骨肉瘤组织中MICA、MMP-9和NF-κB蛋白的表达及相关性

目的 探讨骨肉瘤组织中MHC Ⅰ类链相关蛋白A(MHC class Ⅰ chain-related A,MICA)、MMP-9和NF-KB的表达及相互间的关系,为研究骨肉瘤组织中MICA蛋白表达和脱落机制提供组织学依据.方法 应用免疫组织化学方法检测66例骨肉瘤、11例骨母细胞瘤,8例骨化性纤维瘤和6例正常骨组织中MICA蛋白的表达情况,并检测66例骨肉瘤组织中MMP-9和NF-KB蛋白的表达情况.结果 (1)MICA蛋白在骨肉瘤组织、骨母细胞瘤、骨化性纤维瘤和正常骨组织的表达率分别为51.6%(34/66)、9%(1/11)、0(0/8)、0(0/6),骨肉瘤组织中MICA表达与骨母细胞瘤(P=0.01)、骨化性纤维瘤(P<0.01)和正常骨组织(P<0.05的差异有显著性.(2)骨肉瘤组织中MMP-9和NF-KB表达率分别为55%(36/66)和73%(48/66);NF-KB与MICA(r=0.373,P<0.01)和MMP-9(r=0.536,P<0.01)的表达呈正相关关系.结论 MICA蛋白可作为骨肉瘤诊断的分子标志物;NF-KB可能参与MICA蛋白表达和脱落的分子机制,NF-kB可作为骨肉瘤免疫治疗的候选靶点.     

骨肉瘤中EGFR、HER-2的表达及其临床意义

目的探讨EGFR、HER-2在骨肉瘤组织中的表达及其临床意义。方法采用免疫组化En Vision法检测45例骨肉瘤组织及肿瘤周围正常骨组织中EGFR、HER-2的表达,分析EGFR、HER-2的表达与骨肉瘤临床病理特征的关系。结果骨肉瘤中EGFR、HER-2的阳性率分别为75.6%、20%,显著高于对照组(P0.05)。骨肉瘤中EGFR的表达与肿瘤分化程度、有无转移及TNM分期有关;与患者性别、年龄、肿瘤大小无关(P0.05)。结论骨肉瘤中EGFR的表达与其生物学行为密切相关,可能参与骨肉瘤的发生、发展,并促进骨肉瘤转移,其可以作为骨肉瘤恶性程度的判断指标。     

p53蛋白三维空间结构改变与人骨肉瘤的关系

目的 研究骨肉瘤中p53基因突变及其蛋白三维结构的改变,旨在分析p53基因突变的分子机制及其与骨肉瘤发生、发展的关系.方法 应用PCR-SSCP、DNA序列分析以及相关分析软件重构p53蛋白三维结构等技术对43例骨肉瘤进行研究.结果 将16例p53基因突变病例分为A组(p53基因DNA结合位点发生突变或缺损组)与B组(非DNA结合位点突变组或同义突变),A组的远处转移率明显高于B组(P<0.01),组织学分化低于B组(P<0.05),且中位生存时间(10.3个月)低于B组(34.3个月).结论 p53基因碱基的突变可引起相应氨基酸的改变,导致p53蛋白三维空间构象改变.p53基因DNA结合位点的突变可影响其蛋白的生物学功能,并促进骨肉瘤的发生、发展.     

喉癌中线粒体DNA拷贝数和D-loop区基因突变与临床病理的相关性

目的探讨喉癌线粒体DNA(mitochondrial DNA,mt DNA)拷贝数和D-loop区基因突变与临床病理特征的相关性。方法收集武汉市第一医院诊治的40例喉癌患者,取mt DNA的CoxⅡ和核DNA的β-actin为目的片段进行实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)扩增,对mt DNA D-loop区进行测序。结果喉癌组织中mt DNA拷贝数(0. 925±0. 136)高于其对应癌旁组织(0. 815±0. 096),两者差异有统计学意义(P 0. 05)。mt DNA拷贝数与患者淋巴结转移相关(P 0. 05),但与患者性别、吸烟、饮酒、肿瘤分期以及肿瘤分化无关。40例喉癌中有21例(52. 50%) mt DNA D-loop区共检测17个突变位点、34个突变;发现86个多态性位点;且mt DNA D-loop区基因突变与患者淋巴结转移和肿瘤分化程度相关(P均0. 05),其与患者性别、吸烟、饮酒以及肿瘤分期无关。结论喉癌组织中mt DNA拷贝数高于其对应癌旁组织,且mt DNA Dloop区存在高频基因突变,提示mt DNA拷贝数变化以及D-loop区基因突变与喉癌的发生、发展密切相关。     

线粒体DNA Cyt-b和血管内皮生长因子mRNA在乳腺癌中的表达及意义

目的探讨线粒体DNA(mtDNA)Cyt-b和血管内皮生长因子(VEGF)mRNA的表达在乳腺癌发生、发展过程中的意义。方法采用逆转录-聚合酶链反应(RT-PCR),分别对19位乳腺癌患者的19例乳腺癌组织标本和19例癌旁组织标本的mtDNA Cyt-b、VEGF mRNA的表达进行检测(β-actin作为定量标准物),观察它们在乳腺癌组织和癌旁正常组织中的表达差异以及与临床病理参数的关系。结果19位乳腺癌患者中,乳腺癌组织mtDNACyt-b和VEGF mRNA的表达均高于癌旁组织,两组比较均有明显差异(P<0.01),而乳腺癌组织中VEGF的两个异构体VEGF145和VEGF189的mRNA表达高于癌旁组织,两组比较均有差异(P<0.05),并且mtDNA Cyt-b与VEGFmRNA的表达呈正相关性(P<0.01),与VEGF145、VEGF189的mRNA表达均呈正相关性(P<0.05);淋巴结转移组mtDNA Cyt-b和VEGF mRNA的表达较无淋巴结转移组高,两组比较均有差异(P<0.05);TNM分期Ⅰ期mtDNA Cyt-b和VEGF mRNA的表达较Ⅱ期低,两组比较均有差异(P<0.05);Ⅰ期与Ⅲ期比较两组mtDNA Cyt-b mRNA的表达有明显差异(P<0.01),而VEGF mRNA的表达有差异(P<0.05)。结论乳腺癌中mtDNA Cyt-b和VEGF mRNA的表达上调,并且在肿瘤的能量传递过程起着一定作用。     

p57kip2、cyclin D1在食管癌中的表达及意义

目的研究p57kip2在食管癌中的表达及意义,探讨其与cyclin D1在食管癌中表达的相关性。方法采用免疫组织化学SP法检测50例食管癌组织及15例正常食管上皮组织中p57kip2、cyclin D1的表达情况;应用流式细胞术对这些组织的DNA含量和细胞周期分布进行分析。结果免疫组化:p57kip2蛋白在食管癌组织中阳性表达率(34.0%)低于正常组织(80%)(P<0.01),与食管癌组织分化程度、浸润深度和淋巴结转移情况有关(P<0.05);cyclin D1蛋白在食管癌组织中的阳性表达率(64.0%)高于正常组织(13.33%)(P<0.01),与浸润深度和淋巴结转移情况有关(P<0.05),与食管癌组织分化程度无关(P>0.05)。p57kip2和cyclin D1之间表达负相关(r=-0.429,P<0.01)。流式细胞术:与正常组织相比,癌组织中DNA含量增高,异倍体细胞增加;G0/G1期细胞减少,而S期和G2/M期细胞增多,增殖指数(PI)高于正常组织。结论p57kip2蛋白低表达可能与食管癌的发生发展有关;p57kip2与cyclin D1联合检测可作为评估食管癌恶性程度指标。     

Fascin在骨肉瘤中的表达及意义

目的探讨fascin蛋白在骨肉瘤中的表达变化与临床病理指标及生存时间的关系。方法应用免疫组织化学方法和Western blot方法检测45例手术切除骨肉瘤标本fascin蛋白的表达,将所得fascin蛋白表达的光密度值数据与临床病理指标及生存时间进行统计学分析。结果肿瘤边缘的非瘤骨组织中无fascin蛋白表达,45例骨肉瘤标本中有24例fascin蛋白为中高表达,这种阳性表达与外科分期、化疗反应性呈正相关(P<0.05)。在单因素及多因素分析中,蛋白fascin的高表达的骨肉瘤患者总生存期及无病生存期明显降低(P<0.05)。结论fascin基因可能是骨肉瘤的一个潜在治疗靶点。     

miR-140在骨肉瘤组织中表达降低及其过表达可促进骨肉瘤U2细胞的凋亡

目的探讨骨肉瘤组织中miR-140的表达,以及过表达miR-140后对骨肉瘤细胞生物学行为的影响。方法用实时荧光定量PCR(qRT-PCR)检测40例骨肉瘤患者肿瘤组织与癌旁组织中miR-140的表达。用脂质体法瞬时将人工合成的成熟miR-140片段(miR-140 mimic)转染至骨肉瘤U2细胞中;用qRT-PCR法检测细胞中miR-140的表达,CCK-8法及流式细胞仪检测细胞的增殖及凋亡,Transwell小室检测细胞的侵袭能力,Western印迹法检测miR-140靶基因组蛋白去乙酰化酶4(HDAC4)的表达。结果 72.5%(29/40)的骨肉瘤组织miR-140表达明显低于瘤旁组织(P0.05)。转染miR-140 mimic后U2细胞miR-140的表达水平明显上调(P0.05),细胞增殖能力明显降低(P0.05),凋亡率明显升高(P0.05),侵袭能力明显下降(P0.05),细胞中HDAC4蛋白的表达水平明显下降(P0.05)。结论 miR-140在骨肉瘤中低表达;过表达miR-140后可抑制骨肉瘤U2细胞的增殖和侵袭,并促进其凋亡。这一作用可能部分依赖于miR-140靶向抑制HDAC4的表达。     

KAI1及其等位基因杂合性缺失与胰腺癌转移及预后的相关性

目的 探讨肿瘤转移抑制基因KAI1与胰腺癌转移及预后的相关性,并进一步研究此相关性与其等位基因杂合性缺失(LOH)的关系.方法 收集胰腺癌标本62例,行免疫组织化学Power-vision二步法检测KAI1基因(CD82)的表达;石蜡切片显微切割癌及癌旁正常胰腺组织,提取DNA,变性高效液相色谱法(DHPLC)检测LOH.结果 CD82在胰腺癌原发灶中表达率为76%(47/62),在有淋巴结转移、远处脏器转移病例中表达显著降低(P<0.05),在晚期(Ⅲ、Ⅳ)肿瘤中比早期(Ⅰ、Ⅱ)表达显著降低(P<0.01),CD82表达阳性者的1年生存率显著高于表达阴性者(P<0.05).显微切割标本PCR扩增,微卫星D11S1344有5对、D11S1326有2对存在LOH,发生率为17%.结论 随着胰腺癌的进展,CD82的表达逐渐降低,这种降低与KAI1等位基因的杂合性缺失有关.检测CD82的表达及LOH对判断肿瘤的分期,淋巴转移和远处转移及患者的预后有一定的参考价值.     

肺癌患者RASSF1A启动子甲基化状态及其基因表达水平

目的探讨肺癌RAS相关区域家族1A(RASSF1A)启动子CpG岛甲基化状态和基因表达水平与肺癌发生的关系。方法用甲基化特异性PCR检测RASSF1A启动子CpG岛甲基化状态,实时定量PCR检测RASSF1A mRNA的表达水平。结果在肺癌组织中RASSF1A启动子甲基化频率为53.33%(24/45),在正常肺组织中发生甲基化的频率为13.04%(3/23)(P<0.05)。正常肺组织中RASSF1A mRNA全部表达,肺癌组织中表达缺失率为28.89%(13/45),且表达量低于正常肺组织(P<0.05);不同年龄、性别、肿瘤大小、恶性程度、肿瘤分类中其表达量无显著差异;RASSF1A甲基化与其mRNA表达水平下降密切相关。结论肺癌中RASSF1A基因启动子甲基化频率明显升高,其表达普遍下调或缺失,提示RASSF1A启动子甲基化在肺癌发生、发展中起一定作用。     

Mitochondrial DNA damage as a mechanism of cell loss in Alzheimer's disease

Aging is associated with impaired mitochondrial function caused by accumulation of oxygen free radical-induced mitochondrial (Mt) DNA mutations. One prevailing theory is that age-associated diseases, including Alzheimer's disease (AD), may be precipitated, propagated, or caused by impaired mitochondrial function. To investigate the role of MtDNA relative to genomic (Gn) DNA damage in AD, temporal lobe samples from postmortem AD (n = 37) and control (n = 25) brains were analyzed for MtDNA and GnDNA fragmentation, mitochondrial protein and cytochrome oxidase expression, MitoTracker Green fluorescence (to assess mitochondrial mass/abundance), and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OHdG) immunoreactivity. Brains with AD had more extensive nicking and fragmentation of both MtDNA and GnDNA as demonstrated by agarose gel electrophoresis, end-labeling, and the in situ terminal deoxynucleotide transferase end-labeling (TUNEL) assay, and only the brains with AD had detectable 8-OHdG immunoreactivity in cortical neurons. Increased MtDNA damage in AD was associated with reduced MtDNA content, as demonstrated by semiquantitative PCR analysis and reduced levels of Mt protein and cytochrome oxidase expression by Western blot analysis or immunohistochemical staining with image analysis. The finding of reduced MitoTracker Green fluorescence in AD brains provided additional evidence that reduced Mt mass/abundance occurs with AD neurodegeneration. The presence of increased MtDNA and GnDNA damage in AD suggest dual cell death cascades in AD. Impaired mitochondrial function caused by MtDNA damage may render brain cells in AD more susceptible to oxidative injury and thereby provide a mechanism by which systemic or environmental factors could influence the course of disease.     

Biological characterization of human bone tumors. V. Zonal characterization of osteosarcoma: topological biochemical analysis correlated with morphology

Human osteosarcoma specimens were sliced in a cryomicrotome under strict morphological guidance. Serial sections of ten 10 micron slices each were collected in two groups according to morphologic criteria, one containing mostly undifferentiated tumor tissue, the other predominantly well-differentiated tumor tissue. The two series were analysed chemically for alkaline phosphatase (APase) acid phosphatase (acPase), beta-glucuronidase and proteolytic activities; protein, phosphorus, hydroxyproline, hexosamine, water and collagen contents were also determined. Four different types of osteosarcoma were studied: case 1 was a highly malignant osteoblastic osteosarcoma, case 2 a small cell sclerosing osteosarcoma case 3 a well-differentiated osteosarcoma, and case 4 a highly malignant anaplastic osteosarcoma. The types of cases 1, 2 and 3 are known as osteoid-forming tumors. In their less well differentiated areas APase activity was about twice as high as in better differentiated osteosarcoma. In contrast, no APase was found in the wholly undifferentiated areas of case 4, while the enzyme showed a marked increase in the areas of incipient differentiation of this tumor. The matrix of tumors differs with regard to collagen and hexosamine contents, in accordance with the general state of differentiation. In general, increasing hexosamine contents together with decreasing hydroxyproline contents will reflect the anaplastic, dedifferentiated osteosarcoma. Calcification evident in the better differentiated areas of osteosarcoma is indicated by the phosphorus content, highest in case 2, with cases 3, 1, and 4 following in sequential order.     

胸苷激酶1（TK1）在骨肉瘤中的表达及其临床病理意义

目的　探讨骨肉瘤肿瘤细胞胸苷激酶1（TK1）的表达状况及骨肉瘤患者血清TK1的水平,分析其临床病理意义。 方法 采用免疫组化MaxVision法检测40例骨肉瘤和20例骨巨细胞瘤组织标本中TK1的表达;点印迹免疫酶化学发光法检测20例骨肉瘤患者和20例骨外伤患者血清TK1的水平。 结果　骨肉瘤肿瘤细胞TK1阳性信号位于胞浆及胞核,高级别骨肉瘤阳性率显著高于低级别骨肉瘤（P<0.05）;骨肉瘤肿瘤细胞TK1阳性率亦显著高于骨巨细胞瘤（P<0.05）;骨肉瘤患者血清TK1的水平(2.95±1.33)pmol/L（0.97~7.25 pmol/L）高于外伤除外肿瘤患者(0.89±0.51) pmol/L (0.32~2.58 pmol/L),两者差异具有显著性统计学意义（P<0.05）。 结论　TK1在骨肉瘤与骨的良性病变的鉴别诊断中具有一定的意义。此外,TK1在骨肉瘤患者血清中可被检测到较高的水平,是一项检测骨肉瘤增殖活性较灵敏的指标。     

采用组织块移植法快速建立裸鼠原位骨肉瘤模型

背景：骨肉瘤细胞起源于间叶组织这一特点使得骨肉瘤动物模型的构建存在较多困难,进展缓慢的问题。 目的：采用组织块移植法建立裸鼠原位骨肉瘤模型,并观察和评价其生物学特性。 方法：选用SPF 级 6 周龄裸鼠15只,将制备好的MG-63 骨肉瘤细胞悬液注射到裸鼠右前肢腋下,成瘤后连续传 3 代,待肿瘤生长稳定后将瘤块组织移植到裸鼠下肢胫骨髓腔内,评价其生物学特性,采用X射线观察裸鼠成瘤率和成瘤特点。并以正常裸鼠5只做对照。 结果与结论：15只裸鼠造模过程顺利,造模后通过观察1只成瘤失败,成瘤率为93%;造模后观察发现采用组织块移植法三四周可见裸鼠下肢局部肿瘤状组织形成,四五周后X射线观察可见裸鼠胫骨中上段出现不明显的溶骨以及瘤样骨形成;骨肉瘤模型组碱性磷酸酶的表达水平明显高于正常对照组(P < 0.01)。数据表明采用组织块移植法进行裸鼠原位骨肉瘤造模,其造模方法简单,成瘤率高,瘤体生长速度快,对骨皮质及周围软组织的破坏力较强,接近临床骨肉瘤患者的实际情况。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程      

膜型/可溶型MHC Ⅰ类链相关分子A及其受体NKG2D在骨肉瘤中的表达及其意义

目的探讨膜型／可溶型MHCI类链相关分子A（MICA）及其受体NKG2D在骨肉瘤中的表达及其意义。方法采用免疫组织化学（LSAB法）检测膜型MICA（mMICA）在43例骨肉瘤组织中的表达;用流式细胞仪检测上述病例中的16例患者外周血淋巴细胞NKG2D的表达;用ELISA法检测患者血清中可溶型MICA（sMICA）的水平。结果骨肉瘤细胞广泛表达mMICA（37／43）,外周血淋巴细胞NKG2D的表达普遍下降,两者的表达水平均与骨肉瘤的分化呈正相关（P〈0．05）,与临床分期呈负相关（P〈0．05）。检测16例骨肉瘤患者血清中sMICA含量,其中5例升高,且与骨肉瘤的远处转移及临床分期呈正相关（P〈0．05）。结论（1）骨肉瘤细胞表达mMICA,外周血淋巴细胞NKG2D的表达以及血清中sMICA的含量与骨肉瘤的分化及临床分期相关,因此可望作为骨肉瘤病理诊断和估计预后的生物学指标。（2）MICA-NKG2D介导的免疫监视障碍可能促进了骨肉瘤的进展。     

Biological characterization of human bone tumors. IV. Combined biochemical and histological analyses of different osteosarcomas

A new technique was applied to the study of human osteosarcoma. Ten slices of 10 micron were cut serially from 2 X 2 X 6 mm shock frozen blocks of human osteosarcoma for chemical analysis. Before and after each series of 10 slices, one slice of 10 micron was separated for morphological analysis. Four different types of osteosarcoma were investigated: Case 1 was an atypical osteoblastic osteosarcoma, case 2 a small cell sclerosing osteosarcoma, case 3 a well-differentiated parosteal osteosarcoma grade I, and case 4 a highly malignant anaplastic osteosarcoma. Alkaline phosphatase, acid phosphatase, beta-glucuronidase and proteolytic activities were analysed as well as matrix collagen and hexosamine, phosphorus (Pi and Po), protein, DNA, and water content. In accordance with the morphology, the obtained data illustrate the great heterogeneity of osteosarcomas. Although case 1, 2 and 3 all represent calcifying types of the tumor, characteristic differences exist with regard to the matrix and the degree of calcification. In contrast to these three, case 4 presents a noncalcified type of osteosarcoma whose matrix contains relatively high amounts of hexosamine and low amounts of collagen, whereas DNA and water contents are high. The data from the analysis of osteosarcoma were compared with previous results from the calf epiphyseal growth plate in order to define differences and similarities between the formation of tumor bone and the physiological formation of hard tissue.     

抗人骨肉瘤单克隆抗体5D3的特异性鉴定

目的 鉴定抗人骨肉瘤单克隆抗体（mAb)5D3的特异性。方法 用免疫组化染色法检测mAb 5D3与骨肉瘤细胞系、骨肉瘤组织、其它肿瘤组织及正常组织的反应性。结果 免疫组化染色检测发现，mAb 5D3相应抗原在部分骨源性肿瘤，特别是骨肉瘤组织及骨肉瘤细胞系呈高度表达，中度以上染色者占阳性总数的77％以上，主要分布于细胞核上。14种正常组织中未见阳性反应。结论 mAb 5D3具有高度特异性，为骨肉瘤的早期诊断及免疫治疗提供了可能性。     

Phalangeal intraosseous well-differentiated osteosarcoma of the hand

A case of intraosseous well-differentiated osteosarcoma in one phalanx of the hand is reported. A 78-year-old man noticed swelling in the little finger of his right hand approximately 7 years before referral. Imaging disclosed a tumour with a ground glass appearance and irregular mottled calcification occupying almost all of the phalanx marrow and suggested slight invasion into the soft tissue. Open biopsy suggested a diagnosis of well-differentiated fibroblastic osteosarcoma. The finger and its metacarpal bone were amputated and a tumour measuring 3.5x2.2x2.0 cm and with an indistinct soft tissue margin was found in the bone marrow. Histologically, the tumour was composed of fibroblastic cells with few mitoses, and neoplastic bone formation was apparent. Although the tumour appeared to be a fibrous dysplasia, the presence of nuclear atypia, hypercellularity, and the absence of a typical woven bone pattern in addition to the soft tissue invasion indicated otherwise. Ultrastructural examination showed focal myofibroblastic differentiation, and immunohistochemistry revealed smooth muscle actin, vimentin, osteocalcin, osteonectin and MIB1 in the tumour cells. This ultrastructural and immunohistochemical study is believed to be the first detailed report of an intraosseous well-differentiated osteosarcoma of phalangeal bone.     

MicroRNA-539 suppresses osteosarcoma cell invasion and migration in vitro and targeting Matrix metallopeptidase-8

microRNAs (miRNA) are a class of small, non-coding RNA that involved in different cancer-related processes. Previous studies have been indicated miR-539 as a tumor suppressor during tumorigenesis. However, the role of miR-539 in osteosarcoma is still unclear. In this study, we demonstrate miR-539 was downregulated in osteosarcoma tissues compared to adjacent normal tissue. Functional study suggests miR-539 inhabits the osteosarcoma cell proliferation, invasion and migration. We also identified that MMP8 was a direct target of miR-539 by the luciferase activity assay. These findings provide evidence that miR-539 plays a key role in inhibiting osteosarcoma cell invasion and migration and can regulating MMP8 expression in osteosarcoma cells. These strongly suggest that exogenous miR-539 may have therapeutic value in treating osteosarcoma.