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

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