CDglyTK融合自杀基因杀伤喉癌Hep-2细胞的体外实验研究

目的：研究融合自杀基因CDglyTK联合前体药物对喉癌Hep-2细胞的杀伤效应。方法：构建质粒表达载体pcDNA3．1（-）CMV．CDglyTK，XhoⅠ／HindⅢ酶切鉴定，测序分析CDglyTK基因序列。电穿孔法将重组质粒转染Hep-2细胞，400mg／LG418筛选14d获得稳定表达CDglyTK基因的Hep-2细胞，RT—PCR及Western-blotting鉴定CDglyTK基因的表达。以转染空白载体pcDNA3．1（-）的Hep-2细胞为对照，MTT法观察5-FC、GCV、5-FC加GCV对表达CDglyTK基因的Hep-2细胞生长的抑制作用。结果：酶切和基因测序分析证明重组质粒含完整的CD及TK基因，RT—PCR从转染细胞总RNA中扩出707bp的预期片段，Western-blotting检测到该基因表达的分子量为59000的蛋白。表达CDglyTK基因的Hep-2细胞在5-FC、GCV、5-FC加GCV干预下生长受到抑制，5-FC与GCV联合有更强的杀伤效应。结论：CDglyTK融合自杀基因可以成为基因治疗喉癌的有效方法。     

胞嘧啶脱氨酶尿嘧啶-磷酸核糖基转移酶融合基因/5-氟胞嘧啶治疗鼻咽癌的实验研究

目的研究胞嘧啶脱氨酶-尿嘧啶磷酸核糖基转移酶融合基因/5-氟胞嘧啶(fusion cytosine deaminase- uracil phosphoribosyl transferase gene/5-Flurocy- tosine,FCU/5-FC)对鼻咽癌CNE-2细胞的杀伤效应以及对鼻咽癌移植瘤生长的抑制作用。方法构建表达质粒载体pcDNA3．1(-)CMVe·Egr-1·FCU,BamHⅠ酶切鉴定重组质粒,分析FCU基因和融合启动子CMVe·Egr-1的序列。电穿孔法将重组质粒转染CNE-2细胞,300μg/ml～600μg/mlG418筛选14天获得稳定表达FCU基因的CNE-2细胞,RT-PCR鉴定FCU基因的表达。以转染空白载体pcDNA3．1(-)的CNE-2细胞为对照,MTT法观察5-FC对表达FCU基因的CNE-2细胞生长的抑制作用,考察FCU基因的细胞旁杀效应。5×10~6的CNE-2细胞接种裸鼠右腋下,建立鼻咽癌裸鼠移植瘤模型,12天后将裸鼠随机分成3组：CU组,CU+PBS组和CU+5-FC组,观察不同处理对移植瘤生长的抑制作用。结果酶切和序列分析证明重组质粒含完整的FCU基因和Egr-1启动子的核心序列,RT-PCR从转染细胞总RNA中扩出330bp的预期片段。表达FCU基因的CNE-2细胞在5-FC干预下,生长受到抑制。随着表达FCU基因的细胞比例增加,细胞存活率相应地下降。CU组,CU+PBS组移植瘤体积与CU+5-FC组有显著性差异(P＜0．05)。结论表达FCU基因的CNE-2细胞可以被5-FC杀伤,FCU/5-FC系统抑制了移植瘤生长,为治疗鼻咽癌提供了新的策略。     

建立稳定表达融合自杀基因CD／UPRT．UL49的CNE-2鼻咽癌细胞株

目的：建立稳定表达融合自杀基因CD／UPRT．UL49的CNE-2鼻咽癌细胞株。方法：构建表达载体pcDNA3．1（一）E6．BARFlP．cD／UPRT．UL49,经脂质体法转染CNE-2细胞,G418和前体药物5-氟胞嘧啶筛选出阳性克隆细胞并扩大培养。抽提阳性克隆细胞总蛋白质,Western-blotting检测目的基因表达。结果：融合自杀基因CD／UPRT．UL49在CNE2转染细胞内稳定表达。结论：本组通过脂质体转染技术,成功地建立了稳定表达融合CD／UPRT．UL49基因的CNE-2细胞株。     

hTERTp在鼻咽癌中放射敏感性的研究

目的观察放射干预对人端粒酶逆转录酶基因（hTERT）启动子启动活性以及鼻咽癌靶向调控的影响。方法利用基因重组技术构建pGL-3-hTERTp质粒以及pcDNA3．1．hTERT．CD／UPRT质粒,脂质体介导重组质粒pGL-3-hTERTp转染鼻咽癌CNE-2细胞以及人成纤维皮肤细胞（HDF）,并给予不同剂量放射干预,双荧光素酶活性分析其启动活性变化。pcDNA3．1．hTERT．CD／UPRT质粒转染鼻咽癌CNE-2细胞;运用RT-PCR以及免疫印迹法检测不同放射剂量下自杀基因表达。结果接受放射干预后,hTERT启动子活性明显增强,给予2、6、10Gy放射干预后,其启动活性分别增加了10倍、19倍和34倍,其差异有统计学意义（P〈0．01）。HDF组无论放射与否,其测量值与pGL-3Basic质粒无统计学差异（P〉0．05）。pcDNA3．1．hTERT．CD／UPRT质粒转染CNE-2细胞后,放射剂量增加至6、10Gy检测到目的基因表达。结论放射干预可提高hTERT启动子在鼻咽癌CNE-2细胞中的启动活性,且对其肿瘤特异性无影响。以放疗为主的肿瘤治疗中,hTERT启动子可能为潜在靶向性基因治疗的备选因子。     

pGC-silencer-U6/Neo/GFP/ABCG2真核表达质粒的构建及鉴定

目的:构建靶向人ABCG2基因的pGC-silencer-U6/Neo/GFP/ABCG2真核表达质粒,运用RNA干扰下调ABCG2基因在鼻咽癌细胞系CNE-2中的表达,并筛选出其基因沉默效果最明显的shRNA质粒表达载体.方法:针对ABCG2基因的mRNA序列设计3个干扰靶点,分别构建3个shRNA质粒表达载体和1个阴性对照质粒表达载体,经大肠杆菌扩增,酶切、PCR、测序鉴定,用脂质体法转染鼻咽癌CNE-2细胞,用real-time PCR和Western blot检测ABCG2基因在mRNA和蛋白水平的抑制效果.结果:经测序证实,成功构建pGC-silencer-U6/Neo/GFP/ABCG2真核表达质粒.重组质粒转染CNE-2细胞后,ABCG2基因的mRNA水平及蛋白表达水平明显下调,其中以1号重组质粒效应最强,mRNA水平下调75%,蛋白水平下调68%.结论:成功构建以人ABCG2为靶向的pGC-silencer-U6/Neo/GFP/ABCG2的重组质粒.其对鼻咽癌CNE-2细胞中ABCG2的表达具有显著抑制效应,为进一步研究ABCG2在CNE-2细胞中的功能和鼻咽癌的基因治疗奠定了基础.     

靶向性质粒表达载体pcDNA3.1(-)CMV·Egr-1CDglyTK的构建及转染研究

目的 构建靶向性基因治疗载体pcDNA3.1(-)CMV·Egr—1CDglyTK并进行转染研究。方法采用PCR、RT—PCR、融合PCR、酶切、连接等技术构建pcDNA3.1(-)CMV·Egr—1CDglyTK表达载体,成功转染鼻咽癌CNE—2细胞,绘制细胞相对存活率曲线,初步研究该载体在前体药物干预下对CNE—2细胞生长的影响。结果 表达pcDNAA3.1(-)CMV·Egr—1CDglyTK的CNE—2细胞在5—FC/GCV干预下生长受到抑制。结论pcDNA3.1(-)CMV·Egr—1CDglyTK可作为鼻咽癌基因治疗的载体。     

质粒载体介导CD基因对喉癌细胞的杀伤作用研究

目的构建含酵母菌自杀基因CD的质粒表达载体pcDNA3.1(-)CMV.CD,并利用该载体进行喉癌Hep-2细胞株转染研究,体外实验观察前体药物5-FC对稳定表达CD基因的喉癌Hep-2细胞株的杀伤作用.方法通过RT-PCR从酵母菌RNA内扩增出CD基因全长CDS序列,将其定向克隆到质粒表达载体pcDNA3.1(-)CMV中,重组体质粒经XhoI/HindⅢ双酶切鉴定,并对重组体中的CD基因片段进行序列分析,将鉴定好的阳性重组质粒pcDNA3.1(-)CMV.CD运用电穿孔法转入喉癌Hep-2细胞中.经300～600 μg/mlG418正筛选14 d和10 μg/ml前体药物5-FC负筛选,获得稳定表达CD基因的喉癌Hep-2细胞株,提取该细胞株细胞的总RNA,经RT-PCR鉴定CD基因的表达.MTT法观察不同浓度5-FC对稳定表达CD基因的喉癌Hep-2细胞及转染空白载体pcDNA3.1(-)CMV的对照组喉癌Hep-2细胞的杀伤作用.结果阳性重组质粒pcDNA3.1(-)CMV.CD经XhoI/HindⅢ双酶切后获得5353 bp的片段及496 bp的插入片段,DNA自动序列分析证明重组体质粒含完整的477 bp长的CD基因CDS序列.RT-PCR从转染细胞总RNA中扩出453 bp的预期片段.当添加不同浓度的5-FC时,表达CD基因的Hep-2细胞不同程度地被杀死,而对照组的细胞几乎未受到影响,两组细胞的相对生存率具有显著差异性(P<0.05).结论成功构建了质粒表达载体pcDNA3.1(-)CMV.CD,建立了稳定表达酵母菌CD基因的喉癌表达CD基因的Hep-2细胞株,表达CD基因的Hep-2细胞可以被5-FC杀死.     

自杀基因质粒表达载体pcDNA3.1(-)CMV.CD的构建及进行喉癌Hep-2细胞株转染研究

目的:构建含酵母菌自杀基因CD的质粒表达载体pcDNA3.1(-)CMV.CD,并利用该载体进行喉癌Hep2细胞株转染研究,体外实验观察前体药物5FC对稳定表达CD基因的喉癌Hep2细胞株的杀伤作用。方法:通过RTPCR从酵母菌RNA内扩增出CD基因全长CDS序列,将其定向克隆到质粒表达载体pcDNA3.1(-)CMV中,重组体质粒经XhoI/HindⅢ双酶切鉴定,并对重组体中的CD基因片段进行序列分析,将鉴定好的阳性重组质粒pcDNA3.1(-)CMV.CD运用电穿孔法转入喉癌Hep2细胞中。经300～600mg/LG418正筛选14d和10mg/L前体药物5FC负筛选,获得稳定表达CD基因的喉癌Hep2细胞株,提取该细胞株细胞的总RNA,经RTPCR鉴定CD基因的表达。四甲基偶氮唑盐(MTT)法观察不同浓度5FC对稳定表达CD基因的喉癌Hep2细胞及转染空白载体pcDNA3.1(-)CMV的对照组喉癌Hep2细胞的杀伤作用。结果:阳性重组质粒pcDNA3.1(-)CMV.CD经XhoI/HindⅢ双酶切后获得5353bp的片段及496bp的插入片段,DNA自动序列分析证明重组体质粒含完整的477bp长的CD基因CDS序列。RTPCR从转染细胞总RNA中扩出453bp的预期片段。当添加不同浓度的5FC时,表达CD基因的Hep2细胞不同程度地被杀死,而对照组的细胞几乎未受到影响,两组细胞的相对生存率差异具有统计学意义(P<0.05)。结论:成功构建了质粒表达载体pcDNA3.1(-)CMV.CD,建立了稳定表达酵母菌CD基因的喉癌Hep2细胞株,表达CD基因的Hep2细胞可以被5FC杀死。     

自杀基因载体对体外鼻咽癌细胞杀伤作用的研究

目的检测pcDNA3.1-Egr-CD（简写为pEC）、pcDNA3.1-hTERT-Survivin（简写为pTS）、pcDNA 3.1-Egr-CD-hTERT-Survivin（简写为pEC-TS）真核表达载体对体外鼻咽癌细胞杀伤作用。方法 pEC、pTS、pEC-TS真核表达载体分别转染CNE-2细胞,在放射线照射后用RT-PCR检测和比较胞嘧啶脱胺酶（CD）、Survivin基因的表达,并通过高效液相色谱（HPLC）检测前体药物5-FC转化成5-FU转化效率。结果检测发现用pEC、pEC-TS转染的CNE-2细胞中CD有效表达,HPLC检测前体药物5-FC转化成5-FU;pTS、pEC-TS转染的CNE-2细胞中Survivin基因表达明显降低。结论在CNE-2细胞转染pEC-TS载体后,CD基因能将5-FC转化成5-FU,并通过hTERT启动子特异性降低Survivin基因的表达,有效的提高了CNE-2肿瘤细胞对5-FU的敏感性;为鼻咽癌基因治疗提供了一种新的思路。     

靶向增强型TK表达载体在鼻咽癌细胞中的作用研究

目的探讨hTERT启动子及CMV增强子双调控机制的增强型表达载体pGL3-bas-ic-EGFP-TK-hTERTp-CMV enhancer在鼻咽癌细胞中的靶向杀伤效应。方法构建靶向性增强型hTERT启动子及CMV增强子双调控TK表达载体pGL3-basic-EGFP-TK-hTRETp-CMV enhancer,并以单启动子载体pGL3-basic-EGFP-TK-hTRETp作为对照组,分别转染端粒酶阳性的人鼻咽癌细胞株5-8F、人乳腺癌细胞MCF-7（阳性对照）及正常人血管内皮细胞ECV（阴性对照）。荧光显微镜下观察其TK基因绿色荧光蛋白表达,实时荧光定量PCR方法检测转染细胞中TK基因mRNA定量表达差异,Stretch PCR法检测肿瘤细胞端粒酶活性,MTT法分析杀灭鼻咽癌细胞的效果等作为检验指标。结果①该增强型表达载体转染5-8F细胞及MCF-7细胞后的绿色荧光表达及TK基因的mRNA表达均强于单启动子pGL3-basic-EGFP-TK-hTRETp组;而ECV细胞只有极微弱的绿色荧光和极弱的TK基因的mRNA表达。实时荧光定量PCR显示,增强型载体组A值较对照组单启动子组明显增高,是单启动子组的2～5倍。StretchPCR法检测转染前后5-8F细胞端粒酶活性明显被抑制。②加入GCV后增强载体组对鼻咽癌5-8F细胞及乳腺癌MCF-7细胞体外增殖均有明显抑制作用,均高于单启动子载体组、不加GCV的增强型载体组、空载体组及空白对照组。结论以hTERT启动子及CMV增强子双调控机制介导TK基因的新型靶向增强型表达载体能够高效靶向性杀灭鼻咽癌细胞,这种新型高效靶向增强型载体有可能成为一种针对包括鼻咽癌在内的具有较为广泛抗癌谱的恶性肿瘤临床靶向基因治疗的新策略。     

A Decrease in Maxillary Sinus Pressure,as Seen in Upper Airway Allergy or Infection,Results in an Increase in Upper Airway Nitric Oxide Levels

The paranasal sinuses are connected to the nasal cavity via small osties. Ostial occlusion, caused by mucosal swelling, will result in a slowly increasing negative pressure inside the sinus cavity. In parallel, the oxygen content in the sinus will decrease, resulting in the development of relative hypoxia. Hypoxia is a powerful inducer of nitric oxide (NO) synthase, and inducible NO synthase has been shown to be present in considerable amounts in the upper airways, including the sinuses. The present study was designed to investigate whether a reduction in sinus pressure would affect upper airway NO production. Thirteen healthy volunteers were investigated. A pressure chamber was used to lower the ambient pressure to-4.9 kPa. NO was sampled from one nostril or via a drainage tube inserted into the maxillary sinus before, during and after the hypobaric exposure. When the pressure was decreased, NO levels increased from 256 &#45 15 to 316 &#45 19 ppb ( n =13, p <0.001). The NO levels remained elevated (282 &#45 21 ppb; p <0.05) when measurements were repeated 20 min after leaving the chamber. The nasal airway resistance (V2 tot ) also increased as a result of the chamber session (from 16 &#45 2° before to 21 &#45 3° after; p <0.05). An increase in NO levels was also found when the experiments were repeated with NO sampled directly from the maxillary sinus (225 &#45 6 before and 265 &#45 9 ppb after; n =6, p <0.001). For control purposes the nasal analyses were repeated again, this time under hyperbaric conditions (+4.9 kPa). This resulted in a slight decrease in the NO levels (from 273 &#45 22 to 241 &#45 17 ppb; n =10, p <0.001), but there was no change in the nasal airway resistance. We conclude that a reduction in sinus pressure, as seen in upper airway allergy or infection, may result in an increase in upper airway NO production.     

One‐stop neck lump clinic: phase 2 of audit. How are we doing?

One‐stop neck lump clinic: phase 2 of audit. How are we doing? Regular monitoring and audit of a service are integral to ensuring maintenance of efficiency and standards. This is particularly important where the quality of the service is operator dependent, as is the case in the clinical diagnosis of neck lumps and fine needle aspiration cytology. The one‐stop neck lump clinic has now been running in the department for more than 20 months. A previous article described the results of the first phase audit carried out at 6 months and had identified a waiting time to be seen that was longer than that recommended by the British Association of Otorhinolaryngologists, Head and Neck Surgeons. Measures were implemented to reduce this waiting time and a second audit was carried out after another 10 months with the aims of assessing if modification of the means of referral reduces waiting time and if the outcomes of clinical performance in phase 1 could be maintained or improved. We discuss the results of phase 2 in the audit spiral.     

Physiologische Grundlagen einer Hörprothese

Zusammenfassung Im Bemühen, eine Hörprothese zu entwickeln, die ein Sprachverständnis erlaubt, erscheint es zumindest fürs erste am zweckmäßigsten, durch künstliche elektrische Reizung des Hörnerven die natürlichen Verhältnissen so gut als möglich zu imitieren. Der normale Hörnerv enthält etwa 30000 Nervenfasern, die sich qualitativ gleich, quantitativ jedoch unterschiedlich verhalten, wobei über die Eigenschaften der von den ÄHZ kommenden Spiralfasern im Augenblick sichere Aussagen nicht möglich sind (siehe 2.3). Die quantitativen Unterschiede zwischen den einzelnen Hörnervenfasern beziehen sich auf deren Frequenzabstimmung, Frequenzselektivität, Schwellen, Intensitätsfunktionen und — wichtig insbesondere für das Vorhaben einer künstlichen elektrischen Reizung — in Zeitunterschieden in den Aktivitätsmustern, die durch Laufzeitunterschiede auf der Basilarmembran bedingt sind (2.3). Diese Zeitunterschiede in der Aktivität einzelner Fasern liegen im Bereich mehrerer ms (2.3.6; 2.3.7). Die durch Schallreize im normalen Hörnerven ausgelösten Aktionspotentiale haben überdies einen probabilistischen Charakter, d. h. ihr Auftreten ist keineswegs streng determiniert. Es versteht sich von selbst, daß man bei künstlicher, elektrischer Reizung nicht alle verbliebenen Nervenfasern selektiv reizen kann. Somit wird eine Reizelektrode immer eine Gruppe von Nervenfasern erregen müssen. Bei jeder denkbaren elektrischen Reizung wären alle Fasern im Reizbereich einer Elektrode synchron und streng deterministisch aktiviert, was einen außerordentlich ernstzunehmenden Unterschied zu natürlichen Verhältnissen darstellt (3.2).Um die Zahl der zum Sprachverständnis mindestens notwendigen Reizkanäle abzuschätzen, wird man, in Ermangelung anderer experimenteller Daten, von psychoakustischen Untersuchungen an Normalhörenden auszugehen haben. Diese haben gezeigt, daß das Gehör neben einer außerordentlichen Fähigkeit verschiedene Tonhöhen zu unterscheiden, andererseits die Fähigkeit besitzt, bestimmte Frequenzgebiete zu sogenannten Frequenzgruppen zu integrieren. Die in eine solche Frequenzgruppe fallende Schallenergie wird zu einem einheitlichen Höreindruck verarbeitet. Es scheint also sinnvoll, die für einen Prothesenbau notwendige Zusammenfassung von Gruppen von Fasern des Hörnerven in verschiedene Reizkanäle entsprechend diesen Frequenzgruppen vorzunehmen (3.1). Demnach müßte der Sprachbereich in 15 Reizkanäle aufgeteilt werden, was wiederum, wenn man in der Cochlea reizen will, 1,2 mm Abstand von Kanal zu Kanal erlauben würde. Dabei müßte der Reizerfolg sauber auf die einzelnen Kanäle beschränkt bleiben, d.h. eine optimale Kanaltrennung erreicht werden. In Anbetracht der groben Abweichungen der neuronalen Aktivität vom normalen Verhalten, die bei künstlicher, elektrischer Reizung unvermeidlich sind, ist freilich unsicher, ob die angegebene Zahl ausreichen würde. Andererseits ist es in Anbetracht der zu erwartenden Stromverteilung im Sprachbereich kaum vorstellbar, mehr als die angegebene Zahl von Kanälen realisieren zu können.Was die Kodierung der Schallparameter innerhalb eines Elektrodenkanals betrifft, wird vorgeschlagen, die Frequenzkodierung nach dem Ortsprinzip optimal auszunutzen, und im Hinblick auf die Periodizitätsanalyse und die Lautheitskodierung sich soweit als möglich den natürlichen Verhältnissen anzunähern (3.3). Dabei wären Laufzeitunterschiede zwischen den Kanälen und der probabilistische Charakter der neuronalen Entladungen soweit als möglich einzuführen, um die Dominanz eines periodicity pitch zu vermeiden.Eine für Sprachverständnis ausreichende Prothese ist auch nur denkbar, wenn eine Prothese die zur Sprachübertragung notwendige Übertragungskapazität besitzt. Ergebnisse der Kanal-Vocoder-Technik zeigen, daß Sprache noch mit 1500 bit/s befriedigend übertragen werden kann. Eine Abschätzung der möglichen Leistungsfähigkeit einer 15-kanaligen Prothese (3.4), basierend auf der Zahl der möglichen unterscheidbaren Unterschiedsstufen der Hörempfindung, ergibt, daß diese Übertragungskapazität knapp erreicht werden könnte. Allerdings ist damit noch nicht gesagt, daß das Zentralnervensystem die angebotene Information auch im Sinne einer Phonemanalyse auswertet und damit für ein Sprachverständnis maximal ausschöpft. Nur für diesen Fall wäre ein Sprachverständnis zu erwarten.Als Reizort erscheint in erster Linie die Cochlea (5.1) geeignet. Für den Fall einer Degeneration der primären afferenten Fasern des Hörnerven ist aufgrund physiologischer Überlegungen auch der Nucleus cochlearis ventralis (5.5) interessant, allerdings würde so nur der ventrale Anteil der Hörbahn stimuliert. Doch besitzen auch andere Reizorte spezifische Vorteile (5.2–5.4).Theoretische Überlegungen (6.1) und experimentelle Messungen an implantierten Elektrodensätzen (6.3) zeigen, daß die Forderung der Kanaltrennung nur schwer zu erreichen sein wird. Deswegen wird der dynamische Bereich (im Hinblick auf Veränderung des Reizstromes) eines nach den obigen Kriterien konstruierten Reizkanals auf maximal 3 dB zu beschränken sein, so daß Erregungsausbreitung auf weitere Bereiche der Cochlea durch Ansteuerung von mehreren Reizkanälen zu imitieren wäre.Die Chancen, eine Prothese zu verwirklichen, die befriedigendes Sprachverständnis auf der Basis einer quasinatürlichen Reizung des Hörnerven erlaubt, wird von uns in Anbetracht der geschilderten mannigfaltigen Schwierigkeiten als sehr niedrig angesehen. In Anbetracht des großen Nutzens, der andererseits eventuell resultieren könnte, halten wir die Erforschung des Problems jedoch für angebracht.Es wird von uns vorgeschlagen, auch zu untersuchen, ob sich für eine prothetische Versorgung vorverarbeitete Sprache besser eignet (7.). Für Prothesen, die ein Sprachverständnis nicht anstreben, halten wir eine Implantation in die Cochlea für überflüssig. Hier erscheint uns die Implantation von Reizelektroden am runden Fenster (Douek et al., 1977; Fourcin et al., 1978; s. a. 1. und 7.) wegen des geringeren Risikos der überlegenere Weg.Die zitierten eigenen Arbeiten der Autoren wurden mit Unterstützung der DFG durchgeführt (DFG-K1 219).     

The expression of vascular endothelial growth factor (VEGF) and VEGF‐C in early laryngeal cancer: relationship with radioresistance

The expression of vascular endothelial growth factor (VEGF) and VEGF‐C in early laryngeal cancer: relationship with radioresistance Angiogenesis is essential for tumour growth and invasion. Vascular endothelial growth factor (VEGF) is a prime mediator of tumour angiogenesis. VEGF‐C is a closely related protein that effects lymphatic endothelial cells and may be important in the process of lymphatic metastasis. The purpose of this study was to evaluate the expression of these cytokines in patients with T1 and T2a glottic, squamous cell carcinoma, in comparison with normal epithelial control tissue, to ascertain any association with radioresistance. Twenty‐two tumours treated by radiotherapy (13 radiosensitive, nine radioresistant) and seven normal control tissues were studied. The minimum follow‐up was 2 years after radiotherapy. Expression of VEGF and VEGF‐C was evaluated by immunohistochemistry of formalin‐fixed, paraffin‐embedded biopsy specimens. Analysis was carried out using a quantitative computer image analyser. Both VEGF and VEGF‐C were detectable in tumour and normal control specimens. There was increased expression in tumour specimens of both VEGF (P = 0.03) and VEGF‐C (P < 0.001). In addition, the expression of VEGF‐C was associated with tumours of higher histological grade (P = 0.021). There was, however, no difference in VEGF and VEGF‐C expression between radioresistant and radiosensitive tumours. The expression of VEGF and VEGF‐C is increased in early laryngeal squamous cell carcinoma (SCC). However, measuring the expression of these proteins cannot predict radioresistance in this tumour group.     

Limited Surgery for Cancer of the Larynx and Hypopharynx: Options and Consequences

We reviewed surgical options for laryngeal preservation (limited surgery) in laryngeal and hypopharyngeal cancers and the consequences of the options. Of 44 patients with laryngeal cancer, 11 (25%) received limited surgery and 33 (75%) received total laryngectomy. The survival rates were 91% for the limited surgery group and 73% for the total (radical) surgery group. Of 31 patients with hypopharyngeal cancer, 7 (23%) received limited surgery and 24 (77%) received total laryngopharyngectomy. The survival rates were 53% for the limited surgery group and 40% for the total (radical) surgery group. The survival rates associated with limited surgery were thus better than those for total (radical) surgery for cancers of both the larynx and hypopharynx. This was attributed to the limited surgery group comprising well-selected patients with confined lesions. Organ preservation surgery should be technically simple, reliable in terms of its functional impact and, above all, should not jeopardize the patient's survival. Supracricoid subtotal laryngectomy with cricohyoidoepiglottopexy or cricohyoido-pexy has great potential for laryngeal preservation and will become the major limited surgery modality for treating cancer of the larynx. Limited surgery, however, needs to be performed with great care and is indicated only for very well-selected patients with cancer of the hypopharynx.     

Treatment of Maxillary Sinus Carcinoma: Clinical Results Using the Kitasato Modality

The conventional therapeutic regimen for maxillary sinus carcinoma consists of dissection of the maxilla, full-dose irradiation and extensive chemotherapy. However, the results obtained with this treatment are often poor. Even when patients recover, their quality of life is significantly reduced as a result of deformity of facial structures and swallowing and articulation dysfunctions. A retrospective analysis of 68 patients with maxillary sinus carcinoma treated with the Kitasato modality between 1975 and 1999 was conducted. All patients underwent pergingival maxillary sinus surgery combined with pre- and postoperative irradiation therapy with standardized total doses of 16 Gy; the postoperative irradiation was given in combination with regional intra-arterial infusion chemotherapy administered via the superficial temporal artery. All visible tumor lesions were removed where possible in order to preserve or facilitate cellular immunity after surgery. The cumulative 5-year survival rates were 85.7% for Stage II patients, 88.1% for Stage III, 76.6% for Stage IVA and 75.0% for Stage IVB.     

Cervical Reflex Induced by Click Stimuli in Cats

We studied click-evoked potentials in the anterior horn of the spinal cord in 17 cats. A concentric needle electrode was inserted into the anterior horn of the spinal cord at levels C3-C6. Potentials evoked with 105 dB SPL clicks were recorded with a peak latency of 4.89-5.10 ms only at the C3 level. These responses were observed 45-60 dB SPL above the auditory brainstem response (ABR) threshold, and no potentials were evoked by stimulation of the contralateral ear. Average was performed 100 times with changes in stimulation frequency of 1-20 Hz. The amplitude of the potentials decreased with increasing stimulus frequency, but there were no changes in ABRs. The responses disappeared after destruction of the medial vestibulospinal tract at the obex level, but ABRs were still recorded. The spinal nucleus of the accessory nerves was located in the anterior horn of the spinal cord at levels C1-C6, and the sternocleidomastoid muscle motoneurons were found at levels C1-C3. The click-evoked potentials recorded in this study reflect responses of the spinal nucleus of accessory nerves through the vestibulospinal tract to click stimulation. The responses have the same characteristics as vestibular-evoked myogenic potentials that can be recorded using surface electrodes over the sternocleidomastoid muscles of humans.     

A simple objective evaluation and grading for facial paralysis outcomes

Matrix metalloproteinase (MMP)-2 and -9 degrade type IV collagen, which is one of the major components of the basement membrane in normal tissue and expressed in the surroundings of the cancer nest in squamous cell carinoma. The degeneration of type IV collagen is an essential step in the metastasis to lymph nodes and distant organs. In this study, we examined MMP-2 and -9 levels of cancer tissue and serum obtained from patients with head and neck squamous cell carcinoma (HNSCC) in order to evaluate the relationship between the clinicopathologic features and MMPs. We examined the production of MMP-2 and -9 in cancer tissue homogenates of 73 patients who had HNSCC and the serum MMP levels of 16 patients with HNSCC and 8 healthy volunteers. We also studied the localization of MMP-2 in the carcinoma using an immunohistochemical approach. The concentrations of MMP-2 and -9 in the tissue homogenates and serum were measured by means of a sandwich enzyme immunoassay using a monoclonal antibody. Immunohistochemical analyses were performed with monoclonal antibody to MMP-2. The concentration of MMP-2 in the tumor tissue homogenates was unrelated to tumor size, but that in patients with lymph node metastases was significantly higher than in those without lymph node metastases. The concentration of MMP-9 was unrelated to lymph node metastasis and tumor size. The levels of both MMP-2 and -9 in serum were unrelated to lymph node metastasis. Immunohistochemistry indicated that MMP-2 was mainly expressed in cancer cells. Because MMP-2 degrades type IV collagen, the level of MMP-2 in carcinomas may be a useful indicator of the degree of invasion and metastasis.     

A case of cortical deafness and anarthria

The nasal epithelium protects the underlying tissue from damage. Epithelial cell growth is controlled by epidermal growth factor (EGF) and is possibly affected by toxic proteins, e.g. eosinophil cationic protein (ECP). The aims of this study were to examine nasal fluid epithelial cell counts and their relations to EGF, eosinophils and ECP in 23 patients with seasonal allergic rhinitis and 20 healthy controls. Nasal fluid epithelial cell counts were lower in patients than in controls. EGF levels did not differ between patients and controls, and correlated with epithelial cell counts in controls but not in patients. Eosinophils and ECP were higher in patients than in controls, but did not correlate with epithelial cell counts. The role of growth factors, such as EGF, in regulating epithelial cells merits further study.     

Cochlear Function in Ears with Immunomediated Inner Ear Disorder

The aim of the present study was to evaluate the performance of ears with inner ear disorder, responsive to immunosuppressive drugs, in advanced tests designed to assess primary cochlear functions (temporal integration, frequency selectivity, cochlear mechanics). The results of this study suggest that immunomediated inner ear disease results, in the acute clinical stage, in the development of endolymphatic hydrops, which increases the stiffness of the vibrating structures within the inner ear and causes dysfunctions of the outer hair cells. Our patients presented with upsloping or flat sensorineural hearing loss, absence of evoked otoacoustic emissions and distortion-product otoacoustic evoked emissions and abnormal temporal integration, frequency selectivity and cochlear mechanics. Following immunosuppressive treatment, hydrops recovered, hearing subsequently returned to normal, the audiometric curve became flat at low-to-middle frequencies and primary cochlear function tended to normalize. This study seems to support the usefulness of testing primary cochlear functions in order to monitor the clinical course of immunomediated inner ear disorders.