喉癌及喉正常黏膜组织蛋白质组差异表达的初步研究

目的 研究喉癌及癌旁喉正常黏膜组织蛋白质组的差异表达。方法 用固相PH梯度双向凝胶电泳分离喉癌及癌旁喉正常黏膜组织总蛋白质，银染显色、PD(HYEST2DE软件分析所获得的双向凝胶电泳图谱，找出差异表达的蛋白质点。取差异表达的蛋白质点进行质谱鉴定。结果 获得分辨率和重复性均较好的双向凝胶电泳图谱，并鉴定出与喉癌发生密切相关的12种蛋白质。其中有10种蛋白质在喉癌组织中特异表达，分别是An-giopietin-2，sprouty homologues，Inhibitor of apoptosis-1ike protein2(ILP-2)，urokinase type plasminogen activator re-ceptor uPAR，Wnt-3a protein，癌基因Transforming protein N-Ras、P21以及c-Met．等.connexin31．1，原肌球蛋白Tropomyosin两种蛋白质在癌旁喉正常黏膜组织中特异表达。结论 本研究利用固相PH梯度双向凝胶电泳技术分离喉癌及癌旁喉正常黏膜组织总蛋白质，首次建立了重复性较强的喉癌及喉正常黏膜组织蛋白质组表达图谱，且两者蛋白质的表达明显不同，差异表达的蛋白质分别参与细胞的癌变、细胞间的信号传导、肿瘤血管生成，这对研究喉癌的癌变机制，寻找与喉癌诊断和治疗相关的肿瘤标志物相当重要。     

慢性鼻-鼻窦炎鼻息肉与正常鼻黏膜差异蛋白质的检测及其意义

目的采用蛋白质组学技术筛选慢性鼻-鼻窦炎、鼻息肉和正常鼻黏膜组织之间差异表达的蛋白质,初步鉴定出慢性鼻-鼻窦炎和鼻息肉的候选蛋白质标记物。方法应用固相pH4~7胶条行双向凝胶电泳,胶体考马斯亮蓝染色后,扫描2-DE胶,应用PDquest图像分析软件比较慢性鼻-鼻窦炎、鼻息肉和正常鼻黏膜组织2-DE图谱,得到三组之间差异表达的蛋白质点。经MALDI-TOF-MS鉴定后获得这些差异表达蛋白质的肽质量指纹图谱,用Mascot软件查询NCBInr和SWISS-PROT数据库,得出被测蛋白质的鉴定结果。结果所获双向凝胶电泳图谱分辨率高、重复性好。测得鼻息肉、慢性鼻-鼻窦炎及正常鼻黏膜组织的蛋白质平均斑点数分别为1020±40、1112±10和1008±25;平均匹配率分别为(93±2)%、(95±1)%和(90±3)%。三组之间共计有13个明显差异表达的蛋白质点。初步筛选出角蛋白8和阿朴脂蛋白AI作为鼻息肉的候选标志物,以及PLUNC蛋白、超氧化物歧化酶、自然杀伤细胞促进因子B、垂体腺苷酸环化酶激活肽为慢性鼻-鼻窦炎的候选标志物。结论用蛋白质组学技术能高通量筛选慢性鼻-鼻窦炎、鼻息肉和正常鼻黏膜间存在的差异表达蛋白质,这将为慢性鼻-鼻窦炎、鼻息肉分类、分型分期和预后判断标准寻找新的客观参考指标。     

蛋白质组学方法鉴定喉鳞状细胞癌中的肿瘤相关蛋白

目的利用蛋白质组学方法，建立人喉鳞状细胞癌（简称鳞癌）组织及其癌旁正常黏膜组织的双向凝胶电泳图谱，并识别鉴定其差异表达的蛋白质。方法利用固相pH梯度分离人喉鳞癌组织及癌旁正常黏膜组织的总蛋白质，用图像分析软件比较，以识别差异表达的蛋白质；应用质谱仪得到相应的肽质指纹图谱，’然后搜索数据库鉴定部分差异蛋白质点。结果获得分辨率和重复性均较好的双向凝胶电泳图谱，并鉴定出可能与喉癌发生密切相关的13种蛋白质。其中有10种蛋白质在喉癌组织中上调，分别是切丝蛋白1（cofilin-1），核体蛋白SP140（nuclear body protein SP140），内质网素，热休克蛋白90（heatshockprotein90，HSP90），谷胱甘肽S转移酶P（glutathioneS-transferase，GSTP1—1），锰-超氧化歧化酶（superoxidedismutase[Mn]），亲环素A（cyclophilinA），蛋白酶活性复合体2（proteasome activator complex subunit2），载脂蛋白A-I前体（apolipoproteinA．Iprecursor），钙调素（CaM—like protein）。3种蛋白质在喉癌组织中下调，分别是银屑病相关脂肪酸结合蛋白（fatty acidbinding protein，epidermal，E—FABP），钙粒蛋白A（calgranulin A），钙粒蛋白B（calgranulin B）。结论本研究鉴定出了可能与喉癌的发生密切相关的13种蛋白质，提示多种蛋白分子可能同时成为应对这种疾病的有效策略的靶分子，为理解喉癌的癌变机制和改善临床诊治方法提供理论基础。     

鼻内翻性乳头状瘤与鼻息肉和对照组鼻黏膜组织差异蛋白分析

目的 应用蛋白质组学技术,分析比较鼻内翻性乳头状瘤(nasal inverted papilloma,NIP)与鼻息肉、对照组鼻黏膜组织的差异蛋白质,筛选具有特异性表达的蛋白.方法 分别采集3例NIP组织、3例鼻息肉组织以及3例单纯鼻中隔偏曲患者(对照组)的中鼻甲黏膜组织标本,应用双向凝胶电泳(two-dimensional gel electrophoresis,2-DE)技术分离各组织的总蛋白质,利用GS-800Calibrated Densitometer凝胶成像系统获取图像,识别差异表达蛋白质点,查询数据库,鉴定差异表达蛋白质.结果 鉴定了6个NIP与鼻息肉组织有明显差异表达的蛋白质点,分别是半乳糖凝集素1、锰-超氧化物歧化酶、半乳糖凝集素7、曲古抑菌素A、抑制素、转铁蛋白.这6个蛋白质点在NIP中的表达均明显增强.结论 应用蛋白质组学研究方法可以对鼻黏膜组织进行差异蛋白分析,鉴定出6个差异表达蛋白质可能与NIP的发病相关.     

c-Cbl在喉癌中的表达及临床意义CSCD

目的探讨c-Cbl在喉癌组织中的表达及临床意义。方法应用免疫组化(SP)法检测40例喉癌组织、15例癌旁组织和8例喉正常黏膜组织中c-Cbl蛋白的表达情况。应用流式细胞术定量检测40例喉癌组织、13例癌旁组织和5例喉正常黏膜组织中c-Cbl的含量。结果免疫组化示喉癌组织、癌旁组织及喉正常黏膜组织中c-Cbl的表达强度呈下降趋势,3组相比有显著性差异。应用流式细胞术检测c-Cbl蛋白在喉癌组织、癌旁组织及喉正常黏膜组织中的表达量分别为1.8424±0.1814、1.6350±0.3889、1.1026±0.0181,3组相比有显著性差异。在喉癌组织中c-Cbl蛋白表达与其病理分级有关。结论 c-Cbl蛋白与喉癌的发生发展有关。     

鼻息肉、鼻息肉病及对照鼻黏膜蛋白质双向电泳图谱的建立

目的　建立鼻息肉、鼻息肉病及对照鼻黏膜组织蛋白质双向凝胶电泳 (2 DE)图谱。方法　收集并- 80℃冻存鼻息肉、鼻息肉病及鼻黏膜组织样本各 3例 ,双向凝胶电泳分离样本总蛋白、银染凝胶显色 ,扫描图像 ,ImageMaster 2 DEElite4 .0 1软件分析。结果　鼻息肉、鼻息肉病和鼻黏膜组织同一样本三块凝胶平均蛋白质点数分别为 82 5± 78个 ,891± 6 7个和 936± 6 2个 ;平均匹配点数分别为 6 82± 96个 ,76 7± 83个和 82 1± 78个 ,其平均匹配百分率分别为 82 .7%、86 .1%和 87.7% ;位置重复性分析 ,IEF方向平均偏差为 1.13± 0 .16mm ,SDS PAGE方向平均偏差为 1.4 5± 0 .2 1mm。鼻息肉、鼻息病和鼻黏膜三种组织各 3例样品的平均蛋白质点数分别为 876± 95个 ,95 3± 84个和 10 16± 79个。三种组织的 2 DE平均凝胶图谱 :鼻息肉、鼻息肉病和鼻黏膜蛋白质点数分别为116 2个、12 74和 1336个 ,平均匹配点数为鼻息肉与鼻黏膜之间 75 7个 ;鼻息肉病与鼻黏膜之间为 82 5个 ;鼻息肉与鼻息肉病之间为 883个。结论　本实验建立了图像清晰、分辨率高和重复性好的鼻息肉、鼻息肉病及鼻黏膜组织的固相PH梯度 2 DE图谱 ,有助于进一步识别鉴定三者差异表达的蛋白质。     

鼻息肉病与正常鼻黏膜组织的蛋白质组差异分析

目的:建立分辨率高和重复性好的鼻息肉病及对照鼻黏膜双向凝胶电泳(2DE)图谱,筛选鉴定差异表达蛋白质。方法:收集鼻息肉病(鼻息肉病组)及对照鼻黏膜标本(对照组)各7例,固相pH梯度2DE、凝胶银染,扫描图像,ImageMaster2DEElite4.01软件分析,识别差异表达蛋白质,质谱仪得到相应肽质指纹图谱(PMF),PeptIdent软件查询SwissProtandTreMBL数据库,鉴定差异表达蛋白质。结果:①鼻息肉病组和对照组各自随机挑选的1个样本3次重复2DE,平均蛋白质点数分别为891±67和936±62;匹配点数为767±83和821±78,平均匹配百分率为86.1%和87.7%;同一鼻息肉病的3块胶在蛋白质点位置上有较好的重复性,不同胶间蛋白质点在等电聚焦电泳(IEF)方向偏差为(1.13±0.16)mm,在SDS聚丙烯酰胺凝胶电泳(SDSPAGE)方向偏差为(1.45±0.21)mm。分析2种组织各7例样本的2DE图谱的平均凝胶图谱,鼻息肉病组和对照组蛋白质点数为1532和1617,匹配点数为1065个。②质谱分析差异蛋白质点20个,获取16张PMF,查询数据库鉴定出差异蛋白质11个。结论:本研究建立了分辨率高和重复性好的鼻息肉病及鼻黏膜的2DE图谱,识别鉴定出一些与鼻息肉病变相关的差异表达蛋白质。     

成纤维细胞生长因子2型受体在喉癌中的表达及临床意义

目的　探讨成纤维细胞生长因子2型受体（fibroblast growth factor 2,FGFR2）在喉癌中的表达及临床意义。方法　应用免疫组织化学（SP法）检测FGFR2蛋 白在40例喉癌组织、15例癌旁组织和8例正常喉黏膜中的表达情况。应用流式细胞术的方法定量检测40例喉癌组织、13例癌旁组织和5例正常喉黏膜组织中FGFR2的含量。结果　喉癌组织、癌旁组织及正常喉黏膜中FGFR2蛋白的表达强度呈下降趋势,差异有统计学意义（H =11.4573,P <0.01）。FGFR2蛋白在喉癌组织、癌旁组织及正常黏膜中的表达量分别为1.8776±0.1683、1.1815±0.2710和1.0100±0.1341,差异有统计学意义（F =93.1797,P =0.0000）。喉癌组织中FGFR2蛋白的表达量明显高于癌旁组织及正常喉黏膜组织,而癌旁组织与正常喉黏膜相比无显著性差异。喉癌组织中FGFR2蛋白表达与病理分级有关,而与其临床分期、淋巴结转移、临床分型、肿瘤大小、吸烟量、年龄和性别均无关。结论　FGFR2蛋白在喉癌中的发生、发展有一定的相关性。     

圆锥角膜组织与正常角膜组织差异蛋白的表达

目的运用比较蛋白质组学技术,比较圆锥角膜组织和正常角膜组织的差异表达蛋白,筛选出圆锥角膜特异性分子标记物,探讨其与圆锥角膜发病机制的关系。方法收集12例圆锥角膜组织和4例正常人角膜组织,提取角膜组织总蛋白,行固相PH梯度双向凝胶电泳,分析凝胶电泳图谱,筛选差异表达蛋白,采用基质辅助激光解吸离子化一串联飞行时间质谱(MOLDITOF/TOFMS)和数据库检索对差异蛋白质进行鉴定。结果圆锥角膜组织和正常角膜组织的双向电泳代表胶的蛋白点数分别为1035和1070,匹配点数为869个,匹配率为84%。选取有明显表达差异的7个蛋白质点,其中6个蛋白质点在圆锥角膜组织中表达下调,1个蛋白质点表达上调。这7个蛋白质点分别为转化生长因子B诱导的细胞外基质黏附蛋白(TGFBIp/βigh3)、VI型胶原蛋白α1链前体(collagenalpha1(VI)chainprecursor)、αA晶体蛋白(alphacrystallinA)、βB晶体蛋白(alphacrystallinB)、βB2晶体蛋白(betacrystallinB2)、谷氨酰胺合成酶(glutaminesynthetase)、碳酸酐酶(carbonicanhydrase),除VI型胶原蛋白α1链前体在圆锥角膜组织中表达上调外,其余6个蛋白成分在圆锥角膜组织中均表达下调。结论TGFBIp/βigh3、alphacrystallinA、alphacrystallinB、betacrystallinB2、glutaminesynthetase、collagenalpha1(VI)chainprecursor6种在圆锥角膜中差异表达的蛋白质成分可能与圆锥角膜发病相关。     

喉癌上皮细胞钙黏素和α-连环素的表达与临床因素的关系

目的探讨上皮细胞钙黏素（E-cadherin,E-cad）、α-连环素（α-catenin,α-cat）与喉癌各临床因素及预后的相关性.方法采用免疫组化检测79例喉癌患者肿瘤组织和10例正常喉黏膜的E-cad、α-cat表达.结果正常喉组织中E-cad、α-cat蛋白均呈阳性表达,喉癌中E-cad、α-cat阳性表达率分别为34.18%和40.51%,与正常喉组织比较有显著性差异（P=0.000）.喉癌E-cad、α-cat阳性表达组的5年生存率分别为63.80%、81.74%,而阴性表达组的5年生存率分别为50.68%和41.09%,阳性表达组的5年生存率均明显高于阴性表达组（P＜0.05和P=0.000）.α-cat蛋白表达与喉癌分型、颈淋巴结转移、临床分期、T分期、复发相关（P＜0.05）.结论E-cad、α-cat异常表达在喉癌的恶性进展过程中可能发挥重要作用,检测α-cat对喉癌患者预后评估有一定意义.     

DNA content in laryngeal keratosis, laryngeal cancer and normal laryngeal epithelium

The DNA content in laryngeal precancerous lesion, laryngeal keratosis, laryngeal carcinoma and normal laryngeal epithelium had been measured in order to study the relationship between histopathologic picture and DNA content. The results showed that the DNA content in keratotic tissue increased to varying degrees as compared with the normal tissue. The DNA content in laryngeal cancer increased distinctly, being much higher than that in normal and keratotic tissues.     

Reflex control on laryngeal functions--vibration effect of the laryngeal mucosa on recurrent laryngeal nerve reflexes

Electromyographic (EMG) responses of the intrinsic laryngeal muscle have been investigated to clarify reflexogenic laryngeal controls from a viewpoint of its functional significance during phonation. Twenty-five adult cats were anesthetized with intraperitoneal injection of 4ml/kg of a mixture of 10% urethane and 1% alpha-chloralose. Either the internal branch of the superior laryngeal nerve (ISLN) or the recurrent laryngeal nerve (RLN) was carefully dissected and central end of the dissected nerve was electrically stimulated. EMG of the contra-lateral Thyro-Arytenoid muscle (TA muscle) to the stimulation was recorded using a hooked-wire electrode inserted through the laryngeal mucosa. EMG of the TA muscle evoked by the stimulation of the ISLN were analyzed with respect to its latency and discharge pattern inter-collicular brainstem transsected. Together with the stimulation of the RLN, vibratory stimuli were given mainly to the subglottic mucosa as conditioning stimuli. The vibratory frequency was changed from 50Hz to 400Hz step-wisely. Following results were obtained. 1. EMG response of the contra-lateral TA muscle to the stimulation of the ISLN showed two different kinds of latency, approximately 8-10msec, and 40-60msec. 2. After inter-collicular brainstem transsection, evoked response of the latter disappeared. This result indicates that the ISLN-RLN reflex loop consisted of more than two routes, different in the number of synaptic junctions. 3. The vibratory stimuli given to the laryngeal mucosa had facilitatory effect on the reflexive EMG response evoked by the stimulation of the RLN. 4. This facilitatory effect of the vibratory stimuli disappeared after topical anesthesia of the laryngeal mucosa. 5. The facilitatory effect on the reflex responses was partially increased depending on the vibratory frequencies applied. In conclusion, vibratory stimuli to the laryngeal mucosa reflexively modulate the activity of the intrinsic laryngeal muscles.     

保留功能的喉癌手术70例报告

目的 :探讨喉部分切除术治疗声门型、声门上型喉癌的远期疗效和功能恢复。方法 :对 1978年 7月～1998年 8月间手术的 70例临床资料进行总结和随访。其中声门型喉癌 6 0例、声门上型喉癌 10例 ;施行喉裂开声带切除术 2 2例 ,垂直半喉切除术 2 2例 ,Majer- Piquet手术 17例 ,水平半喉切除术 7例 ,Arslan手术 2例。结果 :1、3及 5年生存率分别为 98.5 3%、87.0 4%和 78.2 6 %。拔管率为 10 0 %。全部病例恢复经口进食 ,一经拔管均能发音。并发症发生率和术后复发率各为 15 .71%和 13.0 4%。结论 :喉部分切除术是功能保全性喉部恶性肿瘤根治的有效术式。它在切除肿瘤、延长生命的同时可以较好地保留喉的生理功能 ,提高患者术后的生活质量     

Three-dimensional laryngeal model for planning of laryngeal framework surgery

CONCLUSION: The three-dimensional prototype model was useful for planning of laryngeal framework surgery. OBJECTIVE: To discuss the usefulness of a three-dimensional laryngeal model for laryngeal framework surgery. MATERIALS AND METHODS: A three-dimensional laryngeal model was created based on the postoperative helical computed tomography (CT) data of the larynx (case 1) which underwent lateral cricoarytenoid muscle (LCA) pull surgery. LCA pull surgery is a kind of arytenoid adduction for unilateral vocal cord paralysis. A three-dimensional model of case 1 larynx was prototyped using a selective laser sintering method. In case 1, the patient's voice did not improve after LCA pull surgery. The three-dimensional model revealed that the original surgical procedure was not appropriate to obtain optimal arytenoid adduction. According to the analysis of this three-dimensional model, we changed the surgical approach and performed this new refined LCA pull surgery on another patient with unilateral vocal cord paralysis (case 2). RESULTS: We were able to pull LCA precisely in case 2. Three-dimensional CT of case 2 after refined LCA pull surgery allowed the correct pulling of LCA and complete adduction of arytenoid. The postoperative voice improved remarkably.     

Magnesium content of laryngeal precancerous conditions and laryngeal cancers

The content of magnesium in tissues, erythrocytes and serum was analysed by atomic absorption spectrometry. A higher concentration of magnesium was found in malignant laryngeal tissue and in lymph nodes of the neck compared to homologous pre-cancerous tissue. In pre-cancerous lesions of the larynx the magnesium concentration of the erythrocytes and serum lay at the lower limit of normal. In patients with cancer of the larynx, the magnesium concentration was considerably below the normal levels and continued to decline as the disease progressed.     

Three-dimensional laryngeal model for planning of laryngeal framework surgery

Conclusion. The three-dimensional prototype model was useful for planning of laryngeal framework surgery. Objective: To discuss the usefulness of a three-dimensional laryngeal model for laryngeal framework surgery. Materials and methods. A three-dimensional laryngeal model was created based on the postoperative helical computed tomography (CT) data of the larynx (case 1) which underwent lateral cricoarytenoid muscle (LCA) pull surgery. LCA pull surgery is a kind of arytenoid adduction for unilateral vocal cord paralysis. A three-dimensional model of case 1 larynx was prototyped using a selective laser sintering method. In case 1, the patient's voice did not improve after LCA pull surgery. The three-dimensional model revealed that the original surgical procedure was not appropriate to obtain optimal arytenoid adduction. According to the analysis of this three-dimensional model, we changed the surgical approach and performed this new refined LCA pull surgery on another patient with unilateral vocal cord paralysis (case 2). Results. We were able to pull LCA precisely in case 2. Three-dimensional CT of case 2 after refined LCA pull surgery allowed the correct pulling of LCA and complete adduction of arytenoid. The postoperative voice improved remarkably.     

喉返神经麻痹的肌电特点及相关研究

目的：研究喉返神经麻痹患者不同时间和损伤程度的喉肌电特点。方法：根据发病时间将87例喉返神经麻痹患者分为7组,分析其喉肌电特点,并与30例正常受试者进行对比研究。结果：①发病2周时,最早5d在受累甲杓肌、环杓后肌和环杓侧肌即可出现纤颤波和正锐波等失神经电位;2周-3个月失神经电位增多,可见再生电位;3个月后失神经电位渐少,再生电位渐多;36个月后失神经电位消失。②受累喉肌肌电募集减弱甚至无明显干扰相,干扰相波幅和转折数显著低于正常对照组,其中转折数减低更为明显,未受累喉肌募集电位明显增大。③部分患者受累喉肌诱发电位消失,其他患者可见波幅小、潜伏期长的诱发电位。结论：喉肌电图对于喉返神经麻痹的诊断和鉴别具有重要意义,失神经电位和再生电位特点与神经损伤的程度和时间相关,异常的诱发电位可提示神经损伤的程度。     

Effect of deficits in laryngeal sensation on laryngeal muscle biochemistry

Swallowing deficits in elderly people are significant clinical problems and may be associated with impaired pharyngolaryngeal sensation. However, the extent to which sensory innervation affects the motor system is unclear. Our purpose was to examine differences in biochemical properties of laryngeal muscles following sensory nerve ablation. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to evaluate laryngeal muscles of young and old Fischer 344/Brown Norway rats, and rats that underwent sensory ablation via bilateral section of the superior laryngeal nerve, internal branch (SLNi), or mixed sensory-motor nerve ablation via left-sided recurrent laryngeal nerve (RLN) section. In lateral thyroarytenoid muscle, a reduction was found in the proportion of the most rapidly contracting myosin heavy chain isoform (type 2B) with SLNi section, RLN section, and aging. Section of the SLNi did not alter the proportion of any myosin heavy chain isoform within the lateral cricoarytenoid or posterior cricoarytenoid muscles, but RLN section resulted in a reduction in the proportion of type 2B. Accordingly, alteration in biochemical properties of the lateral thyroarytenoid muscle alone was demonstrated following sensory ablation. We conclude that sensory changes may affect properties of laryngeal muscles, and may thus have an impact on motor control during critical functions, such as airway protection during swallowing.     

Spasmodic laryngeal dyspnea: a rare manifestation of laryngeal dystonia

We describe clinical experiences in the management of three patients with laryngopharyngeal dystonia causing severe breathing problems. In contrast to spasmodic dysphonia, which presents with action-induced involuntary spasms of laryngeal muscles during speaking, all three patients showed laryngopharyngeal spasms primarily during respiration. In analogy to spasmodic dysphonia we propose the term spasmodic laryngeal dyspnea for this rare condition. Localized unilateral botulinum toxin injected into the thyroarytenoid muscle and /or ventricular folds reduced the quantity and quality of spasms and led to a pronounced improvement of breathing problems.