Omi/HtrA2在腺样囊性癌的表达及其临床意义

目的 探讨Omi/HtrA2在不同分型腺样囊性癌中的表达差异,并评价其对腺样囊性癌的临床意义.方法 纳入确诊为腺样囊性癌的患者100例,应用免疫组织化学染色方法,检测Omi/HtrA2在不同分型腺样囊性癌中的表达;并检测腺样囊性癌转移组和非转移组患者中Omi/HtrA2表达情况.结果 Omi/HtrA2表达主要位于细胞胞质中,在腺样囊性癌组织中其阳性率为83％;腺样囊性癌低级组与高级组间表达情况具有统计学差异(P＜0.05),但不同分型的腺样囊性癌组织中其表达差异无统计学意义(P＞ 0.05);Omi/HtrA2在转移组腺样囊性癌中阳性率为93.1％,在非转移组中其阳性率为46.2％,两组比较具有统计学差异(P＜0.05).结论 Omi/HtrA2与腺样囊性癌的发病密切相关,可作为临床判断腺样囊性癌的辅助性指标,对临床的诊治及预后的判断具有一定的指导价值.     

喉癌相关抗原的表达及特性研究

目的 探讨喉癌相关抗原的生物学特性及在喉癌组织中的表达。方法 应用抗人喉鳞状细胞癌单克隆抗体Ｌｃ９、Ｌｃ１１、Ｌｃ１２三株混合,以免疫组化ＡＢＣ法观察与喉癌组织、喉癌前病变及喉正常粘膜的结合情况。将手术切除的３例人喉癌组织研磨匀浆后,混合,高速离心得到喉癌粗提抗原,经Ｓｅｐｈａｄｅｘ－Ｇ－７５凝胶过滤层析技术得到纯化的喉癌抗原。抗原经胰蛋白酶消化,高碘酸钠、甲醇及加热处理,进行间接ＥＬＩＳＡ检测,     

Omi/HtrA2对前列腺癌细胞株PED/PEA-15表达及PC-3细胞凋亡的影响

背景与目的：促、抑凋亡因子间的相互作用与肿瘤的发生、发展密切相关。Omi／HtrA2是新近发现的一种凋亡调节因子,PED／PEA—15是一种广泛表达的抗凋亡蛋白。本研究旨在探讨Omi／HtrA2对PED／PEA—15表达和前列腺癌细胞PC-3凋亡的影响。方法：构建Omi／HtrA2的表达载体和siRNA载体。并用脂质体法分别将两载体转染至PC-3细胞中,Westernblot和ELISA法检测Omi／HtrA2对PED／PEA—15表达和细胞凋亡的影响;Caspase-8检测试剂盒检测PED／PEA-15对Caspase-8活性的影响;Westernblot、RT—PCR法检测Omi／HtrA2特异siRNA序列对其转录、翻译的影响,流式细胞仪检测siRNA导致Omi／HtrA2基因沉默后PC一3细胞凋亡的变化。结果：酶切和DNA测序证实Omi／HtrA2的表达载体和siRNA载体构建成功。通过转染Omi／HtrA2表达载体高表达Omi／HtrA2可抑制PED／PEA-15表达,并增加肿瘤细胞的凋亡率;抑制PED／PEA—15的表达可提高Caspase-8活性。siRNA沉默Omi／HtrA2基凶后PC-3细胞对顺铂的敏感性降低。结论：Omi／HtrA2可通过抑制抗凋亡蛋白PED／PEA—15表达而在PC-3细胞凋亡中发挥重要作用。     

喉鳞癌中p-STAT3和Skp2的表达及其相关性研究

目的:检测喉鳞癌中信号转导和转录激活因子3的活化和Skp2 蛋白的表达,探讨其临床意义及相关性.方法:采用免疫组织化学法检测p-STAT3和Skp2蛋白在56例喉鳞状细胞癌和30例喉全(或近全)切除者癌旁(＞2.0 cm)正常黏膜中的表达,应用Metamorph/DP10/BX41显微图象分析系统测定阳性表达的平均光密度值,并进行统计分析.结果:与癌旁正常黏膜组相比较,p-STAT3和Skp2蛋白在喉鳞癌组织中过表达(P＜0.01);p-STAT3和Skp2蛋白过表达与喉鳞癌的分化程度、临床分期、颈淋巴结转移有关(P＜0.05或P＜0.01);在喉鳞癌组织中p-STAT3蛋白和Skp2蛋白的表达呈正相关关系,相关系数r=0.8041(P＜0.01).结论:p-STAT3和Skp2蛋白过表达与喉鳞癌的发生、发展和转移等生物学行为有关,两者的表达呈正相关.     

喉鳞癌中P-STAT3和Skp2的表达及其相关性研究

目的：检测喉鳞癌中信号转导和转录激活因子3的活化和skp2蛋白的表达,探讨其临床意义及相关性。方法：采用免疫组织化学法检测P—STAT3和skp2蛋白在56例喉鳞状细胞癌和30例喉全（或近全）切除者癌旁（〉2．0cm）正常黏膜中的表达,应用Metamorph／DPIO／BX41显微图象分析系统测定阳性表达的平均光密度值,并进行统计分析。结果：与癌旁正常黏膜组相比较,P—STAT3和Skp2蛋白在喉鳞癌组织中过表达（P〈0．01）;P—STAT3和skp2蛋白过表达与喉鳞癌的分化程度、临床分期、颈淋巴结转移有关（P〈0．05或P〈0．01）;在喉鳞癌组织中P—STAT3蛋白和skp2蛋白的表达呈正相关关系,相关系数r=0．8041（P〈0．01）。结论：P—STAT3和Skp2蛋白过表达与喉鳞癌的发生、发展和转移等生物学行为有关,两者的表达呈正相关。     

E2F3在喉鳞状细胞癌中的表达及临床意义

目的：探讨喉鳞癌中E2F3的表达与临床意义,为喉鳞癌的诊断、评估及治疗寻找辅助生物学指标。方法：采用免疫组化方法检测喉鳞癌组织及癌旁正常切缘组织中E2F3表达,结合喉鳞癌各临床参数,用SPSS16.0软件包进行统计分析E2F3表达情况与喉鳞癌各临床特征之间的关系。结果：E2F3在喉鳞癌与癌旁正常切缘中的阳性表达率分别为90.44%（123/136）和27.85%（22/79）,差异具有显著统计学意义（P〈0.001）;E2F3高表达与喉鳞癌患者年龄、临床分期及分型均相关（P〈0.05）。结论：E2F3核表达可能参与喉鳞癌发生发展过程;E2F3浆表达可能贯穿于喉鳞癌发生的整体过程。     

喉癌组织PTEN和Ang-2表达及其临床意义的研究

目的:探讨抑癌基因(PTEN)和促血管生成素2(Ang-2)在喉鳞状细胞癌、正常喉组织中的表达关系。方法:采用免疫组织化学SP法检测30例喉鳞状细胞癌患者的病理标本及10例正常喉组织例标本中PTEN和Ang-2的表达。结果:PTEN阳性表达灰度值在癌组织中低于正常喉黏膜组织(P<0.05),与组织分化程度有相关性,P<0.031;Ang-2阳性表达灰度值在癌组织中明显高于正常喉黏膜组织(P<0.041),与组织分化程度有相关性,P<0.01。PTEN与Ang-2呈直线负相关。结论:在喉鳞状细胞癌发生的过程中,PTEN和Ang-2可能发生异常表达,对喉鳞状细胞癌发展作用不同,但与喉鳞状细胞癌的进展无关。     

喉鳞状细胞癌中p63基因蛋白表达及其意义

目的：探讨p63基因蛋白表达与喉鳞状细胞癌（laryngeal squamous cell carcinomas,LSCC）的发生、发展的关系和生物学意义。方法：应用免疫组织化学方法,检测30例LSCC、10例不典型增生和10例正常喉黏膜组织中p63基因蛋白的表达水平。结果：全部30例LSCC和10例不典型增生组织中p63染色均为阳性。LSCC分化程度越低,阳性细胞数越高、细胞的表达强度越高。正常喉黏膜组织中仅基底层及上基层细胞阳性。p63表达与LSCC分级和淋巴结转移相关（P〈0.05）。结论：p63可能参与了调控LSCC的发生、发展过程,其高表达预示LSCC预后不良。     

HPV感染及p53与Cdc2蛋白表达对喉癌术后复发的预测价值

目的检测人乳头状瘤病毒(human papilloma virus,HPV)及p53和Cdc2蛋白表达与喉鳞状细胞癌发生及复发的关系。方法选取2003-01-01-2011-12-31唐山市协和医院92例喉鳞状细胞癌手术切除标本,采用PCR-反向点杂交方法检测喉癌组织中HPV感染情况,SP免疫组化方法检测p53和Cdc2蛋白表达,收集临床病理资料并进行随访。结果随访>2年的喉癌患者中有18例出现复发(复发组),74例无复发(未复发组)。92例喉癌组织中HPV的感染率为19.57%(18/92);喉癌组织中p53和Cdc2蛋白的阳性表达率分别为60.87%(56/92)和65.22%(60/92)。HPV感染、p53及Cdc2蛋白表达与喉癌的分化程度、分期均无明显相关性,P值均>0.05;HPV阳性和阴性喉癌患者术后复发率分别为0(0/18)和24.32%(18/74),差异有统计学意义,χ2=4.007,P=0.045;喉癌复发组和未复发组中的p53蛋白表达率分别为72.22%(13/18)和58.11%(43/74),差异无统计学意义,χ2=0.691,P=0.406;喉癌复发组和未复发组中Cdc2蛋白表达率分别为88.89%(16/18)和59.46%(44/74),差异有统计学意义,χ2=4.307,P=0.038。喉癌组织中HPV的感染与p53(χ2=0.316,P=0.574)及Cdc2(χ2=0.574,P=0.487)蛋白表达均无相关性。结论喉癌组织中存在着一定程度的HPV感染及p53、Cdc2蛋白质的表达,但三者与喉癌的分期及分化程度无关;HPV阳性患者术后复发率低于HPV阴性患者;Cdc2蛋白表达阳性者术后复发率高于阴性患者。HPV感染、p53及Cdc2 3种因素可能参与了喉癌的发生、发展及复发过程。     

Gankyrin蛋白在喉鳞癌中表达的实验研究

目的研究Gankyrin蛋白在喉鳞癌组织及喉鳞癌Hep-2细胞系中的表达。方法免疫组织化学方法检测24例喉鳞癌组织、相应癌旁组织及10例正常组织,免疫细胞化学法检测Hep-2细胞系中Gankyrin蛋白的表达与分布,并计算阳性细胞率,进行统计分析。结果喉鳞癌组织及Hep-2细胞系中,Gankyrin蛋白主要表达于细胞核。喉鳞癌组织中Gankyrin蛋白表达阳性细胞率(58.5%)明显高于相应癌旁组织(11.4%)及正常组织(18.1%),且差异有统计学意义(均P0.01)。结论喉鳞癌组织及喉鳞癌Hep-2细胞系中均有Gankyrin蛋白的表达,可为Gankyrin在喉鳞癌中表达的后续相关研究提供理论依据。     

A new function for the serine protease HtrA2 in controlling radiation‐induced senescence in cancer cells

Radiation therapy can induce cellular senescence in cancer cells, leading to short‐term tumor growth arrest but increased long‐term recurrence. To better understand the molecular mechanisms involved, we developed a model of radiation‐induced senescence in cultured cancer cells. The irradiated cells exhibited a typical senescent phenotype, including upregulation of p53 and its main target, p21, followed by a sustained reduction in cellular proliferation, changes in cell size and cytoskeleton organization, and senescence‐associated beta‐galactosidase activity. Mass spectrometry‐based proteomic profiling of the senescent cells indicated downregulation of proteins involved in cell cycle progression and DNA repair, and upregulation of proteins associated with malignancy. A functional siRNA screen using a cell death‐related library identified mitochondrial serine protease HtrA2 as being necessary for sustained growth arrest of the senescent cells. In search of direct HtrA2 substrates following radiation, we determined that HtrA2 cleaves the intermediate filament protein vimentin, affecting its cytoplasmic organization. Ectopic expression of active cytosolic HtrA2 resulted in similar changes to vimentin filament assembly. Thus, HtrA2 is involved in the cytoskeletal reorganization that accompanies radiation‐induced senescence and the continuous maintenance of proliferation arrest.     

放射线诱导的HtrA2基因表达对人葡萄膜黑色素OCM-1细胞的影响

背景与目的：葡萄膜黑色素瘤(uveal melanoma,UM)是成人最常见的眼内原发性恶性肿瘤,其恶性程度高,转移早。放射治疗是多年来葡萄膜黑色素瘤最常用的方法之一,但辐射抗性和受照部位正常组织的损伤是长期困扰UM放疗的问题。本实验运用放射线可调控的HtrA2基因联合疗法,探讨其对UM的抑制作用。方法：含Egr1启动子的pIRES-Egr1-Omi/HtrA2(pEgr1-HtrA2)质粒体外转染入OCM-1细胞,转染后的细胞暴露于不同剂量的放射线中。将移植瘤模型小鼠分为对照组、放射组、基因组和基因放射组,每组10只。应用实时定量PCR(qRT-PCR)和蛋白质印迹法(Western blot)检测HtrA2基因的mRNA表达和蛋白表达。流式细胞仪检测联合处理后的OCM-1细胞凋亡情况。通过克隆形成实验检测HtrA2基因对放射敏感性的影响。在动物实验中测量联合治疗后肿瘤体积。结果：转染pEgr1-HtrA2质粒后,放射组HtrA2 mRNA表达在8 Gy达到高峰,且HtrA2蛋白表达较放射前明显增加(P<0.05),OCM-1细胞凋亡显著增加。克隆形成实验显示,HtrA2基因可增强OCM-1细胞放射敏感性。与对照组相比,基因放射组肿瘤生长被显著抑制(P<0.05)。结论：转染pEgr1-HtrA2质粒后可在放射线调控下增加HtrA2基因表达,并能增强OCM-1细胞对放射的敏感性,抑制肿瘤细胞生长。     

Fhit基因异常表达在原发性喉癌中的意义

背景与目的:研究原发性喉癌组织中的Fhit基因的失活情况,并在RNA水平揭示Fhit基因失活与喉癌生物学行为之间的联系,及环境因素对Fhit基因影响。材料与方法:采用RT-PCR技术检测29例喉癌组织Fhit表达情况,并对异常表达进行测序。比较喉癌组织和非癌组织,及不同临床病理分级间的Fhit异常表达,同时观察环境因素(包括吸烟、饮酒)对Fhit表达的影响。结果:29例喉癌组织中22例Fhit异常表达,其中4例Fhit基因未表达,18例转录本异常。喉癌组织和非癌组织中Fhit异常表达率差异有统计学意义(P<0.01),各临床病理分级之间的Fhit异常表达情况差异无统计学意义(P>0.05),吸烟组和非吸烟组Fhit异常差异有统计学意义(P<0.05)。结论:Fhit基因异常表达在喉癌中频繁发生。Fhit基因改变可能作用于喉癌的发生过程,并不随肿瘤发展而进一步增强。过度吸烟可能通过改变Fhit基因的结构从而诱导喉肿瘤的形成。     

COX-2及Her2/neu在结直肠癌旁组织中表达意义

目的 检测结直肠癌和旁癌组织中的COX-2及Her2/neu的表达情况,探究COX-2及Her2/neu表达与结直肠癌临床各指标的关系,并探讨其临床意义.方法 选取结直肠癌患者200例,收集各研究对象的结直肠癌病理标本和癌旁正常组织各一份,应用免疫组织化学法(S-P法),检测结直肠癌组织和癌旁正常组织中COX-2及Her2/neu蛋白质表达.结果 结直肠癌中Her2/neu表达情况:蛋白质表达阳性占86.0%,明显高于对照组Her/neu阳性表达(24.0%)(P<0.05).结直肠癌中Her2/neu表达与Dukes分期和肝转移相关(P<0.05).结直肠癌中COX-2表达阳性率为89.0%,而癌旁正常组织中其阳性表达率为32.0%,两者差异具有统计学意义(P<0.05),过表达与肿瘤肝转移和Dukes分期有关(P<0.05),与Her2/neu表达有正相关性(P<0.05).结论 采用免疫组织化学法探究蛋白质的表达,结肠癌COX-2及Her2/neu蛋白质的表达率高于癌旁正常组织;COX-2及Her2/neu蛋白质的表达与肝转移和Dukes有关,而且它们之间的表达具有正相关性(P<0.05).     

The tumor suppressor WARTS activates the Omi / HtrA2-dependent pathway of cell death

Drosophila tumor suppressor WARTS (Wts) is an evolutionally conserved serine / threonine kinase and participates in a signaling complex that regulates both proliferation and apoptosis to ensure the proper size and shape of the fly. Human counterparts of this complex have been found to be frequently downregulated or mutated in cancers. WARTS, a human homolog of Wts, is also known as tumor suppressor and mitotic regulator, but its molecular implications in tumorigenesis are still obscure. Here, we show that WARTS binds via its C-terminus to the PDZ domain of a proapoptotic serine protease Omi / HtrA2. Depletion of WARTS inhibited Omi / HtrA2-mediated cell death, whereas overexpression of WARTS promoted this process. Furthermore, WARTS can enhance the protease activity of Omi / HtrA2 both in vivo and in vitro. Activation of Omi / HtrA2-mediated cell death is thus a potential mechanism for the tumor suppressive activity of WARTS.     

Participation of Omi Htra2 Serine-Protease Activity in the Apoptosis Induced by Cisplatin on SW480 Colon Cancer Cells

Abstract

Apoptosis is triggered by two interconnected pathways, extrinsic and intrinsic. The intrinsic pathway is activated by genomic stress promoting mitochondrial release of apoptotic proteins. One of these proteins is Omi/Htra2, a serine protease which inactivates Inhibitor of Apoptosis Proteins (IAPs). In the present work, we assessed the participation of Omi/Htra2 in the cell death induced by the chemotherapeutic drugs 5-fluorouracil (5-FU) and cisplatin (CDDP) in SW480 colon cancer cells. CDDP and 5-FU induced apoptosis mediated by the intrinsic pathway in colon cancer cells, as demonstrated by morphological analyses, mitochondrial cytochrome c release and cleavage of caspase 3. Omi/Htra2 was also released from mitochondria of cells exposed to these drugs, as demonstrated by immunofluorescence and western blot assays of subcellular fractions. Exposure of cells to the Omi/Htra2 serine protease inhibitor UCF-101 prevented death p<0.0001 and partially suppressed reproductive cell death of cells exposed to cisplatin p<0.05, but not to 5-FU p=0.49. From these experiments we show that Omi/Htra2 serine protease activity participates in the cell death induced by CDDP but not of 5-FU in colon cancer cells.     

Serine protease Omi/HtrA2 targets WARTS kinase to control cell proliferation

The serine protease Omi/HtrA2 was initially regarded as a proapoptotic molecule that proteolyses several proteins to induce cell death. Recent studies, however, indicate that loss of Omi protease activity increases susceptibility to stress-induced cell death. These complicated findings suggest that the protease activity of Omi is involved not only in apoptosis but also in cellular homeostasis. However, the targets which Omi uses to mediate this novel process are unknown. Previously, we showed that WARTS (WTS)/large tumor-suppressor 1 mitotic kinase interacts with the protein/discs-large protein/zonula (PDZ) domain of Omi and promotes its protease activity. We now report that WTS is a substrate for Omi protease activity, thus it is not only a regulator but also a downstream target of this protease. Interaction with Omi PDZ domain is required for WTS to be proteolysed. When caspase-9-deficient mouse embryonic fibroblasts (MEFs) were treated with staurosporine, WTS was proteolysed by activated endogenous Omi without induction of cell death. Therefore, protease activity of Omi and proteolysis of WTS are not necessarily required for cell death. We found that depletion of Omi from HeLa cells results in accelerated cell proliferation despite no significant change in the duration of mitosis. The depletion of WTS showed the same effect on S phase progression. Therefore, WTS proteolytic fragment(s) generated by Omi may act as an inhibitor of G1/S progression. Our data reveal a role for Omi-mediated processing of WTS in negative regulation of cell cycle progression at interphase, suggesting a novel function of Omi other than apoptosis.     

肝细胞肝癌组织中RBL2/P130的表达及其临床意义

目的研究人肝细胞肝癌组织中视网膜母细胞瘤蛋白2(RBL2/P130)的表达以及与肝癌的关系,进一步探讨其在临床中的意义。方法采用免疫组织化学方法检测肝癌、癌旁硬化肝组织和正常肝组织中RBL2/P130蛋白的表达,并分析其与肝癌患者临床参数间的关系。结果肝癌组织中RBL2/P130蛋白的阳性表达率为34.1%,显著低于癌旁硬化肝组织及正常肝组织（χ²=11.7,P<0.01）,其表达与有无门脉癌栓、肿瘤分化程度及肝癌临床分期有关,与年龄、性别、癌灶个数、肿瘤大小、肿瘤包膜完整与否、AFP值的大小及乙肝表面抗原阳性与否无关,并且RBL2/P130阳性患者累计生存率显著高于阴性患者（P=0.014）。结论RBL2/P130在人肝癌组织中表达水平降低,与肝癌的分化程度、疾病的进程及侵袭转移有密切的关系。