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.

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

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 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

Molecular technologies are sensitive, fast, and cheap in the study on microbial ecology. However, these methods do not provide information about morphology, number, and spatial distribution of the microorganisms. In contrast, Fluorescence in situ hybridization (FISH) combines the precision of molecular biology with the visual information from microscope, and permits visualization and identification of individual miciobial cells within their natural habitat. FISH not only allows the detection of slow growing microorganisms, but also of yet-to-be cultured. It is useful for many applications in diagnosis and assessment of the population structure of complex microbial communities, and is a powerful tool for molecular ecology studies in microbiology, In this paper, major techniques and progresses of FTSH were described. Its application in microbial ecology, as well as problems , pitfalls, and perspectives of FISH are discussed.

OBJECTIVE: To investigate the changes of laryngeal cancer Hep-2 cells to cisplatin chemosensitivity after the interference of siRNA of β-catenin gene expression. METHOD: Using a small interference RNA (siRNA) technology interfere β-catenin gene of Hep-2 cells . The mRNA and protein levels of β-catenin in the Hep-2 cells of different groups were detected by qPCR and Western blot. It was divided into siRNA-β-catenin-Hep-2 siRNA group, β-catenin-Neg negative control group and blank control group. Cell proliferation inhibition rate of different concentrations of cisplatin on three groups was detected by MTT assay. Calculate the 50% inhibitory effective concentration IC50 value. Check the change of three groups of cells' apoptosis rate by flow cytometry after the same concentrations of cisplatin stimulation. RESULT: β-catenin-siRNA interference fragment can specifically reduce the expression levels of β-catenin mRNA and protein. qPCR illustrated the expression of mRNA in β-catenin-siR-NA-Hep-2 interference group decreased 70% (P < 0.05) compared with the control group, Western blot results showed that the β-catenin protein expression of interference group (0. 545 ± 0.111) decreased significantly compared with blank control group (1.507 ± 0.139) and negative control group (1.429 ± 0.089), P < 0.05. The IC50 calculation software showed that IC50 of cisplatin on β-catenin-siRNA IC50 interference group is (5.81 ± 0.46)μg/ml, the blank control group is (10.10 ± 1.01) μg/ml, the difference between the two groups has statistical signifi- cance (P < 0.01). Cell apoptosis rate of β-catenin-siRNA interference group was (26.15 ± 0.60)%, significantly higher than the control group (14.16 ± 0.05)%, P < 0.05. CONCLUSION To interfere the expression of β-catenin can effectively enhance the sensitivity of laryngeal cancer cells to chemotherapeutic drugs cisplatin. It provides a theoretical support for the reduction of laryngeal cancer chemotherapy drug cisplatin dosage.
Apoptosis ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Drug Resistance, Neoplasm ; Humans ; Laryngeal Neoplasms ; genetics ; RNA Interference ; RNA, Messenger ; RNA, Small Interfering ; beta Catenin ; genetics

Objective To investigate the auxiliary effects of huaier granule on hepatoeellular carcinoma (HCC)patients after liver transplantation.Methods Sixty HCC patients who had undergone liver transplantation from Julv 2004 to June 2006 and met the standard of UCSF were involved in this study.All patients were divided into huaier granule group(n=20),chemotherapy group(n=15),huaier granule+chemotherapy group (n=15)and control group(n=10).The white blood cell count,liver function,cell immunity and immunologieal reiection were detected.The 1-year tumor recurrence rate wag calculated.Results The white blood cell counts in chemotherapy group 1,3,and 6 months after treatment were significantly lower than that before treatment (F=62.053,58.472,49.807,P＜0.05).The changes of white blood cell counts of the other 3 groups before and aftertreatment were small.The difference on the white blood cell counts of the 4 groups had no statistical 8ignincanee(F=102.361,113.412,87.572,P＜0.05).The NK activity,CD4+/CD8+ ratio,IL-2 level in huaier granule group and huaier granule+chemotherapy group 1,3,6 months after treatment were significantly higher than those before treatment,and were significantly higher than those in chemotherapy group and control group(P＜0.05).No immunological rejection occurred in all the groups.Two patients in each group had recurrence and metastasis of HCC within 1 year after the treatment.and the incidence in control group was higher than the other 3 groups(P＜0.05). Conclusions Humer granule can increase the white blood cell count which is decreased after chemotherapy,impmve cellular immunity,and effectively suppress the recurrence and metastasis of HCC at the first year after operation.