[Interference therapy of laryngeal squamous cell carcinoma by p16beta and EGFR-antisense cDNA in signal transduction].

OBJECTIVE: To test the effect of p163 and EGFR-antisense cDNA in signal transduction on Hep-2 laryngeal squamous cell carcinoma in vitro. METHODS: The Hep-2 laryngeal squamous carcinoma cells were transfected by recombinant adenovirus AdEasy-EGFR-antisense and AdEasy-p16beta in vitro. The inhibition of the EGFR expression and cell growth and changes of cell cycle, DNA content, apoptosis and ultramicrostructure of the Hep-2 cells were examined by MTT, Western blotting analysis, Flow cytometry analysis, Immunohistochemistry, and transmission electron microscope respectively. Results The proliferation of the Hep-2 cells was inhibited significantly by the infection of the Ad- Ad-p16beta or Ad-antisense EGFR. The infection also accelerated the apoptosis of the cancer cells. The proport of of cells in G0/G1 phases increased to more than 77.7%. The Ad-antisense EGFR-infected cells showed lower protein expression of EGFR. The P16beta protein over expression was observed in the Ad-p16beta-infected cells. CONCLUSION: The transfection of Ad- Ad-p16beta and Adantisense EGFR into Hep-2 cells leads to over-expression of Ad-pl6beta, and under-expression of EGFR, along with G1-phase arrest and apoptotic cell death. Both EGFR and Ad-p16beta play important roles in the genesis, growth and differentiation of the human laryngocarcinoma cells.

[Construction of the recombinant adenovirus vector with EGFR antisense cDNA of controlling cell cycle].

OBJECTIVE: To recombine the adenovirus vector carrying EGFR sence/antisense cDNA which takes part in control of cell cycle. METHODS: The 1032 bp EGFR sence/antisense cDNA fragment was cloned into the shuttle plasmid pAdTrack-CMV. The resultant plasmid and the backbone plasmid pAdEasy-1 were transferred into E. coli BJ5183 for homologous recombination, and the recombinant adenoviruses were generated in cells. The recombinant adenoviruses were packaged and amplified in the 293 cells. Then the viral titer was detected by GFP. RESULTS: The recombinant adenovirus vector carrying EGFR sence/antisense cDNA to control the cell cycle was constructed successfully. The viral titers were 2.2 x 10(9) efu/mL and 2.5 X 10(9) efu/mL respectively. CONCLUSION: The recombinant adenovirus vector constructed by us could introduce EGFR antisense cDNA into the laryngeal squamous cell carcinoma line or tumor tissue, which would provide an experiment basis to study further the interfered mechanism of signal transdution and the therapies of laryngeal squamous cell carcinoma.

[Study in vitro on p16beta interfering the cell cycle signal conduction of laryngeal squamous cell carcinoma].

OBJECTIVE: To investigate the effect of p16beta interfering with the signal conduction of Hep-2 cell cycle in Vitro. METHODS: AdEasy Vector System was used to construct the recombinant adenovirus vector AdEasy-GFP-p1613. The recombinant adenovirus vector could introduce p16beta gene into HEK 293 cell. Then the purified recombinant adenovirus was used to infect Hep-2 cells in vitro. The protein expression of p16beta, proliferation inhibition, cell cycle arrest, DNA content, apoptosis ratio in Hep-2 cells were examined by MTT, Western blotting analysis, Flow cytometry assay, Immunocytochemistry respectively. RESULTS: The recombinant adenovirus Adeasy-p16beta was constructed successfully and higher titer recombinant adenovirus particle was got. When Adeasy-p16beta was transferred into Hep-2 cells, both the growth inhibition and P14 (ARF) protein overexpression in Hep-2 cells were visible effectively. The most Hep-2 cells were blocked in G1/G0 phase and the cell apoptosis ratio increased. CONCLUSION: The p16beta in Hep-2 cell infected by recombinant adenovirus, which lead to express P14 (ARF) protein effectively and to inhibit cell proliferative activation, effectively interfere the signal conduction mechanisms of culture Hep-2 laryngeal squamous cell carcinoma, and the growth of much cultured cells is blocked in G1/G0 phase.

[Experimental study of cartilage reconstruction using directed inducing MSCs-alginate complex].

OBJECTIVE: To evaluate the practicability of cartilage reconstruction using directed inducing bone marrow stem cells(MSCs)- alginate complex. METHODS: The MSCs were seperated by gradient centrifugation on Percoll, then were induced into chon-drogenic differentiation. After 14 days,immunohistochemical techique was applied to detect the expression of collagen type II. Then, the MSCs and calcium alginate were mixed to form a complex and were planted under the skin of nude mice. Histochemical or immuno-histochemical tests were made at 4 weeks and 8 weeks later. RESULTS: The MSCs showed the positive result of collagen type II. There were chondrcytes and cartilage lacuna at 4 weeks later, and chondrocytes and the collagen secreted by them became more at 8 weeks later. But, the MSCs without directed inducing did not show the character of the chondrocyte. CONCLUSION: The way of using directed inducing MSCs-alginate complex can form cartilage; it is possible to repair the defect of cartilage in clinical practice.

[Establishment of methods of mechanical dissociation and culture of spiral ganglion neurons in postnatal mice].

OBJECTIVE: To conduct a study on the methods for establishing a cytological model of mechanical dissociation and culture of spiral ganglion neurons (SGNs) in postnatal mice. METHODS: The spiral ganglion neurons were taken from 1-6-day old mice for primary culture. The morphology and the process of axon were observed by use of an inverted/phase contrast microscope. Immunocytochemical identification was performed using fluorescent microscopy. RESULTS: In the state of primary culture in vitro, the SGNs were found to have good cell morphology and axon regeneration power, the surviving cells of SGN(5-7 d) amounted to (78.10 +/- 4.33)/cm2 and the survival period was 7-14 d. CONCLUSION: We successfully established the methods of mechanical dissociation and culture of SGN cytological model; the cell amount and survival period of SGNs were able to meet the requirement of cytological experiments.

[Expression of vascular endothelial growth factor C in laryngeal squamous carcinoma and its role in lymphatic metastasis].

OBJECTIVE: To study the expression of vascular endothelial growth factor C (VEGF-C) in laryngeal squamous carcinoma (LSC) and its relationship to lymph node metastasis. METHODS: Paraffin-embedded specimens of primary laryngeal carcinomas from 60 patients subjected to operation were studied. VEGF-C expression was investigated using immunohistochemical staining. RESULTS: Positive immunohistochemical staining for VEGF-C protein was observed in some laryngeal squamous carcinoma cells (45/60), the VEGF-C antigen was identified in the cytoplasm of cancer cells. Furthermore, the expression of VEGF-C in laryngeal squamous carcinoma cells was significantly higher in lymph-node-positive groups than in node-negative groups, and the expression was significantly higher in poorly differentiated LSC tissues than in moderately and well differentiated LSC tissues. CONCLUSION: The above findings indicate a close correlation between VEGF-C expression and lymph node metastasis in laryngeal squamous carcinoma.

[Measurement of sound pressure level at outlet of external auditory canal and eardrum].

OBJECTIVE: To evaluate the sound pressure level at outlet of external auditory canal and eardrum, and to observe the range of maximum frequency of sound effect of outlet ear canal, so as to provide exact data for designing personal noise rating instrument and studying the effect of environmental noise on human body, and to provide basis for calibration of noise sound pressure level. METHOD: Sound pressure level at eardrum and outlet of external auditory canal in 30 young people (60 ears) were measured by Audio Scan RM500 at 50 dB SPL narrow band noise with different frequencies (0.25 - 8.00 kHz). RESULTS: At the same frequency, sound pressure levels at eardrum were less than (0.6 +/- 2.6) dB SPL, at outlet of outlet ear canal less than (0.2 +/- 1.8) dB SPL, there were no significant differences between both sound pressure levels (P > 0.05). There were significant differences in sound pressure levels between both places at 1.50 - 8.00 kHz (P < 0.01). The maximum difference was (10.5 +/- 3.4) dB SPL at 2.00 kHz, and the minimum was (0.5 +/- 6.2) dB SPL at 0.50 kHz. CONCLUSION: The effects of increased sound pressure should be considered in the evaluation of environmental noise and the design for individual noise rating meter when the noise frequency was >or= 1.50 kHz.

[Study on interference therapy induced by epidermal growth factor receptor-antisense cDNA in signal transduction of laryngeal squamous cell carcinoma].

OBJECTIVE: To investigate effect of interference therapy induced by epidermal growth factor receptor (EGFR)-antisense cDNA in signal transduction of Hep-2 laryngeal squamous cell carcinoma in vitro. METHODS: AdEasy Vector System was used to construct the recombinant adenovirus vector sense/antisense-pAdEasy-EGFR. The recombinant adenovirus vector introduced EGFR-sense/antisense cDNA fragment into HEK293 cell. The purified recombinant adenovirus sense/antisense-pAdEasy-EGFR transfected Hep-2 cells in vitro. The inhibition of EGFR protein expression and proliferation of Hep-2 cells, the changes of cell cycle and DNA content in Hep-2 cells were examined by MTT, Western blot analysis, flow cytometry essay, and immunocytochemistry respectively. RESULTS: The higher titre sense and antisense mRNA expression recombinant adenovirus containing 1,032 bp EGFR-cDNA was constructed and prepared successfully. When antisense-pAdEasy-EGFR was transferred into Hep-2 cells the inhibition of cell proliferation and EGFR protein expression in Hep-2 cells were investigated effectively. CONCLUSION: The antisense-pAdEasy-EGFR effectively interfere the Hep-2 signal transduction pathway and induce apoptosis which results in inhibiting proliferation of laryngeal squamous cell carcinoma.

Aberrant branch of the superior laryngeal artery passing through the thyroid foramen.

This study examines the microsurgical anatomy of the aberrant superior laryngeal artery (ASLA) which passes through the thyroid foramen. It defines the location of the ASLA and its main branches. The aberrant superior thyroid artery was looked for in 20 randomly selected Chinese adult cadavers, and when found, was dissected and measured under an operating microscope (7-30x magnification). The ASLA passed through the thyroid foramen, which only occurred in the presence of the artery. The foramen was observed in six of the 20 cadavers: one on the right, three on the left, and two on both sides. The study provides detailed information concerning the ASLA, which we hope will help explain the arterial bleeding that may occur during laryngeal surgery. The results might be helpful in improving surgery to the larynx and other neck operations. Incidental intraoperative injury of the aberrant artery could be avoided by understanding details of its course in relation to surrounding anatomic landmarks.

[Clinical anatomy measurement of accessory nerve in neck dissection].

OBJECTIVE: To investigate the relations between accessory nerve and its surrounding structures. METHODS: One hundred and thirty six patients were divided into two groups: has or has no neck surgical history. Neck dissection were performed and the four distance were measured simultaneously. The distance of accessory nerve and the great auricular nerve going out the posterior edge of sternocleidomastoid muscle; the distance of the point accessory nerve going out the posterior edge of sternocleidomastoid muscle to clavicular midpoint; the distance of the point accessory nerve going out the posterior edge of sternocleidomastoid muscle to sternoclavicular articulation; the distance of the point accessory nerve enter trapezius muscle to clavicular midpoint. RESULTS: In no neck dissection group, the point accessory nerve going out sternomastoid muscle were supra the point of great auricular nerve going out the sternomastoid muscle, the average length of two points is (0. 61 +/- 0. 35) cm , the significance has not observed between genders (P > 0.05), however, there has significant difference between two groups of has or has no neck surgical history (P < 0.05). 88.2% (112/127) accessory nerve going out supra the great auricular within 1.0 cm, 11.8% (15/127) within 1.0 approximately 2.0 cm. 67.7% (86/127) accessory nerve adopt branch from cervical plexus before entering trapezius. The distances of the point accessory nerve going out the posterior edge of sternocleidomastoid muscle to clavicular midpoint and to sternoclavicular articulation were significant relative not with before neck surgical history but gender. The distance of the point accessory nerve enter trapezius muscle to clavicular midpoint is (4.96 +/- 0.78) cm, it has no difference both before neck surgical history and gender (P > 0.05). CONCLUSION: In no neck surgical history group,both of the distance that accessory nerve and the great auricular nerve going out the posterior edge of sternocleidomastoid muscle and the point accessory nerve enter trapezius muscle to clavicular midpoint were helpful for search accessory nerve in surgery. But in patients who have neck surgical history or great auricular have been injured, accessory nerve could be looked for associating with the distances of the point accessory nerve going out the posterior edge of sternocleidomastoid muscle to clavicular midpoint and to sternoclavicular articulation; the distance of the point accessory nerve enter trapezius muscle to clavicular midpoint.

[Construction of recombinant adenovirus vector carrying cell cycle controlling gene-p14ARF].

OBJECTIVE: The recombinant adenovirus vector carrying p14ARF gene was constructed for using in the interference therapy in signal transduction of laryngeal squamous cell carcinoma. METHODS: The total cDNA fragment of p14ARF was cloned into the shuttle plasmid pAdTrack-CMV, with the resultant plasmid and the backbone plasmid pAdEasy-1, the homologous recombination took place in the E.Coli BJ5183 and the recombinant adenoviral plasmid was generated. The adenoviruses were packaged and amplified in the 293 cells. Then the viral titer was checked by GFP. RESULTS: The recombinant adenovirus vector carrying p14ARF was constructed successfully. The viral titer was 2.3 x 10(9). CONCLUSION: The recombinant adenovirus vector could introduce p14ARF gene into the laryngeal squamous cell carcinoma line or tumor tissue effectively, which would provide experimental basis for the mechanisms and further study of the interference therapy in signal transduction of laryngeal squamous cell carcinoma.

[Anatomic study of pterygopalatine fossa under endoscope].

OBJECTIVE: To provide endoscopic anatomic bony structures of pterygopalatine fossa for skull base surgery. METHODS: The bony structures of the pterygopalatine fossa were observed in ten dry skulls under endoscope. RESULTS: The pterygopalatine fossa showed a long and narrow cleft composed of the body and pterygoid process of sphenoid bone, the lamina perpendicular of palatine bone, and the posterior wall of maxillary sinus. The pterygopalatine fossa is (21.4 +/- 0.8) mm x (5.2 +/- 0.3) mm x (3.2 +/- 0.3) mm, with seven paths communicating with nasal cavity, mouth cavity, pharynx, orbit, infratemporal fossa and middle cranial fossa. Under endoscope,the whole pterygopalatine fossa could be observed. CONCLUSIONS: Endoscopic anatomic study of the pterygopalatine fossa is important to endoscopic endonasal skull base surgery. Under endoscope,the whole pterygopalatine fossa can be observed.

[Microsurgical anatomy of epiglottic artery and its clinical implications].

OBJECTIVE: To study the microsurgical anatomy of human epiglottic blood vessel to provide exact and reliable data and morphological properties for further studies of laryngeal transplantation, reconstruction and other epiglottis-related diseases. METHODS: Thirty Chinese adult cadavers (27 males and 3 females) were examined for the present study. The cadavers were dissected under magnification along the anatomic planes from skin down to vertebral column. The anterior neck was exposed by a midline incision and extended laterally along the angle of mandible superiorly, and on clavicle inferiorly. After the visualization of laryngeal prominence of thyroid cartilage, strap muscles were resected and superior laryngeal artery and epiglottic blood vessel were exposed under an operating microscope ( original magnification 7 -30). The epiglottic artery was named for the first time. RESULTS: The diameter of superior laryngeal artery was (1. 06+/-0. 16) mm( male: 1. 09 mm+/-0. 12 mm). The diameter of origin epiglottic artery was (0. 79+/-0. 13) mm (male: 0. 81 mm+/-0. 11 mm). The vertical distance between origin epiglottic artery and superior horn of thyroid cartilage was (27. 16+/-3. 85) mm. Epiglottic artery loop was observed in all the cadavers, which could be M-, N-, omega-, or U-shaped and mixed under the thyrohyoid membrane or in the epiglottic vallecula. CONCLUSIONS: These findings could improve the application of epiglottis in laryngeal functional reconstruction after partial laryngectomy, as well as in the prevention of epiglottic artery loop injuries during the operation.

[Specific inhibition of Raf-1 gene expression in HNE1 by RNA interference].

OBJECTIVE: To silence the expression of Raf-1 gene in HNE1 cells using vector-based RNA interference (RNAi) technique. METHODS: The vector containing the human U6 promoter was used for targeted gene silencing when a dsDNA oligonucleotide encoding an appropriate shRNA was ligated into the vector, and 67nt oligonucleotide fragment was inserted into the downstream of the U6 promoter. Plasmids containing different Raf-1 target sequences [ (1) pshuttle-Raf-1-a( 225), (2) pshattle-Raf-1-b ( 358) and (3) pshuttle-Raf-1-c(474)], were transfected into HNE1 cells. Expression of Raf-1 mRNA was assayed by RT-PCR. Apoptosis were determined by cytometry. RESULTS: Vector-based RNAi had advantages over antisense RNA because it could be delivered to the target cell more efficiently, and effect could last longer. Raf-1 expression could be inhibited by plasmid-expressed shRNA. Three different targeting sequences were selected from Raf-1 gene, and the inhibitory effect of pSIREN shuttle-Raf-1-b (358) was biggest. CONCLUSION: Raf-1 expression in HNE1 cells can be inhibited significantly using plasmid-based RNAi.

[Effects of budesonide, desloratadine and dexamethasone on interleukine-4 release and expression from human mast cell line].

OBJECTIVE: Since human mast cell is an important source of cytokines, it is of importance to understand the effects of anti-allergic drugs on cytokines modulation in mast cells. In the present study, we aimed at observing whether IL-4 could be released from human mast cell line (HMC-1) after the stimulation of PMA + A23187, and the effects of systemic glucocorticosteroid, dexamethasone, topical glucocorticosteroid, budesonide and H1 antagonist, desloratadine on IL-4 release and mRNA expression. METHODS: HMC-1 was stimulated with 25 ng/ml phorbol 12-myristate 13-acetate (PMA) and 2.5 x 10(-7) mol/L ionomycin (A23187) and cultured for 6 hours, 12 hours and 24 hours respectively in the presence or absence of 10(-6)-10(-10) mol/L concentrations of test drugs. Culture supernatants were collected and the levels of IL-4 were assayed by enzyme-linked immunosorbent assays (ELISA). The mRNA expression of IL-4 was measured by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: HMC-1 expressed IL-4 mRNA and the resulting protein production of IL-4 released after being stimulated with PMA plus A23187. Dexamethasone, budesonide and desloratadine had potent inhibitory effect on IL-4 release at any concentrations and time points, with significant deference (P < 0.05) compared to the control cells. The inhibitory effect did not show time-dependent and concentration-dependent manner. Desloratadine and budesonide showed neither up-regulatory nor down-regulatory effects on IL-4 mRNA expression at the test concentrations, however, desloratadine could down-regulate IL-4 mRNA expression. CONCLUSIONS: HMC-1 could express and produce IL4 after stimulation. Dexamethasone, budesonide and desloratadine all had inhibitory effects on IL-4 release from HMC-1. In addition, desloratadine could also inhibit the IL-4 mRNA expression.

[Establishment of a modified intranasally ovalbumin induced animal model of allergic rhinitis].

OBJECTIVE: To observe the early and late symptomatic, pathological and immunological changes in an intranasal ovalbumin-induced animal model of allergic rhinitis in guinea pigs. METHODS: Guinea pigs were intranasally sensitized with ovalbumin absorbed on aluminum hydroxide and after 5 days' interval, they were challenged with 1% ovalbumin solution once every 3 days for total 11 times. Two control groups were studied in parallel, the positive treatment control group was treated with antihistamine and the negative control group was sham-sensitized and sham-challenged. Typical symptoms of allergic rhinitis, such as sneezing, nasal scratching, nasal blockage and rhinorrhea were evaluated. Passive cutaneous anaphylaxis reaction (PCA) was performed to measure the levels of IgG1 and IgE. Eosinophils infiltration and goblet cells in nasal mucosa were observed. In addition, the level of histamine and the number of total leukocytes and eosinophils in the nasal lavage fluid were also measured. RESULTS: In the model group, symptoms of sneezing, nasal scratching, nasal blockage and rhinorrhea were induced after ovalbumin challenge. The respiratory rate (RR), which reflected the resistance of upper airway, showed a biphasic change. In the PCA test, IgG1 and IgE levels increased after challenges. Eosinophil infiltration in nasal mucosa was more obvious in active groups in comparison to with the negative control group (P < 0.05 or < 0.01). The histamine, total leucocytes and eosinophils levels in nasal lavage fluid also showed higher in the model group (P < 0.05 or < 0.01). The antihistamine treated animals were also induced out above changes but modest compared with the model group (P < 0.05 or < 0.01). The negative control showed few of above changes with significant difference (P < 0.05 or < 0.01). CONCLUSIONS: Our results implied that the modified animal model of allergic rhinitis was capable of showing satisfactory symptomatic and pathophysiological changes in allergic rhinitis. It showed a biphasic nasal blockage with shorter establishment duration. The model also had good treatment reaction to antihistamine. The animal model we introduced may be useful in the study of allergic rhinitis.

[Research of the electrophysiological properties of acutely dissociated spiral ganglion neurons isolated from mice using patch clamp technique].

OBJECTIVE: To explore the electrophysiological properties of spiral ganglion neurons (SGN) isolated from neonatal mice. METHODS: Ion channel currents of acutely trypsin-dissociated spiral ganglion neurons was recorded and analyzed using whole-cell variation of the voltage clamp technique. RESULTS: In the membrane of SGN, we recorded tetraethylammonium (TEA)-sensitive and 4-aminopyridine (4-AP)-sensitive outside delayed rectifier potassium currents, A-type potassium currents and tetrodotoxin-sensitive inside sodium currents. Ion channel activity had the character of voltage-dependence. Some cells, which did not exhibit sodium currents exhibited, delayed rectifier potassium currents. CONCLUSIONS: The electrophysiological properties of acutely dissociated spiral ganglion neurons could be reference to the research of the mechanics of auditory propagation and the ion channel pharmacology.

[Adenovirus p16(INK4A) gene therapy for laryngeal squamous cell carcinoma].

OBJECTIVE: To assess the anti-tumor effects of p16INK4A gene transfer in laryngeal squamous cell carcinoma. METHODS: A Complete p16INK4A gene was inserted into a replication-defective recombinant adenovirus (Ad-p16) and the tumor cells were infected with Ad-p16. Confirmation of p16INK4A protein expression after Ad-p16 infection was performed by Western Blotting. The therapeutic effects were evaluated both in in vitro and in vivo study. RESULTS: The replication-defective recombinant adenovirus can direct a high level of p16INK4A protein expression in laryngeal squamous cell carcinoma. Studies both in in vitro and in vivo experiments demonstrated that Ad-p16 treatment significantly inhibits the cell growth and the established tumors in nude mice. The mean value of tumor volumes among the Ad-p16, Ad-Lacz and phosphatic buffered saline(PBS) groups was (91.00 +/- 6.32) mm3, (137.00 +/- 9.62) mm3 and (144.00 +/- 13.87) mm3 respectively. Statistic analysis showed that there was a significant difference between the Ad-p16 group and the control groups (P < 0.05). No significant difference was found between Ad-lacZ and PBS groups, which indicated that the antitumor effects was not influenced by the adenovirus. CONCLUSION: These results demonstrate a significant antitumor effect of Ad-p16 against human laryngeal squamous cell carcinoma. This data also further support the potential application of Ad-p16 to treat the human head and neck cancer.