( 18/77 ).The sensitivity,accuracy,false-negative,positive-predicting value of the two methods had been significant different (x2 =32.70- 46.21,all P ＜ 0.01 ).When two methods were combined,the diagnostic efficiency could been improved.ConclusionThe 99Tcm-MDP WBBS and 18F-FDG coincidence imaging has a complementary role in detecting bone metastases.