神经元核心抗原在小鼠嗅球和嗅上皮发育中的表达

目的：探讨神经元核心抗原（NeuN）在嗅球和嗅上皮发育过程中的表达特性和意义。方法：在小鼠胚胎发育9．5、11．5、14．5、17．5d,出生当天和3个月成年个体的头部切片标本中,以免疫荧光染色方法检测NeuN的表达。结果：刚出生小鼠嗅球内层状结构尚不明显,NeuN表达于嗅球周边区域。在3个月大成年小鼠,嗅球的层状结构已清晰可见,NeuN表达阳性的成熟神经元主要聚集于接近嗅球中心的颗粒细胞层。位于嗅上皮的双极嗅感觉神经元在小鼠胚胎和成年个体中均未见NeuN表达。结论：NeuN通常被认为表达于几乎所有部位的成熟神经元。但在嗅球和嗅上皮中,NeuN只表达于成熟嗅球中的颗粒细胞层。小鼠出生到发育为成熟个体的过程中,NeuN表达阳性的成熟神经细胞由周边逐渐向嗅球中心迁移,可能与气味模式的形成有关。     

Survival of Partially Differentiated Mouse Embryonic Stem Cells in the Scala Media of the Guinea Pig Cochlea

The low regenerative capacity of the hair cells of the mammalian inner ear is a major obstacle for functional recovery following sensorineural hearing loss. A potential treatment is to replace damaged tissue by transplantation of stem cells. To test this approach, undifferentiated and partially differentiated mouse embryonic stem (ES) cells were delivered into the scala media of the deafened guinea pig cochlea. Transplanted cells survived in the scala media for a postoperative period of at least nine weeks, evidenced by histochemical and direct fluorescent detection of enhanced green fluorescent protein (EGFP). Transplanted cells were discovered near the spiral ligament and stria vascularis in the endolymph fluid of the scala media. In some cases, cells were observed close to the damaged organ of Corti structure. There was no evidence of significant immunological rejection of the implanted ES cells despite the absence of immunosuppression. Our surgical approach allowed efficient delivery of ES cells to the scala media while preserving the delicate structures of the cochlea. This is the first report of the survival of partially differentiated ES cells in the scala media of the mammalian cochlea, and it provides support for the potential of cell-based therapies for sensorineural hearing impairment.     

C57BL/6J小鼠在不同剂量氨基糖甙类药物暴露下听力损害的特点

目的探讨C57BL/6J小鼠在不同剂量氨基糖甙类药物毒性作用下听力损害的特点。方法采用不同剂量的庆大霉素对成年C57BL/6J小鼠进行腹腔注射,小鼠随机分成五组,药物剂量浓度分别为300mg/kg,200mg/kg,100mg/kg和50mg/kg。对照组为等量的注射用0.9%生理盐水,药物注射连续应用7天。分别于用药前(p0,对照组)以及用药后第2天(P2)、第4天(P4)、第7天(P7)检测动物的听功能(Click&Tonebrust),并进组间差异的比较分析。结果 (1)在50mg/kg组,用药后第7天的听阈值和对照组听阈分别为48.33±10.33(P7)和41.67±11.20(P0),两者相比具有显著性差异(P<0.05)。在100mg/kg组,用药后第4天的听阈值和对照组相比具有显著性差异(P4:48.89±9.16;P0:40.83±10.07,P<0.05)。在200mg/kg和300mg/kg组,用药后第2天的听阈值和对照组相比有显著上升(200mg/kg,P2:50.56±5.39;P0:40.42±9.66,P<0.05),(300mg/kg,P2:50.50±6.85,P0:40.00±11.00,P<0.05)。(2)各剂量组药物在Click和Toneburst(4kHz,8kHz&t16kHz)频率中,听力损害程度存在区别,损害程度依次为:200mg/kg组>300mg/kg组>100mg/kg组>50mg/kg组。(3)在8kHz&16kHz频率上,①50mg/kg与300mg/kg剂量组在给药后第2、4、7天均无明显听力变化(P>0.05)。②在100mg/kg组:与对照组相比,给药后第4天、第7天的听阈出现明显升高(P<0.05)。③在200mg/kg组:给药后第2、4、7天的听阈值亦出现显著升高(P<0.05)。(4)在给药后第7天,各剂量组的听阈均达到峰值。结论本研究表明:(1)随着给药剂量的增加,C57BL/6J小鼠听力损害出现的时间提前,但听力损害的程度并非同步加重;(2)以200mg/kg浓度给药后,小鼠听力在Click&Toneburst上均可以产生明显变化,同时小鼠的生理状态依然保持良好,因而200mg/kg(庆大霉素)浓度可能是C57BL/6J小鼠药物性致聋的理想剂量。     

Terminal differentiation of epithelial cells in middle ear cholesteatoma: investigation of patterns of expression of protein kinase C-delta and protein kinase C-eta

OBJECTIVES: The objective of this study was to elucidate the differentiation mechanism of keratinocytes in cholesteatoma. STUDY DESIGN: To achieve the objective, we analyzed the expressions of various cellular proteins: the delta and eta isoforms of protein kinase C (PKCdelta and PKCeta), which are thought to play key roles in signal transduction in differentiation; cytokeratin 1 (CK1) and cytokeratin 10 (CK10) (cytoskeletal constitutive proteins); and involucrin (a marker of differentiation). METHODS: The materials used in this study were tissue specimens obtained from cholesteatoma epidermis, normal external ear canal skin, normal inguinal skin, and psoriatic skin. Immunohistochemical staining techniques were applied to compare the expressions of the above proteins (i.e., PKCdelta, PKCeta, CK1, CK10 and involucrin) in those various tissues. RESULTS: No clear differences in the patterns of expression of PKCdelta and PKCeta were found between the cholesteatoma epidermis and the normal external ear canal skin. These proteins were expressed mainly in the stratum spinosum and stratum granulosum, and their patterns of expression were almost the same as those of the CK1, CK10, and involucrin proteins. CONCLUSION: The findings of this study indicate that the terminal differentiation of keratinocytes in the cholesteatoma epidermis is the same as in normal skin tissues. It was concluded that the growth of epidermis which has undergone hyperproliferation of keratinocytes because of increased levels of various cytokines is being regulated by means of normal terminal differentiation.     

C57BL/6小鼠内耳形态学和年龄相关性听力损失的研究

目的探讨不同周龄C57BL/6小鼠内耳形态学及其ABR阈值变化。方法取C57BL/6小鼠3周、4周、12周、26周各10只,听性脑干反应(ABR)测试双侧2、4、8、16、20 kHz ABR阈值。采用基底膜铺片MyosinⅥ、Neurofilament免疫组化染色,观察耳蜗毛细胞和神经丝的变化。扫描电镜观察耳蜗毛细胞及其静纤毛随年龄的变化。结果随着年龄增长,C57BL/6小鼠各频率ABR阈值明显提高,顶转和底转内毛细胞缺失逐渐增多,神经丝染色渐淡,毛细胞静纤毛逐渐发生数量减少、增粗融合、倒伏等变化。到26周龄时已达到重度聋,各频率较3周组有显著统计学差异。顶转和底转内毛细胞有连续缺失,外毛细胞完全缺失,内毛细胞只有残存的少量静纤毛,粗细不均,倒伏明显。结论本研究对国产C57BL/6小鼠的内耳形态进行观察,明确了其ABR阈值和内耳毛细胞的变化规律,为用国产C57BL/6小鼠进行老年性聋研究提供了依据。     

CD43CD20及总IgE在鼻息肉组织中表达的研究

目的　探讨CD4 3 、CD2 0 及总IgE在鼻息肉组织中的表达 ,分析其在鼻息肉发病中的作用。方法　采用免疫组化SP法及HE染色对 2 5例鼻息肉和 2 0例中鼻甲粘膜进行研究。结果　鼻息肉组织中CD 43 细胞和CD 2 0 细胞、浆细胞数显著多于中鼻甲粘膜组织 (P <0 0 1) ,总IgE阳性表达率为44 %,显著高于中鼻甲粘膜组织的 10 %(P <0 0 5 )。结论　鼻息肉中存在活跃的细胞免疫和体液免疫 ,鼻粘膜局部变态反应可能参与鼻息肉的发病。     

血管内皮生长因子在鼻咽癌组织中的表达及其与淋巴转移的关系

目的 检测鼻咽癌中血管内皮生成因子C(VEGF-C)的表达及微淋巴管密度(microlymphatic vessel density,MLVD),探讨鼻咽癌淋巴管生成及淋巴转移之间的关系.方法 采用免疫组化SP法检测58例鼻咽癌和20例鼻咽部炎性反应组织中VEGF-C的表达情况,并用淋巴管内皮细胞特异性抗体LYVE-1行免疫组化染色,计数肿瘤内MLVD,并结合临床病理特征进行分析.结果 鼻咽癌组和炎性反应对照组VEGF-C的阳性表达率分别为84.5%(49/58)和15.0%(3/20),差异有统计学意义(X2=32.309,P＜0.01).鼻咽癌组织MLVD为(28.6±6.2)个/视野,鼻咽炎性反应组为(10.5 4±3.0)个/视野,两组差异有统计学意义(t=12.491,P＜0.01).鼻咽癌组织中,有淋巴转移组VEGF-C阳性表达率(87.8%)明显高于无淋巴转移组(76.5%);有淋巴转移组MLVD(30.2 4±6.4)个/视野高于无淋巴转移组(24.8±3.6)个/视野,经统计学分析,差异均有统计学意义(t=3.259,P＜0.01).VEGF-C的表达与MLVD(t=3.512,P＜0.01)、淋巴转移(X2=7.715,P＜0.01)、临床分期(X2=4.250,P＜0.05),与病理分化程度无关(X2=0.000,P＞0.05).VEGF-C的表达与MLVD(t=3.512,P＜0.01)、淋巴转移(X2=7.715,P＜0.01,r=0.712)、临床分期(X2=4.250,P＜0.05,r=0.481)相关,与病理分化程度无关(X2=0.000,P＞0.05).结论 在鼻咽癌组织中VEGF-C呈高表达,VEGF-C表达与鼻咽癌组织MLVD、淋巴转移、临床分期密切相关.VEGF-C可能参与了鼻咽癌发生、浸润和转移的过程.VEGF-C与肿瘤组织微淋巴管密切相关,在鼻咽癌发展过程中起重要作用,可能成为抗肿瘤治疗的潜在靶点.     

不同月龄BALB/c小鼠耳蜗miR-96表达与ABR阈值及耳蜗形态的关系

目的　研究不同月龄BALB/c小鼠ABR阈值、耳蜗形态学改变及miR-96的表达,明确miR-96对老年性聋小鼠听力的调节作用。方法　通过听性脑干反应测试、荧光染色、扫描电镜,观察3、6、12、18月龄BALB/c小鼠8 kHz听反应阈值及耳蜗形态学改变。实时定量PCR定量检测miR-96在各月龄BALB/c小鼠耳蜗内的表达。SPSS13.0统计软件进行 统计分析。结果　3、6、12月龄组小鼠8 kHz听反应阈值分别为（18.5±8.3）、（45.8±7.8）、（85.6±15.6）dB SPL,18月龄组120 dB SPL刺激声基本测不出听觉反应。荧光染色及扫描电镜发现,自6月龄起毛细胞出现显著缺失,静纤毛出现不同程度缺失、倒伏、融合、变短和转位等改变,并随月龄增大病变逐渐加重。miR-96在3、6、12、18月龄BALB/c小鼠耳蜗中的相对表达量（2-△CT）分别为0.0225±0.0073、0.0162±0.0048、0.0116±0.0048和0.0050±0.0014,与3月龄小鼠相比,差异有统计学意义（P <0.05）。结论　BALB/c小鼠听力损失、耳蜗毛细胞缺失及纤毛损害随月龄增长而逐渐加重,小鼠耳蜗中miR-96表达随月龄增加而减少,提示miR-96可能在老年性聋的发病机制中起重要用。     

昆明小鼠耳蜗内脂肪酸转运蛋白4的表达与分布

目的 探索昆明小鼠耳蜗组织中脂肪酸转运蛋白4 (FATP4)的表达与分布。方法 选取健康成年(10周)昆明小鼠8只(16耳),其中2只(4耳)用于免疫荧光染色,6只(12耳)用于Western blot蛋白检测。通过耳蜗石蜡切片免疫荧光染色检测FATP4在耳蜗内各组织中的表达分布,Western blot蛋白检测耳蜗组织FATP4的表达水平。结果 耳蜗石蜡切片免疫荧光染色提示FATP4主要分布于耳蜗螺旋神经节、蜗轴、支持细胞、内毛细胞、外毛细胞、血管纹的中间细胞,Western blot蛋白检测结果进一步验证了FATP4在昆明小鼠耳蜗表达。结论 本研究初步揭示了FATP4在成年昆明小鼠耳蜗中的表达具有一定的特异性,为进一步研究耳蜗内脂肪酸代谢提供了理论依据。     

Characterization and Inflammatory Response of Perivascular-Resident Macrophage-Like Melanocytes in the Vestibular System

A large number of perivascular cells expressing both macrophage and melanocyte characteristics (named perivascular-resident macrophage-like melanocytes, PVM/Ms), previously found in the intra-strial fluid–blood barrier, are also found in the blood–labyrinth barrier area of the vestibular system in normal adult cochlea, including in the three ampullae of the semicircular canals (posterior, superior, and horizontal), utricle, and saccule. The cells were identified as PVM/Ms, positive for the macrophage and melanocyte marker proteins F4/80 and GSTα4. Similar to PVM/Ms present in the stria vascularis, the PVM/Ms in the vestibular system are closely associated with microvessels and structurally intertwined with endothelial cells and pericytes, with a density in normal (unstimulated) utricle of 225 ± 43/mm²; saccule 191 ± 25/mm²; horizontal ampullae 212 ± 36/mm²; anterior ampullae 238 ± 36/mm²; and posterior ampullae 223 ± 64/mm². Injection of bacterial lipopolysaccharide into the middle ear through the tympanic membrane causes the PVM/Ms to activate and arrange in an irregular pattern along capillary walls in all regions within a 48-h period. The inflammatory response significantly increases vascular permeability and leakage. The results underscore the morphological complexity of the blood barrier in the vestibular system, with its surrounding basal lamina, pericytes, as well as second line of defense in PVM/Ms. PVM/Ms may be important to maintain blood barrier integrity and initiating local inflammatory response in the vestibular system.     

喉鳞癌蛋白激酶C与Bcl-2基因表达的相关性

目的 探讨蛋白激酶C(protein kinase C,PKC)及Bcl-2基因在喉鳞癌组织表达及细胞凋亡中的意义。方法 采用免疫组化SP法检测28例喉癌手术切除的组织标本中PKC 、Bcl - 2 基因的表达情况,25例声带息肉作为对照。结果 喉鳞癌组织中Bcl - 2基因的阳性细胞表达率显著高于对照组(P < 0.01), PKC蛋白的阳性细胞表达率显著高于对照组(P < 0.01), 且PKC与Bcl - 2基因的表达呈明显正相关(r = 0.0875)。结论 喉癌组织细胞中PKC表达增多与凋亡抑制密切相关,推测喉癌组织细胞凋亡抑制可能是通过PKC这一信号转导途径实现的。     

二亚硝基哌嗪对转基因小鼠TNF受体相关因子2和p16的表达

目的研究二亚硝基哌嗪(N,N′-dinitrosopi- perazine,DNP)对TgN(p53mt-LMP1)/HT小鼠鼻腔和鼻咽黏膜上皮细胞增殖与TNF受体相关因子2(TNF receptor-associated Factor 2,TRAF2)、p16基因表达的影响及两者关系。方法HE染色法观察转基因小鼠组(TC)、转基因小鼠DNP诱导组(T1)和野生小鼠组(CC)、野生小鼠DNP诱导组(CI)鼻腔和鼻咽黏膜上皮细胞增殖特征;免疫组织化学染色法检测小鼠鼻腔和鼻咽黏膜上皮组织中TRAF2、p16基因的表达水平。结果TI、TC、CI和CC组小鼠鼻腔或鼻咽黏膜上皮癌前病变率分别为90%、10%、0和0。与TC、CI和CC组相比,TI组TRAF2基因表达水平显著增高(P<0.01),而p16基因表达水平显著降低(P<0.01);TRAF2和p16基因表达水平在TC和CC组之间的差异亦有统计学意义(P<0.01)。结论TgN(p53mt-LMP1)/HT小鼠鼻腔和鼻咽黏膜上皮细胞增殖活性增加,DNP可提高其增殖活性,而细胞增殖活性增加与TRAF2基因表达水平上调和p16基因表达水平下调密切相关。     

鼻咽癌患者miR-429、miR-200C表达与预后关系分析

目的 探讨鼻咽癌患者miR-429、miR-200C表达与预后关系.方法 选择2016年1月至2017年5月接受治疗的初治鼻咽癌患者80例,对病理穿刺组织样本行qRT-PCR检测对miR-429、miR-200C相抵表达量进行分析,治疗3个月后进行疗效评价.出院后以电话及门诊复诊的方式进行随访,记录患者生存时间.结果 ...     

细胞色素C在老年SD大鼠耳蜗不同阶段凋亡坏死毛细胞中表达的变化

目的：观察细胞色素c在老年SD大鼠不同阶段凋亡、坏死毛细胞中表达的变化,探讨老年大鼠耳蜗毛细胞死亡的机制。方法：16只SD大鼠分组：青年组8只,2～3月龄;老年组8只,24～26月龄。应用电位反应测听仪检测不同频率短纯音（4、8、16、32和40kHz）诱发的青年组和老年组大鼠双侧ABR。听觉功能测试后,解剖取出双耳听泡,分离耳蜗基膜。分别用细胞核DNA染料碘化丙锭染色不同月龄大鼠耳蜗基膜细胞核,细胞色素C免疫荧光染料标记大鼠耳蜗基膜细胞的细胞色素c蛋白,荧光显微镜下观察不同月龄大鼠耳蜗基膜细胞荧光染色的变化。结果：老年组大鼠的不同频率ABR阈值均高于青年组（P〈0．01）。微弱拘细胞色素c荧光标记物存在于青年组大鼠耳蜗基膜毛细胞质（细胞膜的内侧,线粒体存在部位）,增强的细胞色素C免疫反应物存在于老年组大鼠耳蜗基膜细胞。细胞色素c阳性反应见于：①细胞核型不变;②碘化丙锭染色略有加深,细胞核型不变;③细胞核轻微核固缩;④核肿胀的毛细胞。相反,部分核固缩≤3／4核的毛细胞细胞色素C阳性反应物减少或消失,核缺失区域的毛细胞未见细胞色素c阳性反应物。老年大鼠耳蜗基膜凋亡毛细胞线粒体释放的细胞色素c存在核内转移现象。结论：细胞色素c免疫荧光染色为老年耳蜗基膜毛细胞退行性变早期的细胞生物学标记之一。细胞色素C向细胞核内的转移,可能与老年大鼠耳蜗凋亡毛细胞染色质凝聚相关。     

Inner ear morphology in CBA/Ca and C57BL/6J mice in relationship to noise,age and phenotype

Summary CBA/Ca mice showing moderate hearing losses with onset late in life and C57BL/6J mice with progressive hearing losses starting when animals were young adults were exposed to a 2–7 kHz, 120 dB SPL noise band for 5 min in order to investigate morphological consequences to noise as a function of age and genotype. Permanent threshold shifts were determined by auditory brainstem responses 1 month after noise exposure at 1, 3 and 6 months of age. CBA mice had a decreasing susceptibility to noise with increasing age, while C57 mice remained equally susceptible throughout all ages tested. The threshold shifts were then analyzed in relation to morphological changes of the organ of Corti as visualized by light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). CBA mice exposed at 1 month and sacrificed at 2 months had seemingly normal cochlear morphologies under LM. In these animals SEM findings demonstrated mild stereocilia damage to noise trauma, but not when mice were exposed at 3 and 6 months. There was no visible morphological aging of hair cells found during the period tested. In contrast; the C57 mice had early hair cell changes including bent and fused stereocilia, bulging of the cuticular plates, hair cell loss and swelling of afferent dendrites. These changes became more pronounced throughout the test periods with the variability of damage in this strain more evident over time. This pattern was also aggravated in all C57 age groups tested after noise exposure. Quantification with cytocochleograms demonstrated a statistically different reaction pattern to noise trauma between the two different genotypes of mice.     

鼻腔局部应用布地奈德对鼻息肉中c-fos和c-myc表达的影响

目的:探讨鼻腔局部应用激素布地奈德对鼻息肉组织中c-fos、c-myc表达的影响.方法:采用免疫组织化学染色,观察比较经布地奈德喷鼻治疗10～12周鼻息肉组织(治疗组)和未治疗鼻息肉组织(对照组)中c-fos、c-myc蛋白的表达状况.结果:治疗组鼻息肉组织中c-fos表达率为15%,c-myc表达率为20%;对照组c-fos表达率为80%,c-myc表达率为85%.2组c-fos、c-myc表达率均差异有统计学意义(均P＜0.01),治疗组c-fos、c-myc阳性表达明显低于对照组.结论:布地奈德可能通过减少c-fos、c-myc表达,诱导炎性细胞凋亡而达到治疗鼻息肉的作用.     

CD45RO CD20及总IgE在声带息肉组织中表达的研究

目的 :从免疫病理学角度探讨细胞免疫、体液免疫和局部变态反应在声带息肉发病中的作用。方法 :对 30例声带息肉组织 (息肉组 )和 10例正常声带组织 (对照组 )采用免疫组织化学方法观察两组CD4 5RO、CD2 0及IgE的表达情况并检测其阳性细胞数。 结果 :息肉组CD4 5RO、CD2 0和IgE阳性细胞数显著高于对照组 (P <0 .0 1) ;声带息肉组织中CD4 5RO、CD2 0和IgE的表达之间无相关性 ;男、女患者间的表达差异亦无统计学意义 (P >0 .0 5 )。 结论 :由T淋巴细胞介导的细胞免疫和B淋巴细胞介导的体液免疫在声带息肉中免疫应答活跃 ;声带黏膜局部变态反应参与了声带息肉的发病 ;在声带息肉的发病机制中免疫病理改变起着重要的作用。