hEGF、TGFβ2对兔角膜内皮细胞损伤的作用及相关蛋白 p27Kip1和 Cdk4 的表达

目的：观察人表皮生长因子（hEGF）、转化生长因子β2（TGFβ2）对家兔角膜内皮细胞损伤的作用及相关蛋白 p27Kip1和Cdk4的表达。方法：体外培养家兔角膜内皮细胞传一代融合后，定量损伤直径3.5?mm范围内的细胞，培养液中分别加入不同浓度的hEGF和（或）浓度为10?ng/ml的TGFβ2，对照组不加任何药物。损伤后第1、3、5，7天倒置显微镜下照相并用计算机测量未愈合面积。应用免疫荧光染色法，检测损伤修复不同阶段p27Kip1和Cdk4的含量。结果：一定质量浓度的hEGF可加快体外培养的家兔角膜内皮细胞的损伤愈合并上调Cdk4的表达, 且具有剂量依存性，以10～40?ng/ml为最适宜。外源性TGFβ2抑制角膜内皮细胞损伤修复。hEGF孵育损伤模型24?h后，TGFβ2对此无抑制作用。 TGFβ2作用损伤模型24?h后，可抑制hEGF的促修复作用。结论：一定质量浓度的hEGF能促进体外培养的家兔角膜内皮细胞的损伤修复并上调Cdk4的表达。外源性TGFβ2可抑制家兔角膜内皮细胞的损伤修复并上调p27Kip1的表达。     

表皮生长因子及其受体在中耳慢性鼓膜穿孔病变中的作用

目的　探讨表皮生长因子及其受体在中耳慢性鼓膜穿孔病变中的作用。方法在豚鼠慢性创伤性鼓膜穿孔的动物模型上 ,观察应用表皮生长因子治疗的鼓膜愈合率。在具有典型鼓膜后皱襞处穿孔的胆脂瘤型慢性化脓性中耳炎患者的病理标本上 ,应用免疫组化SP染色方法和计算机图像分析系统 ,检测 10例鼓膜穿孔部位邻近皮肤表皮生长因子受体的表达情况。结果　在动物实验中表皮生长因子治疗组鼓膜愈合率明显高于对照组 (χ2 =9 92 3,P <0 0 1)。未发现有诱发中耳胆脂瘤的病例。在临床研究中鼓膜穿孔邻近皮肤表皮生长因子受体的阳性率为 (39 3± 7 4 ) % ( x±s,下同 ) ,耳道深部正常皮肤为 (2 5 4± 3 7) %。 2组间差异具有高度显著性意义。结论　表皮生长因子有明显促进鼓膜穿孔愈合的能力。表皮生长因子受体在鼓膜穿孔部位邻近皮肤的中等表达 ,表明使用表皮生长因子治疗有一定的理论基础和有诱发中耳胆脂瘤的风险。     

鼻内镜术后局部应用肽类生长因子的临床观察

目的观察鼻内镜术后局部应用肽类生长因子(bFGF)的疗效。方法100例慢性鼻窦炎、鼻息肉患者随机分为两组,观察组术后局部应用bFGF+常规用药;对照组常规用药。观察术腔黏膜上皮化情况。结果上皮化时间观察组48.3±8.8天;对照组77.2±12.2天。两组间差异有显著性意义(t=2.72,P<0.01);总有效率观察组92.0%;对照组82.0%。两组间差异无显著性意义(χ2=2.85,P>0.05)。结论鼻内镜术后局部应用bFGF,能促进术腔上皮化,缩短上皮化时间。     

血管内皮生长因子及其受体对喉癌细胞生长的影响

为探讨血管内皮生长因子（ＶＥＧＦ）在喉癌中的作用及可能的作用方式，用抗ＶＥＧＦ抗体及其受体ｆｌｔ抗体通过微波处理暴露抗原后，进行标准化的ＥｌｉｔｅＡＢＣ染色；将不同浓度的抗ＶＥＧＦ及抗ｆｌｔ抗体分别加入ＨＥＰ－２细胞的培养液中，培养５ｄ后测ＨＥＰ－２细胞的活性（ＭＴＴ法）。结果：免疫组化染色显示ＶＥＧＦ及其受体ｆｌｔ主要在喉癌细胞和血管内皮细胞表达；不同浓度的ＶＥＧＦ抗体及抗ｆｌｔ抗体对ＨＥＰ－２     

γ-干扰素对喉癌细胞血管内皮生长因子-C表达的影响

目的:研究γ-干扰素(IFN-γ)对人喉癌细胞株Hep-2细胞血管内皮生长因子-C(VEGF-C)基因及蛋白表达的影响.方法:将IFN-γ以不同浓度(103、104、105、106、107U/L)、不同作用时间(0、12、24、36、48、60、72 h)作用于Hep-2细胞,用四甲基偶氮唑蓝(MTT)比色法测定细胞增殖;用实时荧光定量PCR(Realtime-PCR)法测定IFN-γ(105U/L)不同作用时间(0、2、6、12、24、36、48、60、72 h)细胞VEGF mRNA含量;用双抗体夹心酶联免疫吸附试验、Western blot法和免疫细胞化学方法测定与Realtime-PCR相同药物浓度和作用时间细胞培养上清液和细胞质中VEGF-C蛋白含量.结果:①细胞生长受到抑制,以高浓度组表现最为明显;②不同浓度(103、104、105、106、107U/L)IFN-γ作用不同时间(0、12、24、36、48、60、72 h)后,从48 h开始,105、106、107U/L的IFN-γ对Hep-2细胞有明显的抑制作用,差异有统计学意义(P＜0.05),但不随着浓度的增加而成正比;③106U/L IFN-γ作用后,在36 h后与对照组比,VEGF-C mRNA表达下调,差异有统计学意义(P＜0.05),特别是48 h(P＜0.01);④104U/L IFN-γ作用36 h后,Western blot显示细胞中VEGF-C蛋白含量显著下降;⑤106U/L IFN-γ作用48 h后,与对照组相比,Hep-2细胞质中的棕色颗粒明显减少.结论:IFN-γ可以在核酸和蛋白质水平下调Hep-2细胞分泌VEGF-C.     

Expression of epidermal growth factor, epidermal growth factor receptor and transforming growth factor-alpha in the human fetal inner ear

The expression of epidermal growth factor (EGF), EGF receptor and transforming growth factor (TGF)-alpha was analyzed in the human fetal inner ear using immuno-histochemical methods. EGF receptor was observed only in 9.5-week-old fetal vestibular epithelia. In 14- and 16-week-old fetuses, EGF receptor could not be detected. TGF-alpha was observed strongly in the 9- and 11-week-old vestibular epithelia, whereas only trace amounts were detectable in the 14- and 16-week-old vestibular epithelia. These findings suggest that EGF and TGF-alpha probably have a mitogenic effect in the sensory epithelia of the fetal inner ear, especially at early stages of development.     

红景天素对老龄大鼠嗅球中成纤维细胞生长因子表达的影响及意义

目的　探讨红景天素对老龄大鼠嗅球中成纤维细胞生长因子（ｆｉｂｒｏｂｌａｓｔｇｒｏｗｔｈ ｆａｃｔｏｒ,ＦＧＦ）表达的影响及意义。方法　将２０ 只老龄大鼠随机分成注射红景天素实验组（１０ 只） 和注射等量生理盐水对照组（１０ 只）。分别取嗅球和嗅粘膜,经石蜡包埋,连续切片,免疫组化染色,光镜观察。结果　实验组嗅球中ＦＧＦ阳性表达率明显高于对照组（ Ｐ＜ ０．０１） ,而２ 组嗅粘膜均未见ＦＧＦ表达。结论　红景天素能够提高老龄大鼠嗅球中ＦＧＦ的阳性表达率,提示其对嗅系统可能具有抗衰老作用。     

VEGF在伴鼻息肉的慢性鼻-鼻窦炎中的表达及克拉霉素对其的调节作用

目的：探讨血管内皮生长因子（VEGF）在伴鼻息肉的慢性鼻-鼻窦炎（CRSwNP）患者鼻黏膜中的表达及克拉霉素对其的调控作用。方法：收集16例CRSwNP组织及21例单纯鼻中隔偏曲患者下鼻甲黏膜,采用实时定量RT-PCR方法检测样本中VEGF的表达。另外收集10例伴鼻息肉的慢性鼻窦炎鼻息肉组织及5例单纯鼻中隔偏曲患者下鼻甲黏膜,采用体外组织块培养及ELISA方法,观察克拉霉素对VEGF表达的调节作用。结果：①VEGF mRNA在CRSwNP组的表达较对照组增高,差异有统计学意义（P〈0.01）;②经克拉霉素刺激前后比较,CRSwNP组织中VEGF蛋白的表达量下降,差异有统计学意义（P〈0.05）。结论：CRSwNP组织中VEGF的表达显著升高,推测VEGF在鼻息肉的发病机制中具有极其重要的作用。克拉霉素可能通过抑制VEGF的表达而达到治疗慢性鼻-鼻窦炎的作用。     

Validating the subcutaneous model of injectable autologous cartilage using a fibrin glue scaffold

PURPOSE: To create and validate an injectable model for autologous in vivo cartilage engineering with ultimate clinical applicability in human subjects. HYPOTHESIS: Cartilage can be generated subcutaneously using fibrin glue and autologous chondrocyte components. BACKGROUND: To date, cartilage engineering studies have been limited by several factors. Immunocompromised animals and nonautologous chondrocytes have been successfully used to create cartilage, but results using identical designs failed in immunocompetent subjects. Recent studies using more biocompatible tissues and matrices have been performed with both in vitro and in vivo steps. Although successful, several problems are notable. In vitro cartilage displays a poor modulus of elasticity, even after in vivo implantation. Variable deformation and volume loss occurs when in vitro specimens are matured in vivo. These concerns limit the clinical utility of these methods. We therefore set out to create autologous cartilage using a model that was clinically feasible, easy to create, and could be performed with very low patient harvest morbidity. MATERIALS AND METHODS: Eight New Zealand white rabbits underwent a unilateral harvest of ear cartilage. Samples were then digested using standard methods. Cell counts and survival assays were performed before implantation. One sample of fibrin glue (Tisseel) and chondrocytes was injected subcutaneously into each donor rabbit and then left in situ for 3 months. A second sample with both basic fibroblast growth factor (b-FGF) and insulin-like growth factor (IGF)-1 in the injection suspension was also assessed (for a total of 16 samples). After harvest, analysis of overall volume, histology, and chondrocyte drop out counts was performed. RESULTS: Cartilage formation occurred in 8 of 14 (57%) specimens that were obtained at the time of sacrifice. Of note, 6 of 7 (85%) non-growth-factor containing samples yielded positive results. Comparison with the success rate using concomitant growth factors (2/7) showed a negative effect on cartilage yield (P = .015). Chondrocyte survival, based on chondrocyte dropout counts, was not effected. Angiogenesis appeared to correlate with cartilage formation in the central regions of the implant. Alcian blue demonstrated the presence of active matrix deposition, and elastin Verhoff-van Geison (EVG) stains were positive, showing an elastic cartilage phenotype. Very limited osteoid formation was seen in successful implants. Failed implants demonstrated avascular necrosis, giant cell reactions, and inflammatory infiltrates. CONCLUSIONS: This study validates the subcutaneous site as a recipient bed for the engineering of autologous cartilage in vivo. It also represents the first subcutaneous implantation of fibrin glue and chondrocytes in an immunocompetent host as well as the first published report of elastic cartilage generation in vivo. Although the model needs to be further streamlined to increase yields and overall volume, this study clearly demonstrates the feasibility of in vivo chondrogenesis (85% success). The addition of FGF and IGF-1 at the concentrations used negatively influenced cartilage yield. However, extrapolation of these results to other combinations or concentrations can not be done, and this issue deserves further investigation.     

靶向血小板衍生生长因子-B siRNA对喉癌Hep-2细胞增殖的影响

目的探讨血小板衍生生长因子(platelet derived growth factor,PDGF)B链mRNA的siRNA质粒转染喉癌细胞株Hep-2细胞,沉默Hep-2细胞中PDGF-B mRNA并检测细胞增殖。方法以脂质体lip2 0 0 0为载体,设计4组针对PDGF-B的siRNA片段瞬时转染喉癌Hep-2细胞,实时荧光定量PCR、Westernblot分别检测各组PDGF-B mRNA、PDGF-B蛋白的表达及干扰效果,噻唑蓝(MTT)法检测各组细胞增殖抑制率,并与阴性对照组和空脂质体组进行比较。结果实时荧光定量PCR检测显示:SiRNA2、SiRNA3、SiRNA4组PDGF-B mRNA表达均有明显抑制,其中SiRNA2抑制率最高;与SiRNA1、阴性对照组及空脂质体组进行比较,差异具有统计学意义(P<0.05)。Westernblot检测显示:SiRNA-1、SiRNA-2、SiRNA-3组PDGF-B mRNA蛋白表达均有下降,其中SiRNA-2下降最明显,而SiRNA-4、阴性对照组及空脂质体组对PDGF-B蛋白表达无明显抑制作用。MTT法检测显示:SiRNA-1、SiRNA-2、SiRNA-3均明显抑制Hep-2细胞增殖,其中SiRNA-2对Hep-2细胞的抑制作用最明显;且与SiRNA-4、阴性对照组及空脂质体组比较,差异具有统计学意义(P<0.05)。结论成功运用siRNA干扰喉癌Hep-2细胞PDGF-B mR-NA,靶向沉默PDGF-B mRNA能有效抑制细胞增殖。     

牛碱性成纤维细胞生长因子在鼻内镜术后术腔愈合中的作用

目的：探讨鼻内镜术后术腔局部应用牛碱性成纤维细胞生长因子(bFGF)对术腔上皮化的影响。方法：选择Ⅰ型3期慢性鼻窦炎患者10例,均于鼻内镜术后1周清理术腔后应用bFGF喷鼻,分别于术后1、1,5、2、3个月取中鼻道上皮作扫描电镜形态学观察。结果：电镜下见术后1个月细胞肿胀、排序紊乱、纤毛脱落;术后1．5～2个月,细胞较前排列整齐,纤毛密度明显增加,但排列紊乱;术后3个月,细胞肿胀完全消退,纤毛密布且朝同一方向摆动。结论：bFGF能够促进鼻内镜术后术腔上皮化,提高术腔上皮的质量。     

表皮生长因子促进鼓膜愈合的临床研究

对４７例长期不愈的鼓膜穿孔患者，于鼓膜成型术中应用表皮生长因子（ＥＧＦ），经观察，用药后鼓膜愈合时，愈合时间约为８．５ｄ，未见不良反应，表明ＥＧＦ局部应用具有促进鼓膜穿孔愈合的作用。     

转化生长因子α在中耳胆脂瘤组织中的表达及意义

目的 :研究转化生长因子 (TGFα)在中耳胆脂瘤中的表达 ,探讨TGFα在胆脂瘤上皮细胞增生中的可能作用。方法 :31例胆脂瘤上皮标本及 10例正常外耳道皮肤组织标本制成石蜡切片 ,应用免疫组化 (S P)染色法检测上述两种标本中TGFα的表达情况 ,并采用多媒体彩色图文分析系统 ,对染色结果进行定量分析。结果 :TGFα阳性表达定位于胞浆 ,胆脂瘤上皮组织均呈中等阳性或强阳性反应 ,2 1例标本呈现从基底层向角质层染色逐渐增强的趋势 ,皮下结缔组织中可见散在的阳性细胞 ,主要为成纤维细胞、单核 巨噬细胞、浆细胞 ,少数为淋巴细胞 ;外耳道皮肤组织的阳性反应集中在表皮层 ,其中 8例标本表现为表皮全层稀疏均一的弱阳性表达。胆脂瘤上皮和正常外耳道表皮的TGFα积分吸光度分别为 2 .4 31± 0 .5 87及 1.4 6 3± 0 .14 7,两者差异有显著性意义(t’ =1.95 ,P <0 .0 5 )。结论 :胆脂瘤组织中TGFα的含量较正常皮肤组织显著增高 ,TGFα可能以自分泌和旁分泌机制参与胆脂瘤上皮增生的调节 ,局部炎症反应可能构成特殊微环境 ,诱发并维持胆脂瘤上皮的高度增生     

甘糖酯对糖尿病大鼠视网膜病变中血管生成因子VEGF表达的影响

目的 探讨甘糖酯对糖尿病大鼠视网膜病变(DR)中血管内皮生长因子(VEGF)表达变化的影响,为将甘糖酯应用于临床上防治DR提供可靠的理论和实验依据。 方法 选取清洁级雄性成年Wistar大鼠随机分为正常对照组,实验对照组,甘糖酯治疗组,其中甘糖酯治疗组分为糖尿病+低甘糖酯组(50 mg/kg甘糖酯)和糖尿病+高甘糖酯组(100 mg/kg甘糖酯)。大鼠采用链脲佐菌素60 mg/kg一次性腹腔内注射制作糖尿病模型,甘糖酯治疗组的给药方法为甘糖酯溶液灌胃,持续12周。给药12周后处死各组大鼠并分离视网膜,取房水、血清,用ELISA法检测大鼠视网膜组织、房水及血清中VEGF蛋白的表达变化情况,免疫组化检测大鼠视网膜VEGF蛋白表达的变化及表达位置,实时定量PCR检测大鼠视网膜VEGF mRNA表达变化情况。 结果 给药12周后大鼠DR模型中,甘糖酯对大鼠的血糖没有影响,ELISA检测结果表明,各组大鼠血清中的VEGF蛋白差异没有统计学意义(P>0.05),而糖尿病组大鼠房水及视网膜中的VEGF蛋白含量与正常对照组比较显著增加(P<0.05),甘糖酯干预后房水及视网膜中VEGF蛋白较糖尿病组表达明显降低(P<0.05)。免疫组化检测表明,正常对照组VEGF蛋白呈低表达,糖尿病组VEGF蛋白呈高表达状态,主要在视网膜神经节细胞层、内从状层及外核层高表达;而给予甘糖酯干预后VEGF蛋白呈弱表达。实时定量PCR检测大鼠视网膜中VEGF mRNA,可见正常对照组VEGF mRNA为低表达状态,糖尿病组VEGF mRNA的表达为正常对照组的2.12倍,甘糖酯干预后视网膜VEGF mRNA表达较糖尿病组明显下降(P<0.05)。 结论 甘糖酯对大鼠早期DR有很好的防治作用,其机制与抑制血管生成因子VEGF的表达与分泌有关。     

表皮生长因子受体及血管内皮生长因子在喉乳头状瘤中的表达

目的探讨表皮生长因子受体（EGFR）和血管内皮生长因子（VEGF）在喉乳头状瘤（IJP）中的表达及意义。方法采用免疫组化二步法检测10例成人型喉乳头状瘤（ALP）、19例幼年型喉乳头状瘤（JLP）石蜡标本中EGFR、VEGF的表达与分布;并以10例声带息肉作为对照组。结果EGFR和VEGF在ALP、JLP组上皮层的表达水平明显高于对照组（P〈0．05）。EGFR在ALP、JLP表皮组织全层均有强阳性表达,VEGF呈现以基底层、棘层细胞显著表达,到颗粒层表达逐渐减弱的模式。VEGF在ALP、JLP和对照组间质的血管内皮细胞、炎症细胞、成纤维细胞中也有表达,但3组问VEGF的表达元显著统计学差异（P〉0．05）。JLP组上皮中VEGF的表达评分结果（7．133±0．061）比ALP组（6．934±0．041）高,两组比较有统计学意义（P〈0．05）。结论VEGF、EGFR在LP组织中的过度表达可能在LP的上皮细胞过度增生和血管大量形成中发挥重要作用,JLP比ALP具有更强的增殖活性。     

Development of speech and hearing of two children with Pelizaeus-Merzbacher disease presenting only waves I and II of the auditory brainstem response

Pelizaeus-Merzbacher disease (PMD) is a white matter dystrophy of the brain. Most children with PMD require comprehensive nursing care. Their speech and language abilities are poor or absent. Therefore, evaluating hearing ability is difficult in children with PMD. We have followed up two patients with PMD since early childhood. Patient 1 is an 11-year-old boy, and patient 2 is a 15-year-old adolescent boy in whom horizontal nystagmus was recognized after birth. Magnetic resonance imaging showed diffuse dysmyelination of the cerebral white matter. Auditory brainstem response recordings showed only waves I and II and the absence of all subsequent components. However, conditioned orientation reflex audiometry showed a threshold of 20-30 dB. Both patients can converse orally and have auditory perception and speech abilities better than those of most patients with PMD in the literature. We report on the development of their hearing and speech abilities.     

重组人表皮生长因子促进鼻内镜术后术腔上皮化

目的观察重组人表皮生长因子(rhEGF)对鼻内镜手术后术腔愈合的影响.方法对131例(235侧)施行鼻内镜手术分析的Ⅱ型2期、3期患者,随机分为两组,A组应用rhEGF,B组不用,随访4个月以上比较同期两组的治愈率.结果同期治愈率A组显著优于B组(P＜0.05).结论 rhEGF能促进术腔早日上皮化.     

Effect of growth factors on matrix synthesis by human nasal chondrocytes cultured in monolayer and in agar

Reconstructive surgery of multiple areas of the body may require replacement bone or cartilage transplants to repair defects or lesions of skeletal tissue. Advances in cell and tissue culture techniques now permit synthesis of autologous human cartilage in vitro. Several growth factors regulate the metabolism and activation of cartilage cells. To enhance culture conditions and effectiveness for in vitro cartilage engineering, the aim of our investigations was to characterize the influence of transforming growth factor (TGF)-beta and basic fibroblast growth factor (bFGF) on human nasal septal chondrocytes. The isolated cells were cultured as monolayers on plastic and in soft agar. The biological effects of the growth factors were assessed by determining synthesis of total protein and proteoglycan. TGF-beta caused a dose-dependent stimulation of total protein as well as glycosaminoglycan synthesis by all chondrocytes cultured. This stimulatory effect of TGF-beta was greater for chondrocytes cultured in soft agar than for chondrocytes cultured on plastic. No stimulatory effects of matrix synthesis was observed for bFGF in either culture condition. Our results show that TGF-beta can be employed to enhance in vitro production of cartilage grafts for reconstructive surgery.     

VEGF和VEGF-C在喉癌中的表达及临床意义

目的 :探讨血管内皮生长因子 (VEGF)和血管内皮生长因子C(VEGF C)在喉鳞癌组织及癌旁组织中的表达及其与喉癌生物学行为之间的关系。方法 :采用逆转录聚合酶链式反应技术 ,对 2 0例新鲜喉鳞癌标本、切缘组织及远离肿瘤的正常喉组织中的VEGF、VEGF C基因表达进行检测。结果 :对同一患者 ,VEGF、VEGF C在肿瘤组织、切缘组织、正常组织中的表达差异有显著性意义 (P <0 .0 5 ) ,在肿瘤组织中 ,VEGF、VEGF C的表达与患者年龄、性别、病变部位、T分期、病理分级无关 ,VEGF、VEGF C表达与喉鳞癌淋巴结转移有关。结论 :VEGF、VEGF C联合检测可望作为判断喉鳞癌患者预后的指标。     

The expression of epidermal growth factor receptors in the oral mucosa of patients with oral cancer

Summary Representative tumor samples and mucosal samples were taken from three different groups of patients and were stained immunohistochemically for their expressions of epidermal growth factor receptors (EGFR). Patients in group 1 had oral squamous carcinoma, with specimens taken from the tumor as well as from the mucosa without tumor invasion. Patients in group 2 had no evidence of tumor but had heavy drinking and smoking habits. Tumor-free patients who do not drink or smoke served as the control group. The findings in the present study showed that the tumor and mucosal samples from groups 1 and 2 had increased EGFR expression while the control group showed significantly less EGFR. These results suggest that EGFR may play a role in the development of premalignant tissue changes, which are probably influenced by chronic toxic irritation.Presented at the XXI Pan American Congress of Otolaryngology, Head and Neck Surgery, Bahia-Salvador, Brazil, 2 November 1988