激光显微切割与蛋白质组学技术在口腔癌中的应用

目的 在蛋白水平研究口腔癌的疾病特征,以利于早期诊断及治疗.方法 通过激光显微切割获取单纯的细胞,再用蛋白质组学技术研究分析这些细胞所表达的蛋白质.结果 许多蛋白质在质和量上的变化,与口腔癌的发生、发展有密切关系.结论 激光显微切割与蛋白质组学技术的应用,将促进口腔癌研究的进一步深入.     

激光捕获显微切割技术及其在肿瘤研究中的应用

激光捕获显微切割技术是近几年才发展起来的技术，国内尚无有关这方面完整的文献报道，本文简要综述其一般情况及在肿瘤学研究中的应用。     

联合应用激光捕获显微切割技术及乙酰化稳定同位素标记定量技术在结直肠癌蛋白质组学研究中的价值

目的应用蛋白质组学技术,建立结直肠癌蛋白质组差异表达谱,筛选结直肠癌发生发展相关差异蛋白质。方法应用激光捕获显微切割技术,获取20例结直肠腺癌及配对的正常黏膜、20例绒毛状腺瘤、8例结直肠癌肝转移灶的靶细胞;然后应用基于乙酰化稳定同位素标记的定量蛋白质组学策略寻找差异蛋白。结果获得具有定量信息的高可信度蛋白873个。其中腺癌与正常黏膜、腺瘤及肝转移灶之间差异显著的蛋白质(定量比值≥2或≤0.5)分别为137、119和77个。结直肠癌与正常黏膜之间的差异蛋白在定位上几乎分布于细胞的各个部位。结论联合应用激光捕获显微切割技术及乙酰化稳定同位素标记定量技术进行结直肠癌蛋白质组学研究,发现的差异蛋白功能上参与多种生物学过程,表明本研究所采取的技术策略有很好的蛋白鉴定覆盖率,可成为研究肿瘤蛋白质组学的有效策略。     

激光捕获显微切割技术应用研究新进展

对疾病发生过程中的组织损害要进行分子水平分析,首要步骤就是纯净目标细胞的分离。近年发展起来的激光捕获显微切割(1aser capture microdissection,LCM)技术可以在显微镜直视下快速、准确地获取所需的单一细胞亚群,甚至单个细胞,从而成功解决了组织中细胞异质性问题。本文介绍了LCM技术的原理及其优缺点,并对其在DNA分析和基因表达分析两个方面的应用研究新进展进行综述,同时对LCM技术的发展和完善提出了可能的方向。     

显微切割法对肝细胞癌抑癌基因杂合性缺失的研究

目的 通过对肝细胞癌（HCC）抑癌基因（tumor suppressor genes,TSGs)杂合性缺失（loss of heterozygosity,LOH)谱系特点的分析，进一步了解HCC发生过程中多基因变异的特点。方法 采用显微组织切割法从石蜡包埋的组织切片中提取基因组DNA直接测序，对33例信息性HCC进行了6种TSGs(APC、DCC、MCC、OGC1、p53和RBI1）的LOH检测。结果 LOH的总体发生率为36.4％,其中p53:80.0%、OGG1：50.0%、APC：41.7%、RB1:20.0%、DCC:0.0%、MCC:0.0%。结论 在HCC的发生和发展过程中有多基因变异的参与，其中p53、OGG1和APC基因在HCC的发生中起重要作用，RB1的作用次之，而DCC和MCC基因的作用可能较小。     

利用显微切割技术构建喉癌差异基因组表达谱

[目的]通过激光捕获显微切割技术（LCM）构建喉部纯化组织差异基因表达谱,筛选与进展期喉癌相关的候选靶基因。[方法]采用激光捕获显微切割技术纯化分离8例声门型喉癌组织标本中的癌细胞及对应癌旁组织的黏膜上皮细胞,结合HGU133.Plus2.0芯片技术,构建16例喉部纯化组织标本差异基因组表达谱,筛选出进展期喉癌相关靶基因;采用QRT-PCR检测特异性的候选靶基因的表达情况。[结果]将不同分期的喉癌组与癌旁正常上皮组基因表达谱分别进行组内、组间比较,共筛选出与喉癌临床进展相关的候选靶基因761个;对部分特异性基因如KRT19、KRT23基因进行mRNA水平的检测,其结果与基因表达谱的结果具有一致性。[结论]利用激光捕获显微切割结合寡核苷酸芯片技术构建喉癌差异基因表达谱,可准确、高效地筛选出与喉癌临床进展相关的候选靶基因。     

免疫组织化学双标记结合激光显微切割技术在霍奇金淋巴瘤诊断中的应用

 目的 探讨IHC双标记技术结合LCM技术在研究HL肿瘤细胞与背景细胞中的应用。 方法 选取10例HL标本，10%中性福尔马林固定，石蜡包埋组织，采用不同的抗原修复方法，通过IHC 双标记与LCM技术获得单一的肿瘤细胞和背景细胞。 结果 在10例HL标本中通过IHC双标记与LCM技术获得单一的肿瘤细胞和背景细胞，二者表现出不同的 颜色以及表达不同的CD分子。 结论 通过IHC双标记与LCM技术获得单一的肿瘤细胞和背景细胞，IHC双标记在HL研究中起着区分不 同细胞的作用。     

肝细胞癌VEGF、uPA和ICAM-1蛋白的表达及意义

转移和复发是肝癌患者术后主要的死亡原因 ,也是影响患者长期存活和治疗效果的主要障碍。我们通过检测血管内皮生长因子 (VEGF)、尿激酶型纤溶酶原激活物 (uPA)、细胞间黏附分子 1(ICAM 1)蛋白及增殖细胞核抗原 (PCNA)在肝癌中的表达 ,探讨与肝癌转移、复发有关的分子生物学机制。一、材料与方法1.组织标本 :12 3例选自同济医院病理科保存的肝外科中心 1998年 1月～ 1999年 12月肝癌患者手术标本。其中 ,临床无转移肝癌 (A组 ) 4 2例 ,肝内转移肝癌 (B组 ) 5 0例 ,伴门静脉主干癌栓肝癌 31例 (门静脉癌栓原发灶标本为C组 ,癌栓标本…     

激光捕获显微切割结合RNA线性扩增技术在膀胱移行细胞癌相关基因研究中的应用

目的当对有间质细胞混杂的膀胱移行上皮细胞与膀胱移行细胞癌进行基因差异表达分析时,激光捕获显微切割技术(lasercap-turemicrodissection,LCM)是不可缺少的,然而从LCM所得的细胞中获取足够RNA量相当困难。本文首次将LCM与RNA线性扩增结合用于膀胱癌相关基因研究,目的是确定一条切实可行的技术路线,将膀胱移行上皮细胞从膀胱粘膜中分离出来,将膀胱癌细胞从肿瘤间质细胞中分离出来,并对其进行RNA体外线性扩增,以备进一步研究。方法采用LCM技术分别从正常膀胱粘膜及膀胱癌组织冰冻切片中获取膀胱移行上皮细胞及膀胱癌细胞,提取RNA,并对微量RNA进行体外线性扩增。然后用RT-PCR验证扩增前、后RNA中β-actin基因表达水平。结果对照实验I证实经LCM后RNA完整性较好;经对照实验Ⅱ初步确定设定条件下LCMshooting次数与可获得RNA量间对应关系。从膀胱粘膜种捕获膀胱移行上皮细胞25万shootings;从膀胱癌组织中获取癌细胞20万shootings。产物RNA扩增后获得片段大小为0.5-2.5kh的aRNA,且β-actin基因表达表达完整。结论LCM结合RNA体外线性扩增技术能成功地获取较为单一的研究目的细胞,扩增后RNA完整性较好,能用于进一步研究中。     

细胞周期蛋白D1在人肝细胞癌中的表达及其意义

探讨人原发性肝细胞细胞周期蛋白Ｄ１的表达及其意义。方法采用免疫组化ＡＢＣ法检测了ｃｙｃｌｉｎＤ１在２９例肝癌组织的表达情况,并分析了其表达水平与肝癌病理分级的关系。结果５８．６％（１７／２９）的肝癌组织癌细胞表达ｃｙｃｌｉｎＤ１,而癌旁非肿瘤性肝细胞的阳性率为１８．２％。ｃｙｃｌｉｎＤ１在肝癌中表达的阳性率随着肝癌细胞分化程度的下降呈升高趋势,并且在Ⅱ级和癌旁肝组织之间Ⅰ级和Ⅲ级之间分别具有显著性     

激光捕获显微切割技术应用于子宫内膜癌相关基因的研究

目的:摸索一条可行的技术路线,运用激光捕获显微切割技术(lasercapturemicrodissection,LCM)获取无间质混杂的人正常子宫内膜腺管上皮细胞与子宫内膜癌细胞,用于进行子宫内膜癌相关基因差异表达的研究。方法:采用LCM技术分别从正常子宫内膜组织及子宫内膜癌组织冰冻切片中捕获子宫内膜腺管上皮细胞及子宫内膜癌细胞,提取微量RNA,并对微量RNA进行纯化及浓缩,用RTPCR验证捕获细胞RNA中β-actin基因表达水平。结果:分别从冰冻切片中捕获子宫内膜腺管上皮细胞及子宫内膜癌细胞各50000Shootings。对照实验Ⅰ、Ⅱ证实经LCM后RNA完整性较好。确定了LCMShooting次数与可获得RNA量间对应关系。捕获细胞所提取的RNA中βactin基因表达完整。结论:通过LCM技术可以从冰冻切片中捕获单一类型的目的细胞,此过程不会造成细胞RNA明显降解,可以应用于下游的实验研究。     

蛋白质组学在原发性肝癌中的研究进展

近年来,蛋白质组学作为一门新兴学科,越来越受到研究者的普遍关注。随着蛋白质组学技术日趋成熟,这种探索蛋白质结构和功能的科研方法深入到了各个疾病领域。原发性肝癌是一种以高死亡率为特征的常见癌症,运用蛋白质组学技术,可以充分了解疾病的发生、发展机制,并在寻找诊断标记物和药物靶向治疗方面产生价值。如今,世界各地的研究人员正在运用不同的蛋白质组学技术对原发性肝癌进行深入研究,并取得了相应进展。     

p53 mutation profiling of multiple esophageal carcinoma using laser capture microdissection to demonstrate field carcinogenesis

Inactivation of the p53 tumor suppressor gene is one of the most frequent genetic alterations observed in human esophageal carcinomas. In patients with esophageal carcinoma, one of the significant pathological features of the tumor is the presence of multiple lesions within the esophagus. However, the molecular mechanisms involved in the occurrence of multiple lesions have remained elusive. To characterize p53 alterations in multiple esophageal carcinomas and to study their roles in carcinogenesis, we performed p53 immunohistochemical and p53 mutation analyses using laser capture microdissection on surgically resected human esophageal carcinomas from 11 patients: 9 patients with multiple esophageal carcinomas, 1 with an intramural metastasis lesion within the esophagus and 1 with an intraepithelial carcinoma lesion contiguous to the main lesion. In each of the patients with multiple esophageal carcinomas, we examined samples from 1 main lesion and 1 representative concomitant lesion. Molecular analyses of samples from fresh-frozen normal tissues and tumor tissues of the main lesion (whole tumor) were also performed by the same method. p53 protein accumulation was observed in 16 (72.7%) of 22 lesions from the 11 cases. No p53 mutation was found in normal esophageal tissues. In the 9 cases of multiple esophageal carcinomas, point mutations were detected in the whole tumor in 1 (11.1%) case, in the microdissected tumor samples of main lesions in 3 (33.3%) cases and in the microdissected tumor samples of concomitant lesions in 3 (33.3%) cases. For the microdissected tumor samples, there was a 54.5% (12/22) concordance rate between the results of immunostaining and molecular analysis. In the 8 cases of whole tumors in which a p53 mutation was not observed, 2 cases revealed p53 mutation in the microdissected tumor samples of the main lesion. All 6 cases of multiple esophageal carcinomas that showed a p53 mutation in the microdissected tumor sample had a discordant p53 mutational status between the main and concomitant lesions. In contrast, both the intramural metastasis lesion and the intraepithelial carcinoma contiguous to the main lesion showed p53 mutational patterns identical to those of the main lesions. In conclusion, the analysis of microdissected DNA by laser capture microdissection is useful for characterizing the heterogeneity of the p53 gene mutation in multiple carcinoma lesions that cannot be accurately analyzed in whole esophageal tumor DNA. The finding of different p53 gene mutations among multiple esophageal carcinoma lesions suggest further evidence of multicentric or field carcinogenesis of the human esophagus.     

ADAR1 mRNA在肝癌及癌旁组织的表达及意义

目的探讨ADAR1mRNA在肝细胞肝癌(HCC)及癌旁组织的表达及意义。方法采用半定量逆转录-聚合酶链反应(RT-PCR)分别检测诊断为HCC41例患者的肝癌及癌旁组织RNA编辑酶ADAR1mRNA的表达。结果所有肝癌组织RNA编辑酶ADAR1均有表达,相对表达量为3.340±0.863;所有癌旁组织有RNA编辑酶ADAR1表达,相对表达量为0.801±0.209;对照的26例正常肝组织及其他肝脏疾病标本RNA编辑酶ADAR1的相对表达量为0.880±0.226。将肝癌组织和癌旁组织的ADAR1mRNA表达量进行成组t检验,两者间存在显著性差异(t=18.30,P<0.001);肝癌组织和对照肝组织的ADAR1mRNA表达量进行成组t检验,两者间亦存在显著性差异(t=17.65,P<0.001);将癌旁组织和对照肝组织检测到的ADAR1mRNA的表达量进行成组t检验,两者间差异无统计学意义(t=1.64,P>0.10)。将从同一患者取材的肝癌组织和癌旁组织的ADAR1mRNA表达量进行配对t检验,两者间存在显著性差异(t=18.53,P<0.001)。结论ADAR1mRNA在肝癌组织的表达增高,可能在肝癌的发生机制中起一定的作用。     

Detection of plasminogen activators in oral cancer by laser capture microdissection combined with zymography

Plasminogen activation is believed to be critical to the progression of oral squamous cell carcinoma by facilitating matrix degradation during invasion and metastasis, and high levels of urokinase plasminogen activator (uPA) and plasminogen activator (PA) inhibitor-1 (PAI-1) in tumors predict poor disease outcome. We describe the development of a novel method for studying PA in oral cancer that combines the sensitivity and specificity of zymography with the spatial resolution of immunohistochemistry. Laser capture microdissection (LCM) was combined with plasminogen–casein zymography to analyze uPA, tissue PA (tPA), uPA–PAI-1 complexes, and tPA–PAI-1 complexes in 11 tumors and adjacent non-malignant epithelium from squamous cell carcinomas of the tongue, floor of mouth, larynx, and vocal cord. uPA was detectable in all tumor samples analyzed, uPA–PAI-1 complexes in three samples, and tPA in nine. PA was detectable in as little as 0.5 μg protein lysate from microdissected tumors. In all specimens, uPA expression was highly increased in tumor tissue compared to adjacent non-malignant tissue. In conclusion, LCM combined with zymography may be excellently suited for analyzing the prognostic significance and causal involvement of the plasminogen activation system in oral cancer.     

Cyclin D1在肝细胞癌中的表达及意义

目的探讨Cyclin D1蛋白表达在肝细胞癌发生、发展中的作用.方法免疫组化S-P法检测40例肝细胞癌(HCC)、15例肝硬化组织和5例正常肝组织石蜡标本Cyclin D1蛋白表达.结果Cyclin D1蛋白在HCC、肝硬化、正常肝组织表达阳性率分别为57.5%(23/40)、46.67%(7/40)、40%(2/5),Cyclin D1蛋白在HCC中表达与肝硬化和正常肝组织中表达比较无显著差异(P＞0.05),Cyclin D1表达仅与HCC的组织学分级有统计学差异.结论Cyclin D1与HCC的发生、发展有一定联系,在判断患者病情预后方面有较重要的价值.     

First attempt of boron neutron capture therapy (BNCT) for hepatocellular carcinoma

A 60-year-old man with multiple hepatocellular carcinomas (HCCs) was enrolled as the first patient in a pilot study for treating multiple liver tumors with boron neutron capture therapy (BNCT). Because of compromised liver function, the multiple tumors in the right liver lobe were treated with BNCT and those in the left lobe with hepatic arterial chemoembolization. The feasibility and clinical outcome of this first case was assessed. During the treatment and follow-up period, no adverse effect as a result of BNCT was observed except for temporary temperature elevation to 38.3 degrees C, and the AST and ALT being higher than 200 IU/l. For 1 month, the tumors treated with BNCT remained stable in size. The BNCT-treated tumors showed regrowth 3.5 months after BNCT and the patient died of liver dysfunction caused by progression of HCC 10 months after BNCT. The feasibility of BNCT for HCC is confirmed in this first case.     

Rsf －1在肝细胞癌中的表达及意义

目的：分析 Rsf －1在肝细胞癌中的表达及其与临床病理因素间的关系。方法：应用免疫组化方法检测肝细胞癌中 Rsf －1的蛋白表达情况。结果：免疫组化结果显示43．1％（28／65）的病例存在 Rsf －1蛋白的过表达,其过表达与肝癌患者的年龄、性别、肝炎、肝硬化和淋巴结转移等未发现有明显的相关性,而与肝细胞癌的术前 AFP 值和高 p －TNM分期呈正相关（P ＝0．026;P ＝0．041）。结论：肝细胞癌中存在 Rsf －1蛋白的高表达,并与术前 AFP 值和高 p －TNM分期相关。     

LRG1 expression indicates unfavorable clinical outcome in hepatocellular carcinoma

Leucine-rich-alpha-2-glycoprotein1 (LRG1) is a novel oncogene-associated protein which has been clarified vital to the progression of human cancers, but its role in hepatocellular carcinoma (HCC) remains unclear. Here, we showed that the expression of LRG1 was noticeably increased in HCC tissues, compared to the nontumorous tissues. High LRG1 expression was significantly associated with tumor size (P = 0.004), tumor differentiation (P = 0.010), TNM stage (P < 0.001) and vascular invasion (P = 0.019). Kaplan-Meier analysis showed that LRG1 expression was closely correlated to overall survival and disease-free survival in a training cohort of 474 patients with HCC. The correlation was further validated in an independent cohort of 303 HCC patients. The prognostic implication of LRG1 was confirmed by stratified survival analyses. Multivariate Cox regression model indicated LRG1 as an independent poor prognostic indicator for overall survival (Hazard ratio = 1.582, 95% confident interval: 1.345–1.862, P < 0.001) and disease-free survival (Hazard ratio = 1.280, 95% confident interval: 1.037–1.581, P = 0.022) in HCC. In vitro data showed that LRG1 markedly promoted cell migration but has no effect on cell proliferation. Collectively, our data show that LRG1 is markedly up-regulated and serves as an independent factor of poor outcomes in HCC. Our study therefore provides a promising biomarker for prognostic prediction in clinical management of HCC.