凋亡抑制基因survivin在多种肺癌细胞株中的表达及其意义

目的:探讨凋亡抑制基因survivin在多种肺癌细胞株中的表达及其意义.方法:分别采用Real-time PCR与Western blot的方法,检测人肺腺癌细胞株A549、肺鳞癌细胞株SK-MES-1、大细胞肺癌细胞株NCI-H460、小细胞肺癌细胞株NCI-H446中survivin mRNA与蛋白的表达水平.结果:Real-time PCR检测发现四种肺癌细胞株中survivin mRNA均呈强阳性表达,且Western blot检测结果亦与 RT-PCR基本一致.结论:Survivin基因与肺癌发生、发展紧密相关,值得进一步深入研究.     

乳腺癌癌基因C—erbB—2蛋白的表达

C-erbB-2位于第17染色体921带上,编码一个分子量为185～190千道尔顿的膜受体,具酪氨酸激酶活性,在结构上与表皮生长因子受体(EGFR)非常相似。有证据表明,在人类多种肿瘤特别是乳腺癌中可检测到该基因的扩增,在一些恶性转化细胞中也有过度表达。作者采用C-erbB-2蛋白抗血清21N,用免疫组化和免疫电镜技术检测乳腺癌中C-erbB-2的表达产物,并探讨其意义。     

人骨肉瘤组织中Ⅰ、Ⅱ和Ⅲ型胶原基因的表达

目的研究人骨肉瘤组织中Ⅰ、Ⅱ和Ⅲ型胶原蛋白和mRNA的表达及其与骨肉瘤分型和分化的关系.方法用饱和苦味酸-天狼星红-偏振光镜、免疫组化LSAB和原位杂交法检测46例人骨肉瘤组织中Ⅰ、Ⅱ和Ⅲ型胶原蛋白和mRNA的表达,EMAL-200真彩图像分析系统进行分析和半定量测定.结果骨肉瘤组织中不仅有Ⅰ型胶原蛋白的表达(阳性率87%,灰度值171.99±14.74),且出现了Ⅱ型和Ⅲ型胶原蛋白(阳性率分别为30.4%和43.4%,灰度值分别为153.07±18.82和168.29±18.36);Ⅱ型胶原在软骨母细胞型和混合型的表达明显高于其它型(P＜0.01),而Ⅰ、Ⅲ型胶原的表达在各型骨肉瘤间差异无显著性;高、中、低三组不同分化程度的骨肉瘤Ⅰ型胶原表达阳性率和灰度值分别为100%,157.38±5.49;83.33%,173.58±12.61;76.92%,186.59±6.92;三组间差异有显著性(P＜0.01),分化越低表达越少;天狼星红与免疫组化染色结果基本一致.Ⅰ、Ⅱ型胶原mRNA原位杂交与蛋白免疫组化所得结论基本一致.结论Ⅱ型胶原可作为软骨母细胞型和混合型骨肉瘤的分型参考指标,Ⅰ型胶原的表达可作为骨肉瘤分化及恶性度判断的参考指标.天狼星红染色是鉴别Ⅰ、Ⅲ型胶原简便、经济的方法之一.     

葡萄胎与正常胎盘绒毛的基因表达谱研究

目的：探讨葡萄胎与正常胎盘绒毛基因表达谱，研究葡萄胎滋养层细胞增生的机制。方法：采用cDNA芯片技术，用Cy3-dUTP标记正常胎盘绒毛mRNA，用Cy5-d UTP标记葡萄胎组织mRNA，制备探针进行芯片杂交，随机抽取3条基因作RT-PCR验证。结果：从4004个有效基因位点中筛选出106个差异表达基因，占2．65％，其中上调基因表达共57条，占1.42％；下调基因共49条，占1．22％。获得了葡萄胎与正常胎盘绒毛基因表达的信息。结论：葡萄胎滋养层细胞增生与多基因激活具有密切的联系，初步揭示了葡萄胎基因调控异常的复杂性。     

nm23基因在肿瘤预后监测中的应用

nm23基因是1988年由Steeg首次从小鼠黑色素瘤细胞中分离出来的-种与肿瘤转移表型抑制相关的基因。人类nm23基因家族有8个，即nm23-H1、nm23-H2、DR—nm23、nm23-H4、nm23-H5、nm23-H6、nm23-H7和nm23-H8，其中研究最多的是主要与肿瘤转移相关的nm23-H1及调控c—mye和PDGF基因转录的nm23-H2。nm23-H1和nm23-H2所编码的蛋白与核苷二磷酸激酶（NDPK）的A、B亚基分别相对应，编码蛋白的氨基酸序列与NDPK的同源性分别为89％和97％，二者之间的同源性为88％。DR—nm23基因又称nm23-H3，其结构与nm23-H1和nm23-H2具有65％～70％的同源性，DR—nm23靶蛋白位于细胞质和线粒体膜上，其表达调控不在转录水平，可能出现在转录后或翻译水平。nm23-H4基因产物是线粒体NDPK，其氨基酸序列与NDPKA和NDPKB有58％～59％的同源性，nm23-H5至nm23-H8基因产物均被证实具有NDPK活性或NDPK样区域。     

p16与Rb蛋白在骨肉瘤中的表达及相关性研究

为探讨骨肉瘤中ｐ１６和Ｒｂ蛋白的表达及其相关性，采用免疫组化ＡＢＣ法，对３２例骨肉瘤手术标本使用抗ｐ１６和Ｒｂ蛋白抗体进行检测。结果显示，ｐ１６和Ｒｂ蛋白阳性表达分别为３７．５％（１２／３２）和５６．３％（１８／３２）。在１２例Ｒｂ蛋白阳性或强阳性表达的骨肉瘤中，ｐ１６蛋白表达阴性或弱阳性表达８例（６６．７％）；相反，在１０例Ｒｂ蛋白阴性或弱阳性表达的骨肉瘤中，ｐ１６蛋白阳性或强阳性表达６例（６０．０％）。结果表明：ｐ１６和Ｒｂ蛋白参与骨肉瘤细胞的增殖过程并与骨肉瘤的进展和预后有关；ｐ１６和Ｒｂ蛋白之间阳性表达的相互抑制，可能是判断骨肉瘤恶性程度的标志之一。     

利用Tet—on可诱导系统构建稳定表达PESl的SKOV3细胞株

目的为了进一步研究PES1的生物学功能,采用Tet—on系统构建可诱导稳定表达PES1的卵巢癌细胞株SKOV3。方法采用PCR技术将PES1克隆到pTRE—Tight载体中,并进行表达鉴定。将调控质粒pTet—on转染卵巢癌细胞,经G418筛选后再转染pTRE—Tight—PES1,然后经潮霉素筛选出阳性克隆。用不同浓度的强力霉素（Dox）进行诱导后,Westernblot确定Dox的最佳诱导浓度。结晶紫实验观察Dox诱导的稳定转染pTRE—Tight-PES1的SKOV3细胞生长速度。结果将成功构建的pTRE—Tight—PES1转染SKOV3细胞后,经过筛选获得Dox可诱导表达PES1的SKOV3细胞克隆。浓度低于2mg／L的Dox可以剂量依赖性地诱导PES1表达,2mg／L可以诱导PES1高表达。Dox诱导的转染pTRE—Tight和未转染任何质粒的SKOV3生长速度无明显差异,而转染pTRE—Tight—PES1的SKOV3细胞比转染pTRE-Tight和未转染任何质粒的细胞在第g天时生长速度明显更快（P=0．001）。结论成功建立了可诱导表达PES1的SKOV3细胞,为研究PES1的生物学功能提供了有效的细胞模型。PES1表达可以增强SKOV3细胞的生长。     

大脑皮质和脑干中c-fos基因表达的图像定量分析

ｆｏｓ基因为即早应激反应基因，我们发现ｃｆｏｓ在正常脑组织中也有表达，且在大脑、脑干细胞中的表达强度不同，我们对大脑皮质、脑干细胞中的ｃｆｏｓｍＲＮＡ进行了原位杂交和图像定量测量，结果显示，大脑皮质细胞中ｃｆｏｓｍＲＮＡ的数量显著多于脑干细胞。我们推测，大脑皮质、脑干细胞中ｃｆｏｓ基因的不同程度表达可能与脑组织的活动密切相关。     

C—myc蛋白和增殖细胞核抗原在人膀胱癌组织中表达的意义

应用增殖细胞核抗原（ＰＣＮＡ）和Ｃ－ｍｙｃ单克隆抗体，以免疫组织化学方法检测正常膀胱组织和膀胱组织中Ｃ－ｍｙｃ蛋白和ＰＣＮＡ的表达水平。结果表明：５例正常膀胱中未发现Ｃ－ｍｙｃ和ＰＣＮＡ阳性表达。阳性的Ｃ－ｍｙｃ和ＰＣＡ均定位于肿瘤的细胞核内。５８例膀胱癌中Ｃ－ｍｙｃ和ＰＣＮＡ蛋白阳性表达分别为４３．１％和６２．１％，并且Ｃ－ｍｙｃ和ＰＣＮＡ阳性表达率与膀胱癌的病理分级、临床分期和患者术后生存率相     

EGFR and KRAS status of primary sarcomatoid carcinomas of the lung: Implications for anti‐EGFR treatment of a rare lung malignancy

Sarcomatoid carcinomas (SC) of the lung are uncommon malignant tumors composed of carcinomatous and sarcomatous cell components and characterized by a more aggressive outcome than other histological subtypes of nonsmall cell lung cancer (NSCLC). Although epidermal growth factor receptor (EGFR)‐targeted therapies have emerged as a promising therapeutic approach in patients with advanced typical NSCLC such as adenocarcinoma, the potential clinical activity of these drugs in lung SC is still unknown. To investigate this point, we have analyzed the status of 4 EGFR pathways biomarkers in a series of lung SC. EGFR protein expression, EGFR gene copy number, EGFR mutational status and KRAS mutational status were assessed in a series of 22 consecutive cases of primary lung SC. EGFR protein overexpression was observed in all the cases. High level of polysomy (≥4 copies of the gene in >40% of cells) was detected in 5 cases (23%). No EGFR mutation was detected. KRAS mutations were found in 8 patients (38%; Gly12Cys in 6 cases and Gly12Val in 2 cases). The consistent EGFR protein overexpression and the high rate of KRAS mutation may contribute to the poorer outcome of lung SC in comparison with typical NSCLC. The rare incidence of increased EGFR gene copy number, the lack of EGFR mutation and the high rate of KRAS mutation observed in our series also suggest that most patients with lung SC are not likely to benefit from anti‐EGFR therapies. © 2009 UICC     

核不均一核糖核蛋白A2/B1在非小细胞肺癌发病机制中作用的探讨

目的 观察核不均一核糖核蛋白A2/B1(hnRNP A2/B1)在非小细胞肺癌(NSCLC)中的表达及其与DNA修复酶O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)、8-羟基鸟嘌呤DNA糖苷酶(OGG1)、氧化还原因子1(Ref-1)、DNA依赖性蛋白激酶复合物DNA-PKcs和Ku mRNA之间的相互作用,并进一步探讨其在NSCLC发病机制中的作用.方法 采用免疫组化、Western blot及荧光实时定量PCR方法,检测NSCLC患者癌组织及正常肺组织hnRNP A2/B1的表达.采用免疫共沉淀结合逆转录聚合酶链反应(RT-PCR)方法,研究人肺鳞癌细胞株中hnRNP A2/B1蛋白是否与上述5种DNA修复酶的mRNA直接结合,然后采用免疫组化及荧光实时定量PCR方法,检测NSCLC患者癌组织及正常肺组织MGMT的表达情况.结果 免疫组化染色显示,hnRNP A2/B1定位于细胞核,hnRNPA2/B1在NSCLC癌组织中的表达阳性率(100%)和蛋白表达评分[(5.3±0.9)分]均显著高于正常肺组织[32%和(2.2±0.7)分,P＜0.01],在Ⅲ～Ⅳ期NSCLC组织中的表达略高于Ⅰ～Ⅱ期(P＜0.05),而与年龄、性别、组织学类型及吸烟状况无关(均P＞0.05).通过RT-PCR方法可以从人肺鳞癌细胞株免疫共沉淀产物中扩增出MGMT mRNA,提示hnRNP A2/B1与MGMT mRNA相结合.进一步的免疫组化染色结果显示,在NSCLC组织中,MGMT的表达阳性率为32.0%,明显低于正常肺组织(78.0%),蛋白表达评分[(2.2±0.8)分]也显著低于正常肺组织[(4.1±1.2)分,P＜0.01].荧光实时定量PCR结果显示,NSCLC组织中MGMT mRNA的表达量为1.8(0.6～3.1),明显低于正常肺组织[9.8(6.8～18.3),P＜0.01].结论 HnRNP A2/B1蛋白及mRNA在NSCLC组织中的表达均升高,hnRNP A2/B1与MGMT mRNA相结合,可能通过对MGMT mRNA的转录后调控参与NSCLC的发生.

Abstract：

Objective To study the expression of heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) in non-small cell lung cancer(NSCLC),and the interaction between hnRNP A2/B1 protein and mRNA of DNA repair enzymes O6-methylguanine DNA-methyltransferase(MGMT),8-oxoguanine DNA glycosylase(OGG1),redox factor 1(Ref-1),DNA-dependent protein kinase(including DNA-PKcs and ku). Methods The expression and distribution of hnRNP A2/B1 were detected by immunohistochemistry and Western blot on 50 NSCLC samples from patients who underwent resection in Zhongshan Hospital.The hnRNP A2/B1 mRNA expression was tested by real-time PCR.Coimmunoprecipitation(co-IP) combined RT-PCR was used to investigate whether hnRNP A2/B1 could be bound with the mRNA of the above mentioned 5 DNA repair enzymes in human lung cancer cell line(HTB-182).Then immunohistochemistry and real-time PCR were used to detect the expression of MGMT in the same group of patients. Results HnRNP A2/B1 protein and mRNA expressions were increased in the NSCLC tissues than that in the corresponding normal lung tissues.HnRNP A2/B1 was expressed predominantly in the nuclei of tumor cells.The positive rate and immunohistochemistry score of hnRNP A2/B1 in tumor tissue were significantly higher than that in normal tissue(P ＜0.01).In stage Ⅲ-Ⅳ NSCLC,hnRNP A2/B1 expression was higher than that in stage Ⅰ-Ⅱ.There was no significant differences of hnRNP A2/B1 expression among patients of different age,sex,histological type,and smoking history.The results of co-IP combined RT-PCR suggested that hnRNP A2/B1 is bound with MGMT mRNA,and MGMT expression is decreased in tumor tissue of NSCLC. Conclusions The results of this study show that hnRNP A2/B1 protein and mRNA are highly expressed in NSCLC,and hnRNP A2/B1 is bound with MGMT mRNA,which indicate that it might be one of the mechanisms of hnRNP A2/B1 participating in the pathogenesis of NSCLC.     

成纤维细胞活化蛋白在非小细胞肺癌组织中的表达及其临床意义

目的： 检测人非小细胞肺癌（non-small cell lung cancer,NSCLC）组织中成纤维细胞活化蛋白（fibroblast activation protein,FAP）mRNA的表达,探讨FAP mRNA与NSCLC临床特征及预后之间的关系。 方法： 2004年6月至2006年12月选取天津医科大学附属肿瘤医院组织库中247例NSCLC患者瘤组织标本和对应的48例癌旁组织标本,患者随访至2011年12月1日。采用实时荧光PCR检测NSCLC组织和正常肺组织中FAP mRNA。临床病理参数与FAP mRNA相对表达量的相关性分析采用Mann-Whitney U检验,以Kaplan-Meier法绘制生存曲线,用log-Rank检验比较患者的生存差异,采用Cox回归模型评估独立的预后因素。 结果： FAP mRNA在NSCLC组织中过表达。NSCLC组织中FAP mRNA表达水平与NSCLC患者KPS评分成正相关（P<0.05）,而与肿瘤原发部位、肿块大小、淋巴结转移、临床分期、组织学类型等其他病理特征无明显关系（P>005）。高表达FAP mRNA 的NSCLC患者与低表达者相比,中位总生存时间（overall survival,OS）无明显差异（43 vs 39个月,P>0.05）。进一步以组织学类型分层分析显示,肺腺癌患者FAP mRNA表达量与临床分期成负相关（P=0.031）,与KPS评分成正相关（P=0.041）。高表达FAP mRNA的肺腺癌患者与低表达者相比,中位OS时间显著延长（42 vs 26个月,P<005）,多因素分析进一步证实FAP mRNA表达是影响肺腺癌患者预后的独立因素。 结论： FAP在NSCLC组织中高表达,FAP mRNA高表达与肺腺癌的临床分期呈负相关,并与肺腺癌患者的预后密切相关。     

Up‐regulation of interleukin‐17 expression by human papillomavirus type 16 E6 in nonsmall cell lung cancer

BACKGROUND:

Human papillomavirus (HPV) 16/18 infection is associated with nonsmoking lung cancer. In this study, the authors investigated a putative correlation between interleukin (IL)‐17 expression and HPV infection in clinical nonsmall cell lung cancer (NSCLC) tissues and examined the effects of HPV infection on a human NSCLC cell line.

METHODS:

IL‐17 expression was investigated in 79 NSCLC tumor tissues by immunohistochemistry. Growth rate, IL‐17 mRNA, and secreting protein levels were also examined in HPV 16/18 E6‐transfected H1299 human NSCLC cells.

RESULTS:

Immunohistochemical data showed that 48.1% of lung tumors had IL‐17 staining, which was significantly associated with patients' sex (P = .03), HPV infection (P = .002), and tumor stage (P = .03). Significant correlations of IL‐17 with IL‐6 (P < .001) and IL‐17 with Mcl‐1 (P < .001) expression were also observed. Cell growth rate was increased, and IL‐17/Mcl‐1 expression levels were elevated in HPV 16 E6‐transfected H1299 cells. The transfected E6 oncoproteins can significantly up‐regulate expression levels of IL‐17 and antiapoptotic protein Mcl‐1.

CONCLUSIONS:

The study suggests that HPV infection‐induced IL‐17 levels can stimulate Mcl‐1 expression through the PI3K pathway and promote lung tumor cell progression through a p53‐ and IL‐6‐independent pathway. Cancer 2010. © 2010 American Cancer Society.     

TAZ在非小细胞肺癌组织中的表达及其临床意义

背景与目的:TAZ(具有PDZ结合域的转录共刺激因子)是由400个氨基酸构成并与14-3-3蛋白结合,内含一个WW结构域和PDZ结合基序的转录共刺激因子,其在正常机体内参与基因转录调节和细胞信号转导。然而,近年研究发现其在多种肿瘤中表达升高,并参与肿瘤的发生、发展。本研究检测非小细胞肺癌(non-small cell lung cancer,NSCLC)组织中TAZ mRNA及蛋白表达情况,并探讨其临床意义。方法:取58例NSCLC患者癌组织、远癌组织(距肿瘤边缘>5 cm)和20例肺良性病变组织,并将远癌组织和肺良性病变组织作为对照组与肺癌组比较,采用实时荧光定量PCR法(real-time qPCR)和Western blot检测显示TAZ mRNA及其蛋白表达。结果:实时荧光定量PCR法检测显示,肺癌组织中TAZ mRNA的表达高于远癌及肺良性病变组织,差异有统计学意义(P<0.05);Western blot检测显示,肺癌组织中TAZ蛋白表达显著高于远癌及肺良性病变组织;NSCLC患者癌组织中TAZ mRNA的表达量与其肿瘤大小、分化程度和p-TNM分期相关(P<0.05)。结论:NSCLC患者癌组织中TAZ参与肿瘤的发生、发展,TAZ可作为NSCLC预后评价指标。     

miR‐203 inhibits cell proliferation,invasion, and migration of non‐small‐cell lung cancer by downregulating RGS17

Involvement of the RGS17 oncogene in the promotion of non‐small‐cell lung cancer (NSCLC) has been reported, but the regulation mechanism in NSCLC remains unclear. MicroRNAs (miRNAs) negatively regulate gene expression, and their dysregulation has been implicated in tumorigenesis. To understand the role of miRNAs in Regulator of G Protein Signaling 17 (RGS17)‐induced NSCLC, we showed that miR‐203 was downregulated during tumorigenesis, and inhibited the proliferation and invasion of lung cancer cells. We then determined whether miR‐203 regulated NSCLC by targeting RGS17. To characterize the regulatory effect of miR‐203 on RGS17, we used lung cancer cell lines, A549 and Calu‐1, and the constructed miR‐203 and RGS17 overexpression vectors. The CCK8 kit was used to determine cell proliferation, and the Transwell® assay was used to measure cell invasion and migration. RT‐PCR, western blots, and immunofluorescence were used to analyze expression of miR‐203 and RGS17, and the luciferase reporter assay was used to examine the interaction between miR‐203 and RGS17. Nude mice were used to characterize in vivo tumor growth regulation. Expression of miR‐203 inhibited proliferation, invasion, and migration of lung cancer cell lines A549 and Calu‐1 by targeting RGS17. The regulatory effect of miR‐203 was inhibited after overexpression of RGS17. The luciferase reporter assay showed that miR‐203 downregulated RGS17 by direct integration into the 3′‐UTR of RGS17 mRNA. In vivo studies showed that expression of miR‐203 significantly inhibited growth of tumors. Taken together, the results suggested that expression of miR‐203 inhibited tumor growth and metastasis by targeting RGS17.     

非小细胞肺癌组织中性粒细胞明胶酶相关载脂蛋白表达及其临床意义探讨

目的探讨中性粒细胞明胶酶相关载脂蛋白（neutrophil gelatinase-associated lipocalin,NGAL）在非小细胞肺癌（non-small cell lung cancer,NSCLC）组织中的表达及其临床意义。方法收集江西省胸科医院2009-03-17-2012-03-23经手术切除的65例NSCLC及癌旁组织,其中鳞癌35例,腺癌30例。实时荧光定量PCR检测NGAL mRNA的表达;免疫组化技术检测NGAL蛋白的表达,并分析其与临床病理特征的相关性。结果鳞癌组织NGAL mRNA检测结果与β-actin的ΔCt之差为-2.3±1.2,腺癌组织为-2.8±1.5,差异无统计学意义,P〉0.05;但两者与癌旁组织的表达水平-1.6±1.3比较,差异均有统计学意义,q值分别为3.568和5.811,P值均〈0.05。腺癌组织NGAL蛋白表达阳性率为80.0%（24/30）,鳞癌组织为57.1%（20/35）,癌旁组织为32.3%（21/65）,3组样本两两比较差异均有统计学意义,P值均〈0.05。但其蛋白表达与患者性别、年龄、吸烟和有无淋巴结转移差异无统计学意义,P值均〉0.05。结论 NGAL在NSCLC组织内高表达,并且与其分型有关,可能成为NSCLC早期诊断的标志。     

WT1在非小细胞肺癌中的表达及其与肺癌细胞增殖的关系

目的:探讨人非小细胞肺癌及其相应癌旁组织中WT1基因(Wilms' tumor gene 1,WT1)的表达,体外研究其与肺癌细胞增殖的关系.方法:应用实时荧光定量PCR法检测79例非小细胞肺癌组织及其相应癌旁组织中WT1 mRNA的表达情况.构建重组质粒pLV-GFP-WT1,并通过脂质体LipofectAMINE 2000将其瞬时转染至非小细胞肺癌H1299细胞中,应用蛋白质印迹法检测转染后H1299细胞中WT1蛋白的表达,CCK-8 (cell counting kit-8)法检测细胞的增殖情况.结果:非小细胞肺癌组织中WT1 mRNA的表达水平高于相应的癌旁组织,差异有统计学意义(P＜0.01).重组质粒pLV-GFP-WT1成功构建,并将其成功转染至H1299细胞中;转染后的H1299细胞中,WT1蛋白的表达水平上调;在pLV-GFP-WT1转染后24、36和48 h时,过表达WT1的H1299细胞增殖率高于对照组,差异有统计学意义(P＜0.05).结论:WT1 mRNA在非小细胞肺癌组织中的表达水平明显高于相应癌旁组织,高表达WT1基因能促进非小细胞肺癌H1299细胞的增殖,提示WT1在非小细胞肺癌中扮演癌基因的角色.