【作者】 王萍；

【导师】 王铁；

【作者基本信息】 中国医科大学 ， 耳鼻咽喉科学， 2004， 硕士

【摘要】 前言 近年来研究发现，热休克蛋白Heat shock protein70(HsP70)与肿瘤细胞的发生、发展及恶变密切相关，并在肿瘤免疫中发挥重要作用。热休克蛋白作为分子伴侣，在肿瘤细胞不断增殖过程中协助蛋白质的合成和稳定，帮助其适应或耐受不利的生长环境；同时，热休克蛋白的抗原呈递作用和并不具备免疫原性又被用来制备多肽复合物疫苗用于肿瘤的特异性治疗。热休克蛋白在许多肿瘤组织中表达增高，在头颈部上皮性肿瘤如喉癌、鼻咽癌中已有报道，而在喉乳头状瘤Laryngeal papilloma(LP)中尚未见报道。因此，我们选择30例喉乳头状瘤应用免疫组化法检测HsP70的表达水平并分析其可能的临床意义。 实验材料和方法 1．标本来源 随机选取我院1989年1月至2002年8月喉乳头状瘤病理标本30例。其中男16例，女14例；年龄在20岁到72岁之间。平均年龄46岁。选择30例声带息肉及11例恶变喉乳头状瘤作为对照组。以上病例均经病理证实，且术前均未接受任何药物及其它方法治疗。 2．主要试剂及仪器 鼠抗人HsP70单克隆抗体 SP试剂盒(均购自福建迈新公司) PBS缓冲液，10％正常山羊血清，DAB显色剂，100％—70％不同浓度的酒精，冰箱，温箱。显微图象采集系统。 3．实验方法 本实验采用常规免疫组化SP法：将切片常规脱蜡至脱水，3％过氧化氢溶液10分钟去除内源性过氧化物酶。正常山羊血清20分钟封闭；滴加一抗HsP70 4℃过夜；滴加二抗(生物素化山羊抗兔IgG)，室温孵育20分钟后滴加SP复合物37℃反应20分钟；二氨基联苯胺显色，光镜下控制反应时间，蒸馏水终止显色，梯度酒精脱水，二甲苯透明，中性树胶封片，光镜下观察。空白对照用PBS代替一抗，其余步骤不变。用卡方检验法分析其结果的统计学意义。实验结果 30例喉乳头状瘤组织中Hs种O阳性者9例，阳性率为30%，30例声带息肉组织中阳性者1例，阳性率为3.33%，两组间具有显著差异性(P<0.01);恶变喉乳头状瘤n例阳性8例，阳性率为72.7%，与喉乳头状瘤组间具有显著差异性(P<0.05)。讨论 热休克蛋白是一组普遍存在于生物细胞中的高度保守的蛋白质，受到各种理化因素(病毒感染，缺氧，乙醇等)刺激后，可被诱导表达。HsP7O与肿瘤细胞的发生、发展及恶变密切相关。我们在实验中发现，在喉乳头状瘤组织及恶变喉乳头状瘤组织中，HsP7O的表达显著升高，这可能与肿瘤组织营养缺乏和慢性缺氧有关;同时与肿瘤细胞生长活跃，蛋白质合成增加需要HsP7O作为分子伴侣来稳定和调节这一异常增殖过程有关。热休克蛋白可以使上皮细胞继续增殖并向恶性转化，如此反复使肿瘤细胞得以增殖。结论HsP7O在喉乳头状瘤的发生、发展及恶变中可能具有重要作用。更多还原

【Abstract】 It is found that HsP 70 ( Heat shock protein 70 ) has a relationship with the development of neoplasm and plays an important role in the neoplasm - immune. HsP is a kind of molecular companions . It can help the protein construct and stablize in the course of hyperplasia to adapt the bad enviroments . The expression of HsP in malignant tumor is higher than that in the others. But there is not any report about it in larynegeal papilloma (LP) .We chose thirty cases of LP, used immunohistochemistry method to test the expression of HsP 70 in it and analyses the clinical value.Materials and MethodsMaterials; Choose thirty cases of LP in CMU 2 from 1989 to 2002. Among these, male 16 cases, female 14 cases. Age from 20 to 72 years old, average 46 years old. At the same time , choose 30 cases of vocal polyps and 11 cases of vicious larynegeal papilloma as control.Main reagents and instruments; The antibody we used is HsP70 single - leaf antibody (mouse antihuman) ; And the SP - box is a chemical reagent box which came from FU JIAN MaiXin company. The other reagents and instruments inclde PBS buffer; 10% normal goat serum;DAB colour reagents;the different concentration ethyl from70 - 100% ; fridge; lukewarm box and photograph collect system.Methods; Use immunohistochemistry method to examine the expression of HsP 70 in larynegeal papilloma. Conventional getrid of paraffin wax and water. After immersed in 3% H202 and normal goat serum,the sections were incubated with antibody to HsP70 at 37℃ overnight. A biotinylated anti -rabbit antibody was used as the second antibody. For negative controls the primary antibody was omitted on one of the mounted sections on all slides. Processing was ultimatelyperformed with a SP and DAB. The reaction was observed under a light - microscope and was finally stopped by application of PBS. The specimens were dehydrated in baths of ascending ethanol concentration. Analysis and photograph of the sections under Olympus light - microscope . Thex2 - test was used to a-nalysis the results in statistics.ResultsNine cases are positive in 30 cases of LP (30% ) ; one case in 30 cases of vocal polyps (3. 33% ) is positive . There is a difference between them in statistics. Eight cases are positive in 11 cases of vicious larynegeal papilloma (72. 7% ). There are differences between vicious larynegeal papilloma and LP (p < 0.05).DiscussionHsP is a kind of proteins which exists in many cells. After stimulated by alcohol and virus infection and so on , it can be induced to express. HsP 70 has a closely relationship with the development of tumor cell. We found from tests that in larynegeal papilloma and vicious larynegeal papilloma, the expression of HsP 70 is higher clearly, which may have to do with the lack of nutrition of tumor cells and have an association with the HsP 70 as a molecular companion to stablize the abnormal hyperplasia in rapid grow of tumor cells. HsP can make the epithelial cells grow continuously and turn to molignant ones.ConclusionsHsP 70 may have an important role during the development of larynegeal papilloma.更多还原