Effects of RNA interference targeting four different genes on the growth and proliferation of nasopharyngeal carcinoma CNE-2Z cells / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To explore the effects of RNA interference (RNAi) targeting four different genes (VEGF, c-myc, survivin, hTERT) on the growth and proliferation of nasopharyngeal carcinoma (NPC) CNE-2Z cells.</p><p><b>METHODS</b>Plasmid-1 targeted all four genes, plasmid-2, 3, 4 and 5 targeted VEGF, c-myc, survivin and hTERT respectively. These plasmids were transfected separately into human NPC CNE-2Z cells and xenograft tumors in nude mice. The expressions of plasmids in NPC CNE-2Z cells and xenograft tumors were observed. Cell proliferation was detected with MTT assay. The inhibitory effects on target genes were evaluated with RT-PCR and Western blot respectively. The effects of the plasmids on the biological behavior of CNE-2Z cells were observed with Transwell invasion chamber model. Apoptosis was determined with flow cytometer. The inhibitory effect of the plasmids on xenograft tumors were observed in nude mice.</p><p><b>RESULTS</b>CNE-2Z cell proliferation was significantly inhibited and in vitro invasion ability was significantly decreased in the plasmid-1 group compared with those in the plasmid 2 - 5 groups (all P < 0.05). mRNA and protein expressions of all four genes decreased in the plasmid-1 group. The apoptosis rate in the plasmid-1 group was higher than that in the plasmid 2 - 5 groups (all P < 0.05). Growth of xenograft tumors in nude mice were significantly inhibited in the plasmid 1 - 5 groups, particularly in the plasmid-1 group.</p><p><b>CONCLUSION</b>RNA interference targeting multiple genes can effectively inhibit NPC proliferation and induce apoptosis, which provides an experiment basis for NPC gene therapy.</p>

Effect of silencing VEGF, c-myc, survivin and hTERT by multiple shRNA expression vectors on the xenografted human nasopharyngeal carcinoma in nude mice / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the effects of multiple short hairpin (shRNA) expression vectors, targeting VEGF, c-myc, survivin and hTERT, genes on the xenografted human nasopharyngeal carcinoma (CNE-2Z) in nude mice.</p><p><b>METHODS</b>The shRNA expression vectors were constructed and subsequently transfected by direct injections into the tumors formed by CNE-2Z cells implanted in nude mice. The expressions of the targeted genes in tumor tissues and the apoptosis of tumor cells were evaluated.</p><p><b>RESULTS</b>NPC CNE-2Z cells were successfully inoculated and subcutaneous tumor was formed in all nude mice. Under fluorescence microscope, tumor tissues showed the expression of each vector with green fluorescence. The expression of multiple shRNAs led to the decreases in the expressions of VEGF, c-myc, survivin, hTERT mRNA and proteins. Multi-gene silencing was better than single gene silencing in inducing the apoptosis of tumor cells. Tumor growth curves showed that the tumors treated with the shRNAs, including VEGF, c-myc, survivin, hTERT or the combination of 4 shRNAs, grower slowly obviously compared with control tumors. Inhibited rates of tumor growth by VEGF-, c-myc-, survivin- and hTERT-shRNA were 46.2%, 48.5%, 51.9% and 46.8% respectively. The combined application of 4 shRNA produced the more significant inhibitory rate (82.4%) than single shRNA application.</p><p><b>CONCLUSIONS</b>The application of vector-based RNAi targeting multiple genes is a promising therapeutic modality in the gene therapy of nasopharyngeal carcinoma and multi-gene silencing is a new strategy for tumor therapy.</p>

Clinical features of parathyroid space-occupying lesions / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the clinical manifestation, diagnosis, treatment of parathyroid occupying lesions.</p><p><b>METHODS</b>The clinical data of 42 patients with parathyroid occupying lesions were retrospectively analyzed, including the clinical symptoms and signs, laboratory results, pathologic and imaging results and treatment.</p><p><b>RESULTS</b>The number of males and females were 8 and 34, with females: males ratio being 1:5.25. The median age was 39 years. There were 2 cases of parathyroid cancer, 29 cases of parathyroid adenoma, 11 cases of parathyroid cysts in this study. The symptoms were as follows: 40 cases of neck lump, 34 cases of osteoporosis/osteitis fibrosa cystica, 29 cases of urinary symptom, 7 cases of voice hoarseness, 4 cases of peptic ulcer, 3 cases of dyspnoea and dysphagia, 3 cases of thoracic cavity lump, 2 cases of enhanced amylase activity. Serum calcium ion level and serum parathyroid hormone (PTH) level were examined qualitatively before operation. Ultrasonography, ECT-99mTc-MIBI, CT, MRI were used in diagnosing and locating parathyroid occupying lesions before operation. Twenty nine cases of parathyroid adenoma were treated with operation, 28 patients achieved complete remission, 1 suffered relapse after 23 months postoperative follow up. Eleven cases of parathyroid cysts were treated with operation and the outcome was no recurrence. Two cases of parathyroid cancer survived with out recurrence during follow up for 28 months and 50 months after operation.</p><p><b>CONCLUSIONS</b>Examination of serum calcium and PTH level together with ultrasonography, ECT-99mTc-MIBI, CT, MRI is helpful to diagnose parathyroid occupying lesions. Surgery should be done as primary treatment. Tumor resection can be performed for parathyroid cysts, intraoperative exploration of bilateral neck is indicated for parathyroid adenoma, and a radical resection should be performed primarily for the parathyroid cancer.</p>

Effects of sodium butyrate on growth, apoptosis and telomerase activity in Hep-2 cells / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To study the effects of sodium butyrate (SB) on growth, apoptosis and telomerase activity in Hep-2 cells.</p><p><b>METHODS</b>Growth inhibition effect of SB on Hep-2 cells was assessed by methyl thiazolyl tetrazolium (MTT) assay. Morphological alterations were observed by electronic microscope. Cell apoptosis was confirmed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) method, DNA fragmentation and flow cytometry (FCM). Cell cycle was analyzed by FCM. Telomerase activity was examined by telomeric repeat amplification protocol (TRAP)-silver staining. The expression status of telomerase subunits was analyzed by reverse transcription-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>A time-and dose-dependent inhibition was detected in cells treated with SB. Typical morphological changes of apoptotic cells were observed under electronic microscopy. The characteristic DNA fragmentation of apoptotic cells was detected by agarose gel electrophoresis. Apoptosis and the changes of cell cycle were confirmed by TUNEL method and FCM. The apoptosis indexes of the cells before treatment and at 72 h after SB (2.5 mmol/L) treatment were 2.27 +/- 1.18 and 33.50 +/- 2.75 respectively, the apoptosis rates were 2. 86% and 31. 28% respectively, the proportion of the cells at G0/G1 stage were 50.38% and 70.88% respectively, the proportion of the cells at S stage were 27.40% and 8.20% respectively, and the proliferation indexes of the cells were 49.62% and 29.12% respectively. Telomerase activity and expression level of human telomerase reverse transcriptase (hTERT), the key subunit of telomerase, decreased after SB treatment. No significant changes were observed in the expression of human telomerase RNA (hTR) and human telomerase associated protein (hTP1), the other two subunit of telomerase.</p><p><b>CONCLUSION</b>SB could inhibit growth of Hep-2 cells and induce apoptosis in the cells, and inhibit telomerase activity by decrease expression level of hTERT.</p>

Observation of auditory brainstem response and distortion product otoacoustic emission on the animal model of autoimmune auditory neuropathy / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To set up an animal model of autoimmune auditory neuropathy and to observe the auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) in guinea pigs.</p><p><b>METHODS</b>The spiral ganglion and the cochlear nerve were obtained and purified by electrophoresis from 250 normal guinea pigs. The purified cochlear nerve antigen was mixed with an equal volume of complete Freunds adjuvant for immunization. Seventy guinea pigs were divided into three groups: experiment group (50 guinea pigs), control group (10 guinea pigs), normal group (10 guinea pigs). ABR, DPOAE, serum IgG levels, and morphological changes of spiral ganglion cells and the cochlear nucleus were observed. The protein expressions of the antigen were examined by immunohistochemistry and the super-structure of the auditory nerve were observed.</p><p><b>RESULTS</b>The threshold of ABR response increased ranged from 10 to 25 dB in 32% (32/100 ears) of the guinea pigs. The peak latencies of waves I , III and the interpeak latency I approximately III were prolonged in the hearing loss group of guinea pigs. Prolonged peak latency of wave III was noted in hearing loss group at 2 and 3 weeks post immunization and slowly decreased to normal peak latency. The amplitude of DPOAE was no difference in the guinea pigs. The levels of serum IgG increased significantly compared with those of the control group. Inflammatory cell infiltration was observed in the cochlear nerve and the number of spiral ganglion cells detected. On the contrary, inflammatory cell infiltration was not observed in the cochlear nucleus. The cell densities and the across-sectional areas of neurons in anteroventral cochlear nucleus and posteroventral cochlear nucleus were no difference in the guinea pigs. The antigen protein distributed strictly in cochlear nerve and the spiral ganglion. Some demyelinated areas in cochlear nerve was observed in this group. The threshold of ABR response in 68% guinea pigs (68/100 ears) did not increase. The data of DPOAE and the serum IgG levels show no difference compared with the control group. There were not pathological observation in spiral ganglion cells, cochlear nucleus and cochlear nerve.</p><p><b>CONCLUSION</b>An animal model of autoimmune auditory neuropathy has been set up successfully and the character of the ABR and DPOAE was observed.</p>

Structure and function influence of cochlear after dexamethasone intratympanic application / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To study the changes of the structure and function of cochlear after dexamethasone intratympanic application.</p><p><b>METHODS</b>Forty-five guinea pigs were divided into three groups as normal group, 0.9% sodium chloride injection group and 0.5% dexamethasone group. By using the auditory brainstem response (ABR), the superoxide dismutase (SOD) activity, the nitric oxide (NO) value and cochlear surface preparation technique, the changes of structure and function of cochlear after intratympanic Dexamethasone application has been investigated in this study.</p><p><b>RESULTS</b>There was not significant difference among three groups in ABR thresholds of wave III (F = 0.5, P = 0.5). There was not significant difference among three groups in SOD activity (F = 2.45, P = 0.96). There was not significant difference among three groups in NO value (F = 3.1, P = 0.3). There was not significant difference among three groups in necrotic values of hair cells on the cochlear surface specimens in 15 mm from basal turn (F = 0.93, P = 0.45).</p><p><b>CONCLUSION</b>There is no change of cochlear construction and function after intratympanic Dexamethasone application.</p>

Distribution of basic fibroblast growth factor in inner ear of guinea pigs injected by different drug-approaches / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the approach of basic fibroblast growth factor(bFGF) entering inner ear, as well as its the protective mechanism to inner ear and nerve tissue in pathological situation.</p><p><b>METHODS</b>125I-bFGF was injected into guinea pigs body via the lateral ventricle and muscle under physical situation as well as pathological situation. Then the per minute gamma-radioactive in blood, liver, thyroid gland, brain, cochlear and perilymph fluid was counted, and the distribution and metabolism of bFGF in the inner ear and autoradiography of the cochlea were also observed.</p><p><b>RESULTS</b>Gamma-radioactive cpm of blood and liver increased significantly, while it did not change in brain, cochlea and perilymph after 125I-bFGF intramuscular injections. Gamma-radioactive cpm in blood, liver, brain, perilymph and cochlea had increased and autoradiography granules was found in the cochlea in 30 min after 125I-bFGF injected into CSF. In brain, perilymph and cochlea, a maximal value of gamma-radioactive cpm was obtained between 2 h and 4 h, while that in 8 h decreased significantly. Autoradiography granules still were seen in 8 h. gamma-radioactive cpm in 12 h was still higher than that in control group, but autoradiography granules can't be seen. The result in 24 h was similar to that in control group. The time course of cpm in the blood, cochlea and perilymph always parallel changed.</p><p><b>CONCLUSIONS</b>bFGF has some difficulties in getting across blood-labyrinth barrier (BLB) and blood-brain barrier (BBB) under physical and pathological situation, such as acute anoxia, aminoglycoside-induced deafness. bFGF can reach inner ear, perilymph fluid, brain tissue and blood rapidly when it is injected into CSF and excreted slowly in those tissues. Permeability of BBB and BLB to bFGF is similar and has orientation.</p>

Microsatellite instability and loss of heterozygosity of fragile histidine triad gene in laryngeal squamous cell carcinoma / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the loss of heterozygosity (LOH) and microsatellite instability (MSI) of fragile histidine triad (FHIT) gene in laryngeal squamous cell carcinoma (LSCC).</p><p><b>METHODS</b>Using polymerase chain reaction-simple sequence length polymorphism (SSLP) -silver staining technique, 41 samples of LSCC were detected for LOH and MSI with microsatellite sites D3S1234 and D3S1300.</p><p><b>RESULTS</b>LOH was found in 44.4% (16/36) and 36.4% (12/33) of informative cases at D3S1234 and D3S1300 respectively, while MSI at the two loci was found in 19.4% (7/36) and 24.2% (8/33) of informative cases respectively. In 41 cases of LSCC, 38 cases were informative at either locus. 52.6% (20/38) of cases had LOH while 28.9% (11/38) had MSI. The rate of LOH was related to the tumor TNM stage, pathological grade, lymph node metastasis and tumor relapse (P < 0.05). The rate of MSI was related to the tumor lymph node metastasis (P < 0.05 ).</p><p><b>CONCLUSION</b>The frequencies of LOH and MSI in the two microsatellite sites, D3S1234 and D3S1300, of FHIT gene might play a role in carcinogenesis and development of LSCC and might provide some new approaches for early gene detection for LSCC.</p>