HMGB1在胶质瘤中的表达及对胶质瘤细胞的影响

发布时间：2018-07-11 15:47

本文选题：HMGB1 + U87-MG细胞系　；参考：《郑州大学》2017年硕士论文

【摘要】：研究背景和目的:高迁移率族蛋白1(high mobility group box 1,HMGB1)是存在于真核生物细胞内的一类非组蛋白染色体结合蛋白,参与基因转录、DNA修复、V(D)J重组、细胞外信号转导、核小体的构建、细胞的增殖、分化、迁移及凋亡,肿瘤的发生、生长、转移和浸润等。HMGB1含有215个氨基酸残基,高度保守。HMGB1由三个不同的结构域组成:两个HMG box结构域以及一个由30个氨基酸组成的C末端。HMGB1能与DNA结合,通过调节染色质架构(染色体中由非组蛋白构成的结构支架)进而调控翻译、修复和重组。在细胞受到刺激、细胞死亡、凋亡,组织缺氧及缺血再灌注时,HMGB1可以被释放到细胞外,参与炎症、细胞迁移、分化和血管发生。许多研究显示:HMGB1可参与肿瘤的发生和发展,其可能的机制包括HMGB1的高表达引起某些基因表达失调,从而导致细胞具有肿瘤表型;此外,HMGB1的高表达还可使获得肿瘤表型的细胞免于凋亡,最终导致肿瘤的发生。在结肠癌、前列腺癌、肺癌及食道癌等多种肿瘤组织中均发现了HMGB1的高表达,同时也伴随有晚期糖基化终末产物受体(receptor for advanced glycation end products,RAGE)的表达。RAGE是HMGB1的受体,通过JAK/STAT信号转导通路对HMGB1的表达产生调节效应。胶质母细胞瘤(Glioblastoma,GBM)是颅内最常见的恶性肿瘤,具有很高的致死率,其主要原因是由于肿瘤细胞的高浸润性和转移性。根据世界卫生组织(World Health Organization,WHO)所定标准,胶质瘤的病理分型可分为四级:I级是毛细胞型星形细胞瘤、II级是弥漫性星形细胞瘤、III级为间变型星形细胞瘤、IV级为多形性GBM,其中I级和II级为低级别,属良性肿瘤,III级和IV级为高级别,属恶性肿瘤。近些年来,针对胶质瘤的治疗虽然在手术、放化疗方面取得了很大进步,但由于肿瘤细胞的侵袭性、血脑屏障的不通透性以及对肿瘤细胞来源和其生长机制的不明确性,大大局限了治疗的有效性。有研究通过检测人脑胶质瘤组织中HMGB1基因的表达水平,探讨其与胶质瘤发生发展的关系,结果显示:HMGB1 m RNA的表达水平在胶质瘤组明显高于对照组,但在低级别组与高级别组中的表达水平无显著差异。也有研究显示:高表达HMGB1的肿瘤细胞或者坏死的肿瘤细胞可将HMGB1释放到细胞外,促使周围肿瘤细胞的增殖,同时诱导小血管的再生,从而促使肿瘤的不断生长;给予HMGB1或RAGE抑制剂,可抑制肿瘤细胞的增殖。目前对于HMGB1在人胶质瘤组织中的表达与定位及其作用尚不完全清楚,如:HMGB1在各级胶质瘤中的表达程度是否相同;表达在胞核还是胞浆;表达在何种类型细胞中;HMGB1是否影响胶质瘤细胞的增殖、存活、凋亡和自噬,以及具体的机制是什么等问题尚待解决。因此,本课题的研究目的是阐明HMGB1在各级胶质瘤组织中的表达程度,细胞定位与表达的细胞类型,初步探讨HMBG1对胶质瘤细胞线粒体的形态、细胞的凋亡与自噬的影响,为胶质瘤的临床治疗提供理论依据。研究材料:选自中国人民解放军第153中心医院病理科提供的各级胶质瘤组织的石蜡标本,这些标本均来自该院神经外科胶质瘤患者手术切除的组织,其中低级别标本(I~Ⅱ)12例,高级别标本(Ⅲ~Ⅳ)15例。细胞实验所用到的细胞系为U87-MG胶质瘤细胞系,购置于中国科学院细胞库。研究方法:1.免疫组化单标记或双标记染色,观察HMGB1在各级别胶质瘤中的表达程度、细胞定位及表达的细胞类型。2.质粒的构建及抽提:通过上海生工生物公司合成过表达HMGB1的质粒,HMGB1连接在p EGFP-C1载体上(HMGB1与EGFP融合表达),对照载体是p EGFP-C1空载体。使用BIOMIGA无内毒质粒DNA小量提取试剂盒抽提质粒。3.质粒转染:应用Simple-Fect Transfection Reagent转染试剂盒将过表达HMGB1的质粒和空对照质粒转染U87-MG细胞,1)提取细胞蛋白,进行Western Blotting(WB)实验,观察构建的质粒能否过表达HMGB1;2)用细胞免疫荧光法观察过表达HMGB1是否影响线粒体的形态、细胞的凋亡或自噬。4.用促炎因子LPSIFN-γ联合刺激U87-MG细胞,提取蛋白,进行WB实验,检测炎症是否影响HMGB1的表达。5.统计学分析:数据采用SPSS17.0或Origin70软件分析,具体分析方法为卡方检验以及单因素方差分析(One-Way ANOVA),数据采用均数±标准误(Mean±SE)表示。结果:1.高级别胶质瘤标本中,HMGB1高表达的标本比例显著高于低级别标本中的高表达比例(χ2=7.56,P=0.01);对于HMGB1中等程度表达的标本比例(χ2=1.17,P=0.18)以及低表达的标本比例(χ2=1.93,P=0.13),两个级别间未出现显著差异。2.HMGB1的阳性信号主要定位在胞核,偶见在一些细胞的胞浆中也有HMGB1的阳性信号。在低级别和高级别组织中,HMGB1在胞核中表达的细胞比例均显著高于在胞浆中表达的细胞比例(低级别P=0.00797;高级别P=0.00130);而将低级别和高级别在胞核与胞浆的相应数据进行对比,均未检测到有显著差异(胞核P=0.79254;胞浆P=0.44251)。此外,随着胶质瘤级别的增加,HMGB1的表达出现不规则形态或分页形态,而在低级别胶质瘤组织中,HMGB1的表达为均匀的圆形或椭圆形。3.各级胶质瘤组织中,在胶质瘤相关星形胶质细胞(GFAP标记)、胶质瘤相关干细胞(Nestin标记)、小胶质细胞(Iba1标记)、血管内皮样细胞以及神经元样细胞中均有HMGB1的阳性表达。4.高级别胶质瘤中先天免疫细胞小胶质细胞表现出明显的活化状态(突起回缩,胞体增大和变圆),此外,炎性因子IL-6的表达明显增加(P=0.0178)。采用促炎因子LPS和IFN-γ联合刺激U87-MG细胞,HMGB1的蛋白表达量增加。5.HMGB1在U87-MG中,主要表达在胞核,核仁分布更为明显。6.HMGB1过表达导致U87-MG细胞中线粒体发生断裂;未发现HMGB1过表达对LC3B以及Caspase3表达的影响。结论:1.随着胶质瘤级别的增加,HMGB1的表达增加;HMGB1主要表达在胞核,并且随着级别增加,HMGB1出现多形性分布;HMGB1在胶质细胞、免疫细胞、肿瘤干细胞、血管样细胞以及神经元样细胞中均有表达。2.在胶质瘤细胞中,HMGB1在炎症刺激下表达增加;HMGB1主要定位在胞核中,有较明显的核仁定位。过表达HMGB1导致胶质瘤细胞线粒体断裂。
[Abstract]:Background and purpose: high mobility group protein 1 (high mobility group box 1, HMGB1) is a kind of non histone binding protein that exists in eukaryotic cells, participates in gene transcription, DNA repair, V (D) J recombination, extracellular signal transduction, nucleosome construction, cell proliferation, differentiation, migration and apoptosis, tumor occurrence, and growth. Metastasis and infiltration of.HMGB1 contain 215 amino acid residues, and highly conserved.HMGB1 consists of three different domains: two HMG box domains and a C terminal.HMGB1 composed of 30 amino acids can be combined with DNA to regulate translation, repair and repair by regulating the chromatin structure (the structural scaffold made up of non egg white in chromosomes). Recombinant. When cells are stimulated, cell death, apoptosis, tissue hypoxia and ischemia-reperfusion, HMGB1 can be released to the extracellular, involved in inflammation, cell migration, differentiation and angiogenesis. Many studies show that HMGB1 may participate in the occurrence and development of tumor, and its possible mechanism, including the high expression of HMGB1, causes some gene expression maladjustment, from In addition, the high expression of HMGB1 also makes the cells that obtain the tumor phenotype free from apoptosis and eventually lead to the occurrence of tumor. The high expression of HMGB1 is found in many tumor tissues such as colon, prostate, lung and esophagus cancer, and also with the advanced glycosylated end product receptor (receptor for a). The expression of dvanced glycation end products, RAGE) is the receptor of HMGB1, which regulates the expression of HMGB1 through the JAK/STAT signal transduction pathway. Glioblastoma (Glioblastoma, GBM) is the most common malignant tumor in the brain and has a high mortality rate. The main reason is the high invasion and metastasis of the tumor cells. According to the standards set by the WHO (World Health Organization, WHO), the pathological classification of glioma can be divided into four stages: I grade is hairy astrocytoma, II grade is diffuse astrocytoma, III is an astrocytoma, IV level is polymorphic, and I and II are low grade, benign tumor, III class and grade are advanced. In recent years, the treatment of glioma has made great progress in surgery and radiotherapy and chemotherapy, but the effectiveness of the treatment is limited by the invasiveness of the tumor cells, the inability of the blood brain barrier and the unknown origin of the tumor cells and the mechanism of its growth. The expression level of HMGB1 gene in the tumor tissue and the relationship with the development of glioma showed that the expression level of HMGB1 m RNA was significantly higher in the glioma group than in the control group, but there was no significant difference between the low level group and the advanced group. There were also studies showing that the tumor cells with high expression of HMGB1 or the tumor necrosis of the tumor were fine. The cell can release HMGB1 to the extracellular, promote the proliferation of peripheral tumor cells, induce the regeneration of small blood vessels, and induce the growth of the tumor. HMGB1 or RAGE inhibitors can inhibit the proliferation of tumor cells. At present, the expression and location and role of HMGB1 in human glioma tissues are not completely clear, such as: HMGB1 at all levels Whether the expression of glioma is the same, expressed in the nucleus or cytoplasm, and in what type of cells, whether HMGB1 affects the proliferation, survival, apoptosis and autophagy of glioma cells, and what the specific mechanism is still to be solved. Therefore, the purpose of this study is to clarify the expression of HMGB1 in glioma tissues at all levels. Degree, cell location and expression of cell types, preliminary study on the morphology of mitochondria, apoptosis and autophagy of glioma cells by HMBG1, and provide theoretical basis for the clinical treatment of glioma. Research materials: the paraffin specimens from the glioma tissues at all levels from the 153rd Central Hospital of the PLA Hospital, which are provided by the pathology department of the Chinese people's Liberation Army. All the specimens were from the surgical excision tissue of patients with glioma in the Department of Neurosurgery, of which 12 cases were low grade (I~ II) and 15 cases of high grade specimens (III ~ IV). The cell line used in the cell experiment was U87-MG glioma cell line and was purchased in the cell bank of the Chinese Academy of Sciences. Method: 1. immuno histochemical single marker or double marker staining were used to observe the HMGB1 in the cell line. The expression level of glioma at all levels, the construction and extraction of cell type.2. plasmids of cell location and expression: through the synthesis of HMGB1 plasmid in Shanghai biotech company, HMGB1 is connected to P EGFP-C1 vector (HMGB1 and EGFP fusion expression), and the control carrier is p EGFP-C1 empty carrier. DNA small quantity extraction of BIOMIGA non endotoxin plasmid is used. The plasmid transfection of plasmid.3. plasmid: transfection kit with Simple-Fect Transfection Reagent transfection kit transfected HMGB1 plasmid and empty control plasmid into U87-MG cells, 1) extract cell protein, carry out Western Blotting (WB) experiment, observe whether the constructed plasmid can reach HMGB1; 2) observe the HMGB1 expression by cell immunofluorescence. Whether the morphology of mitochondria, apoptosis of cells or autophagic.4. using pro-inflammatory factor LPSIFN- gamma to stimulate U87-MG cells, extract protein, and carry out WB test, detect whether inflammation affects HMGB1 expression.5. statistical analysis: the data are analyzed by SPSS17.0 or Origin70 software, the specific analysis method is chi square test and single factor ANOVA analysis (One-W) Ay ANOVA), the data were expressed with mean + standard error (Mean + SE). Results: in 1. high grade glioma specimens, the ratio of high expression of HMGB1 was significantly higher than that in low grade specimens (x 2=7.56, P=0.01); the proportion of specimens expressed in moderate HMGB1 (chi 2=1.17, P=0.18) and the proportion of low expressed specimens (chi 2=1.93, P=0.13), The positive signals that did not show significant difference between the two levels were mainly located in the nucleus, and the positive signals of HMGB1 were also found in the cytoplasm of some cells. In the low and high level tissues, the proportion of cells expressed in the nucleus of HMGB1 was significantly higher than that expressed in the cytoplasm (low grade P=0.00797; advanced P=0.00130). Compared with the corresponding data of the nucleus and cytoplasm of the nucleus, there were no significant differences (nucleus P=0.79254; cytoplasm P=0.44251). In addition, the expression of HMGB1 appeared irregular or paging with the increase of glioma level, while in the low grade glioma tissue, the expression of HMGB1 was even round or round. In oval.3. glioma tissues, glioma related astrocytes (GFAP markers), glioma related stem cells (Nestin markers), microglia (Iba1 markers), vascular endothelial like cells and neuron like cells all have HMGB1 positive expression in.4. high grade glioma, and the innate microglia in the high grade glioma is obvious. In addition, the expression of inflammatory factor IL-6 increased significantly (P=0.0178). U87-MG cells were stimulated by the combination of proinflammatory LPS and IFN- gamma. The protein expression of HMGB1 increased in U87-MG, mainly in the nucleus, and the distribution of nucleolus was more obvious in the U87-MG cell line. The effect of HMGB1 overexpression on LC3B and Caspase3 expression was not found. Conclusion: 1. as the grade of glioma increased, the expression of HMGB1 increased; HMGB1 was mainly expressed in the nucleus, and as the grade increased, HMGB1 appeared polymorphic; HMGB1 in glial cells, immune cells, tumor stem cells, vascular like cells and neurons. The expression of.2. in glioma cells was expressed in the glioma cells, and the expression of HMGB1 increased under the stimulation of inflammation; HMGB1 was mainly located in the nucleus and had a distinct nucleolus location. The overexpression of HMGB1 led to the rupture of mitochondria in glioma cells.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

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