【作者】 李严；

【导师】 程宏伟； 冯春国； 徐培坤；

【作者基本信息】 安徽医科大学 ， 外科学， 2010， 硕士

【摘要】 目的BNIP3(Bcl-2/adenovirus E1B 19 kDa-interacting protein 3,Bcl-2腺病毒E1B 19 kD结合蛋白)是一种促凋亡的线粒体蛋白,属于Bcl-2蛋白家族成员,具有促进细胞凋亡的功能。国内外研究表明,BNIP3在多种肿瘤的发生、发展中有重要的作用。人髓母细胞瘤是颅内恶性程度极高的神经上皮肿瘤之一,有关BNIP3在髓母细胞瘤中的表达及其对肿瘤发展的作用的报道少见。本文旨在从蛋白质和基因两个水平上检测BNIP3在人髓母细胞瘤细胞中的表达,并采用甲基化抑制剂作用于髓母细胞瘤细胞,研究BNIP3基因和髓母细胞瘤的发生、发展的关系以及其对于诱导髓母细胞瘤细胞凋亡的作用,探讨BNIP3是否可以做为治疗髓母细胞瘤的靶标。方法(1)培养人髓母细胞瘤细胞Med-341(购自中科院细胞所),传代备用,并观察细胞的生长情况;另取10例安徽医科大学第一附属医院神经外科颅脑外伤手术切除的非肿瘤小脑组织,经HE染色证实为非肿瘤脑组织作为正常对照组。用免疫组织化学(IHC)方法检测人髓母细胞瘤细胞和非肿瘤小脑组织中BNIP3的表达。(2)收集培养的Med细胞备用;收集安徽医科大学第一附属医院神经外科颅脑外伤手术切除的非肿瘤脑组织新鲜标本5例,经HE染色证实为非肿瘤小脑组织作为正常对照组;应用逆转录聚合酶链式反应(reverse transcriptase-polymerase chain reaction, RT-PCR)技术检测Med细胞和非肿瘤小脑脑组织中BNIP3 mRNA的表达,以Hep-2细胞作为阳性对照;(3)将Med细胞接种于培养瓶中,待细胞生长良好时给予不同浓度的甲基化抑制剂5-氮杂脱氧胞苷处理,以不加5-氮杂脱氧胞苷为对照,持续作用7d,终止培养。将Med细胞接种于96孔板,用MTT法检测细胞的吸光度值,观察5-氮杂脱氧胞苷对Med细胞增殖的影响。结果(1)免疫组化及RT-PCR实验均表明,BNIP3在Med细胞中有表达,细胞质和细胞核内均有表达;而正常小脑组织中,BNIP3不表达;(2)在观察甲基化抑制剂5-氮杂脱氧胞苷对Med细胞增殖影响的实验中,1.0μmol/L以上的5-氮杂脱氧胞苷连续作用7天能够有效地抑制Med细胞的增殖,而且随着浓度的增加,其抑制作用增强。1μmol/L的5-氮杂脱氧胞苷对Med细胞的抑制率达38%,这种抑制活性5-氮杂脱氧胞苷在一定浓度范围内有剂量依赖关系。结论(1)BNIP3在Med细胞中的表达高于正常小脑组织的表达,提示BNIP3在人髓母细胞瘤的发生中起一定的作用。(2)甲基化抑制剂5-氮杂脱氧胞苷可以抑制Med细胞的增殖,且在一定浓度内随着浓度的增加,其抑制作用增强。提示甲基化抑制剂可能诱导BNIP3的表达,而BNIP3有可能成为治疗人髓母细胞瘤的靶标。更多还原

【Abstract】 Objective BNIP3(Bcl-2/adenovirus E1B 19 kDa-interacting protein 3) is a type of mitochondrial protein,belong to apoptosis Bcl-2 protein family members, promote cell apoptosis. Research shows that, in the development of many tumors,BNIP3 plays an important role.Medulloblastoma is a high-degree malignant of intracranial neuroepithelial tumors,and little was reported about the expression of BNIP3 in medulloblastoma and so was its function of tumor development. The aim of this article is to detect the expression of BNIP3 in human medulloblastoma cell at the levels of proteins and genes, and used the remover of DNA methylation on the medulloblastoma cell, to investigate the relevance of overexpression of BNIP3 ,and the occurrence,development of human medulloblastoma;and investigate its relationship of induce the medulloblastoma cell apoptosis, discusses the BNIP3 whether can be considered as a treatment targets of medulloblastoma.Methods (1)Cultivated medulloblastoma cells(Med-341),and observed the growth of cells; ten normal cerebellum tissues (non-tumor cerebellum tissues) were obtained from Neurosurgery Department of the First Affiliated Hospital of Anhui Medical Universityas as normal control from surgical resections of trauma patients, for whom a partial resection of normal cerebellun tissue was required as decompression treatment for their severe head injuries to reduce increased intracranial pressure. The SP immunohistochemistry method was use to detect the expression of BNIP3 in medulloblastoma cell and 10 cases of non-tumor cerebellum tissue samples.(2) The medulloblastoma cell was collected;five fresh non-tumor cerebellum tissues were collected from the First Affiliated Hospital of Anhui Medical University during the operation of neurosurgery. All tissues were diagnosised as non-tumor cerebellum by two senior pathologists. The expression of BNIP3 mRNA was examined by semi-quantity reverse transcription-polymerase chain reaction (RT-RT-PCR) in medulloblastoma cell and 5 non-tumor cerebellum tissues,Hep-2 cell was chosen as positive control.(3) The medulloblastoma cell was cultivated in bottles, all was gived different concentration of remover of DNA methylation(5-Aza-2-Deoxycytidine) for 7 days , compared with the medulloblastoma cell with nothing.Then cultivated the cell in 96-bore borade, the influence of cell generation was examined by MTT.Rusluts (1)The expression of BNIP3 was positive by immunohistochemical and RT-PCR in medulloblastoma cell;in non-tumor cerebellum tissue samples, no expression of BNIP3 was detected.(2)In the experimentation of observation the influence of remover of DNA methylation (5-Aza-2-Deoxycytidine),we were discoverd that the cell was depressed by the concentration of 5-aza-cytosine deoxyriboside above 1.0μmol/L.This cued that remover of DNA methylation ( 5-Aza-2-Deoxycytidine ) may induse the expreesion of BNIP3.The inhibition rate up to 38% by the 5-Aza-2-Deoxycytidine of 1.0μmol/L.And this inhibitory activity had dose-response relationship in certain concentration.Conclusion (1)The level of BNIP3 in medulloblastoma cell is higher than in non-tumor cerebellun tissue.There was a relationship between overexpression of BNIP3 and the development of human medulloblastoma.(2)Remover of DNA methylation(5-Aza-2-Deoxycytidine)could inhibit proliferation of medulloblastoma cell,and in certain concentration with concentration increases, its effect was enhanced. This cued that remover of DNA methylation (5-Aza-2-Deoxycytidine)could induce the expression of BNIP3,and BNIP3 could be one treatment targets of medulloblastoma.更多还原