Construction of a recombinant eukaryotic vector of Atoh1 and its expression in 293-T cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To clone the coding sequence of Atoh1 cDNA from SD rat and construct a eukaryotic expression vector for its expression in eukaryotic cells.</p><p><b>METHODS</b>The total RNA was extracted from colonic mucosa of SD rats and the double-stranded cDNA of Atoh1 was amplified using RT-PCR. The cDNA coding sequence was then cloned into PMD-19T vector followed by sequence analysis. The digested fragment, after purification, was linked into the eukaryotic expression vector pIRES2-EGFP containing the EGFP gene and the internal ribosomes site (IRES). After identification by enzyme digestion and sequence analysis, the recombinant plasmid was transfected into 293T cells via Lipofectamine, and the expression of the target protein was detected under fluorescence microscope, using PT-PCR and Western blotting.</p><p><b>RESULTS</b>DNA sequence analysis showed that the amplified rat Atoh1 gene, with a length of 1056 bp of the coding sequence which encoded 351 amino acids, had two base mutations compared to the reference sequences in GenBank, possibly as a result of single nucleotide polymorphisms that induced nonsense mutation without affecting the amino acid sequences or the protein expression. The results of enzyme digestion and sequence analysis demonstrated that the Atoh1 gene was correctly inserted in the eukaryotic expression vector plRES2-EGFP. Fluorescence microscopy and Western blotting both confirmed successful expression of Atohl at the mRNA and protein levels in 293T cells 48 h after transfection with the recombinant plasmid.</p><p><b>CONCLUSION</b>The recombinant plasmid harboring the coding sequence of SD rat Atoh1 gene has been constructed successfully and can be expressed in the 293T cells, which provides a basis for functional study of Atoh1 gene and future researches in gene therapy for sensorineural hearing loss.</p>

Long term follow up of midline partial glossectomy and uvulopalatopharyngoplasty for treating obstructive sleep apnea hypopnea syndrome / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To observe the long-term results of midline partial glossectomy with uvulopalatopharyngoplasty (UPPP) in the treatment of obstructive sleep apnea hypopnea syndrome (OSAHS).</p><p><b>METHODS</b>Twenty-four severe OSAHS patients treated with midline partial glossectomy and UPPP from January 2003 to March 2004 were included in this study, the follow-up was 5 years. The median of preoperative lowest arterial oxygen saturation (LSaO(2)) of this group at night (the same below) 0.650, and AHI was 56.5 times/h, UPPP was performed under general anesthesia, no tracheotomy performed. Criteria of curative effects: AHI < 5 times/h was recovery, AHI < 20 times/h and decreased beyond 50% marked improvement, only AHI decreased beyond 50% improvement.</p><p><b>RESULTS</b>Post-operation AHI (6 months, 1 year, 2 years and 5 years after surgery) decreased significantly compared to that before the surgery, and post-operation LSaO(2) was significantly higher than that of preoperative (Wilcoxon's signed rank test, the same below, P < 0.01). The LSaO(2) and AHI were significantly different between 1 year, 2 years, 5 years and 6 months post-operatively (P < 0.01). Six months after surgery, PSG results showed that 21 were recovery, marked improvement for the other 3 cases, the recovery rate was 87.5%. One year after surgery, 18 were recovery, marked improvement in 3 cases, the recovery rate 75.0%. Two years after surgery, 14 cases recovery, marked improvement in 4 cases, the recovery rate 58.3%. Five years after surgery, 6 were recovery, the recovery rate 25.0%. Among 5 cases with hypertension before the surgery, after surgery antihypertensive drugs were not necessary in 4 cases, and the dosage was decreased in 1 case.</p><p><b>CONCLUSION</b>The midline partial glossectomy with UPPP surgery may be an effective treatment for the severe OSAHS, long-term effect is satisfactory.</p>

Construction of expression vector for NT4-ADNF-9 fusion gene / 西安交通大学学报·英文版

Objective: To construct the prokaryotic expression vector bearing fusion gene NT4-ADNF-9 and lay foundation for further study on genetic therapy of neurosensory deafness. Methods: By means of asymmetrical primer/ template, double stranded cDNA of activity dependent neurotrophic factor-9 (ADNF-9) was obtained, which included restriction enzymes sites on the two extremities. ADNF-9 cDNA was ligated to the signal and leader peptides of neurotrophin 4 (NT4), and the fusion gene was named NT4-ADNF-9. Then it was subcloned into prokaryotic expression vector pBV220, and called pBV220/ NT4-ADNF-9. Results: Evidences of DNA sequence analysis and restriction enzymes digestion showed that we recombined ADNF-9 cDNA to the 3′terminal of the signal and leader peptides of NT4, and the fusion gene was subcloned into pBV220 successfully. Bioactivity of the products was proved that it could support the cell survival and neurite growth in the primary cultures of dorsal root ganglia (DRG) of embryonic day-8 chicken neurons as compared to the control. Conclusion: Prokaryotic expression vector pBV220/NT4-ADNF-9 can be constructed successfully and the bioactivity is satisfactory.

Growth retardation in children with obstructive sleep apnea hypopnea syndrome / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To observe changes of growth, body composition and biochemical markers associated with growth (IGF-1) in prepubertal children with obstructive sleep apnea syndrome (OSAHS).</p><p><b>METHODS</b>Thirty-one children aged 3-10 years with OSAHS were followed up for 1 year after the corresponding surgery. During the same period of time, 20 children of similar age without OSAHS (excluded any other diseases that could result growth retardation or hypoxemia) were also followed up for 1 year. PSG, height, weight as well as insulin-like growth factor-1 (IGF-1) were measured during the preoperative period, 3 months, 6 months and 1 year after surgery in patient group. The same indexes were measured before surgery, and only height and weight were recorded after surgery in the control group. Wilcoxon signed- rank test and Mann-Whitney U test are used to analyze the data.</p><p><b>RESULTS</b>The lowest oxyhemoglobin saturation of the patient group (0.88) is significantly lower than that of the control group (0.98), and was found increased at the 6 months post-op follow up (0.97, U = 238.5, P > 0.05), no significant change was found at the 1 year follow up. The post-op AHI (6 months after surgery) of the patient group (6.0/h) decreased to the similar level of the control group (0/h, U = 240.0, P > 0.05), and was similar to 1 year after surgery. Height of the patient group (116 cm), which was lower than the control group (U = 127.0, P < 0.001), significantly increased 1 year (138 cm) after the corresponding surgery (Z = 3.726, P < 0.01), and reached the similar levels of the control group (137 cm) 1 year after the surgery (U = 123.5, P > 0.05). The serum IGF-1 levels of the patient group (33.7 ng/ml), which were significantly lower than those of the controls preoperatively (44.1 ng/ml, U = 206.0, P < 0.05), increased to similar levels with the controls 6 months after the operation (50.3 ng/ml, U = 261.0, P > 0.05), and the 1 year post-op follow up was similar to the control group too (48.6 ng/ml, U = 163.0, P > 0.05).</p><p><b>CONCLUSIONS</b>The cure of OSAHS could accelerate growth in prepubertal children, and the serum IGF-1 levels increases at the same time. The growth retardation is presumed in children with OSAHS.</p>

Theory of local inflammatory infiltration and irritation in the pathogenesis of acquired cholesteatoma / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To explore the deep pathogenesis of acquired cholesteatoma.</p><p><b>METHODS</b>The temporal bone slides of 12 ears with retraction pocket were histopathologically studied under microscope, especially focusing on the location of retraction pocket and inflammatory pathology in the local middle ear cavity next to retraction pockets. The temporal bone slides of 11 ears with acquired cholesteatoma were histopathologically observed and 33 cases diagnosed as acquired cholesteatoma were clinically observed observed in the local middle ear cavity next to the part without retraction pocket of eardrum. The results of pathological observation of the temporal bone slides with acquired cholesteatoma and clinical observation during operation for acquired cholesteatoma show that cholesteatoma invade mainly and occupied the ossicular chain eara of the middle ear cleft.</p><p><b>CONCLUSION</b>In the pathological process of otitis media, the intractable pathological changes in the ossicular chain area can inward adhere posterosuperior quadrant or pars flaccida of the eardrum to form retraction pocket and permanently infiltrate the external squamous epithelial layer of retraction pocket to excessively proliferate and keratinize, leading to formation of acquired cholesteatoma.</p>