Si-10靶点对不同COX-2蛋白表达状态肺癌细胞体外恶性增殖的影响

目的:研究Si-10靶点对不同环氧化酶-2(COX-2)蛋白表达状态肺癌细胞干扰效果及对肺癌细胞恶性增殖的影响.方法:将psi-10及空载体(pEGFP)分别转染到不同COX-2表达状态的肺癌细胞,建立转染细胞株.采用反转录聚合酶链反应和蛋白质印迹方法检测COX-2表达水平的变化.通过细胞生长曲线、集落形成验研究这一靶点对肺癌细胞体外恶性增殖的影响.结果:转染后24、48和72 h,转空载体的细胞均有绿色荧光表达,转染psi-10的801D、A549和LTEP-A2细胞均未观察到绿色荧光.在A549和LTEP-A2细胞中COX-2表达均受到抑制,但抑制效果不同,与A549细胞比较Si-10对LTEP-A2细胞COX-2表达抑制更为明显.与其母系比较,LTEP-A2-10和A549-10细胞COX-2 mRNA表达量分别降低64.2%和61.2%;COX-2蛋白表达量分别降低60.2%和56.2%.LTEP-A2-10和A549-10细胞生长明显减慢,集落形成率减少.与A549-10细胞比较,LTEP-A2-10细胞生长更慢,集落形成率更低,集落更小.Si-10靶点对COX-2不表达的801D细胞生长没有抑制作用.结论:si10对不同COX-2基因表达的肺癌细胞有不同的干扰效果,而不同干扰的效果对细胞的恶性增殖有着不同的抑制作用.     

PCNA表达与肺癌细胞株凋亡的关系

 目的　探讨肺癌细胞株增殖细胞核抗原 (PCNA)的表达与肺癌细胞株凋亡的关系。方法　用三氧化二砷 (As2 O3 )诱导肺癌细胞株GLC 82凋亡。运用细胞培养、MTT、流式细胞技术 (FCM )检测及逆转录聚合酶链式反应 (RT PCR)检测PCNA表达在细胞凋亡过程中的变化及相互关系。结果　三氧化二砷可明显抑制GLC 82细胞增殖 ,使细胞周期阻滞于G2 /M并随后凋亡 ,PCNA表达受到抑制。结论三氧化二砷诱导GLC 82凋亡过程中 ,PCNA表达受到明显抑制。对于耐药性肿瘤 ,PCNA活性的抑制可能代表一种新的化疗策略。     

mTOR和PTEN在非小细胞肺癌组织中的表达及临床意义

背景与目的 mTOR是调节细胞生长和增殖的重要信号转导分子,也是一种蛋白激酶.它通过活化下游的相关的效应蛋白发挥作用.在信号转导通路中PTEN基因可通过对该信号途径的负调控而抑制mTOR的活化.本研究通过分析mTOR信号转导途径中mTOR和PTEN基因在非小细胞肺癌(non-small cell lung cancer,NSCLC)组织中的表达和临床意义.方法 外科手术中获取65例NSCLC组织及30例癌旁组织,RT-PCR技术检测NSCLC组织及癌旁组织中mTOR和PTEN基因的表达水平.结果 mTOR在NSCLC组织中表达量(0.23±0.16)显著高于癌旁组(0.12±0.09)(P<0.01),PTEN在NSCLC组织中表达量(0.19±0.28)显著低于癌旁组(0.53±0.28)(P<0.01).mTOR和PTEN与病人的性别、年龄、病理类型、淋巴结转移情况无关,与病人的肿瘤大小有关.结论 mTOR在NSCLC中被激活,PTEN在NSCLC组织表达缺失或减少,mTOR路的激活和PTEN表达缺失在NSCLC发生发展中起到一定的作用.     

肺癌患者胸水中MOC-31 mRNA的表达及临床意义

目的探讨MOC-31mRNA在肺癌患者胸水中的表达及临床意义。方法应用逆转录-聚合酶链反应（RT—PCR），检测74例肺癌和40例肺良性疾病患者胸水中MOC-31mRNA的表达，并与常规细胞学诊断进行对比。结果肺癌组胸水中MOC-31mRNA的阳性表达率为91．9％（68／74），肺良性疾病组胸水中MOC-31mRNA的阳性表达率为10．0％（4／40），差异有统计学意义（Χ^2=74．83，P〈0．01）：肺癌组胸水中MOC-31mRNA的表达与病理分型无关（P〉0．05）；MOC-31mRNA的检测对恶性胸水诊断的敏感度和准确度均明显高于常规细胞学检查（P〈0．01），但两种诊断方法的特异度差异却无统计学意义（P〉0．05）。结论MOC-31mRNA可作为检测肺癌患者胸膜微转移的分子标志物，是判断肺癌患者胸水中是否存在脱落癌细胞和肺癌临床分期的辅助参考指标。     

腋淋巴结阴性乳腺癌外周血CK19mRNA表达与临床病理因素相关性的探讨

目的:观察腋淋巴结阴性乳腺癌患者外周血中CK19 mRNA的表达情况,探讨其表达与临床病理指标间的关系及意义.方法:用RT-PCR方法检测113例乳腺癌患者和30例健康志愿者外周血中CK19 mRNA的表达,并分析CK19mRNA的表达情况与乳腺癌患者的年龄、肿瘤大小、月经状态、肿瘤组织学分级、病理类型、临床病理分期(TNM分期)、雌激素受体表达、孕激素受体表达、Ki-67增殖表达和HER-2表达等指标之间的关系.结果:113例乳腺癌患者的外周血中有41例患者的CK19 mRNA阳性表达,阳性率为36.3%;30例健康志愿者的外周血中有1例CK19 mRNA阳性表达,阳性率为3.3%.两者相比差异有统计学意义,P=0.000.乳腺癌患者的外周血中CK19 mRNA的表达与乳腺癌患者的年龄、月经状态、雌激素受体表达和孕激素受体表达等均无相关性,P>0.05;但与肿瘤大小、肿瘤组织学分级、病理类型、临床病理分期、HER-2表达和Ki-67增殖表达等指标均有相关性,P<0.05.结论:腋淋巴结阴性乳腺癌患者外周血中的CK19 mRNA表这是一个判断微转移的敏感指标,肿瘤的大小、组织学分级、TNM分期、病理类型、HER-2表达及Ki-67增殖表达可评估该类患者外周血微转移的风险.     

肺癌患者Th1和Th2类细胞因子的基因表达及临床意义

目的观察肺癌患者体内Th1和Th2类细胞因子的基因表达及化疗对其表达的影响。方法采用逆转录聚合酶链反应（RT—PCR）技术，检测肺癌患者在化疗前、化疗后外周血单个核细胞（PBMC）Thl和Th2类细胞因子mRNA表达。结果正常对照及良性肺部疾病组Th1和Th2类细胞因子基因表达呈阴性或基本不表达；肺癌组IL4、IL．6、IL-10的mRNA表达水平显著高于良性肺部疾病组和正常对照组（P值均〈0．001）；IFN-γ无阳性表达（0／52），IL-2则仅有2例阳性表达，均较正常对照组及良性肺部疾病组明显减少（P值分别为0．006，0．007；0．004，0．003）；且Th1和Th2类细胞因子基因表达水平与肺癌的病理分型及临床分期无相关性（P值分别为0．925，0．752，0．868，0．780，0．949）。化疗后IL-4、IL-6、IL-10的阳性表达较化疗前显著减少（P值分别为0．003，0．001，0．002）。而IFN-γ、IL-2阳性表达较化疗前有显著增加（P值均〈0．001）。结论 肺癌患者Th1类细胞因子表达受抑，Th2类因子呈强势表达；化疗可使肺癌患者Th2类细胞因子的强势表达向Th1类逆转。     

高转移性人肺癌细胞系p53基因突变的研究

ＰＧ癌系为来自人巨细胞型肺癌的体外细胞系，具有生长快和成瘤率高的特性。裸小鼠移植后，肺和淋巴结的自发转移性频率高，而且稳定。为了深入了解其生物学特性的分子基础，对其细胞和分子生物学改变作了系统的分析。结果表明：免疫组化发现ｐ５３蛋白的异常高表达；用非同位素ＰＣＲ－ＳＳＣＰ法检测到第７外显子的单链多态性变化，提示有碱基组成的改变。对该外显子的两条链用热循环ＤＮＡ测序法分析后，证实第２４８密码子有ＣＧＧ→ＣＴＴ的碱基颠换；同时以上述方法检测了Ｋｉ－ｒａｓ癌基因的点突变，未发现ｋｉ－ｒａｓ突变。结合文献探讨了ｐ５３基因突变的生物学意义，这一研究为进一步研究ｐ５３基因与ＰＧ癌细胞生物学特性的关系奠定了基础。     

非小细胞肺癌患者外周血CK19 mRNA的表达及与临床的关系

目的：研究非小细胞肺癌（NSCLC）患者外周血中细胞角蛋白19（cytokeratin 19，CK19）mRNA的表达变化及与临床的关系。方法：采用逆转录多聚酶链反应（RT—PCR）法进行外周血CK19mRNA表达的测定。结果：NSCLC组患者外周血CK19mRNA的阳性率为60．7％。显著高于良性病变组的3．3％和健康对照组的0（P〈0．01）；Ⅲ和Ⅳ期NSCLC患者的阳性率显著高于Ⅰ和Ⅱ期患者的阳性率（P〈0．05）；而不同病理类型NSCLC患者外周血CK19mRNA阳性率之间无显著性差异（P〉0．05）。结论：外周血CK19mRNA的检测有助于我们对NSCLC患者的病情、预后和治疗进行判断．且这并不受病理分型的影响。     

Aurora-A激酶在人类肺癌细胞株中的表达

目的:探讨aurora -A基因在正常肺组织与不同肺癌细胞中的表达。方法:采用半定量RT- PCR、Western blot方法,检测Aurora- A在高转移的肺癌细胞PG、肺腺癌细胞A549、大细胞肺癌细胞NCI H460 三种肺癌细胞株的表达情况结果:RT -PCR结果显示,三种肺癌细胞与正常肺组织比较aurora -A mRNA均高表达,经图象分析,PG、A549、NCI H460 与正常肺组织中aurora A/βactin比值分别为1 14、1 16、0 84 和0 26Western blot结果经图象分析发现, A549、NCI H460 与PG 的aurora A/βactin 比值分别为21 .13、6 .43 与8 .96。三者比较,A549 最高, PG其次, NCI H460最低。结论:aurora -A在三种肺癌细胞中其mRNA及蛋白水平均呈高表达,不同细胞株其表达水平不同。aurora -A基因在肺癌细胞中高表达的结果提示,作为一种新的癌基因,aurora- A有可能成为肺癌新的肿瘤标记物及分子治疗靶点。     

Expression of glutathione S-transferase isoenzymes in human small cell lung cancer cell lines

A glutathione S-transferase (GST) isoenzyme having common antigenicityto rat placental form (GST-P) and human placental form (GST-)has recently been suggested may be a marker of carcinogenesis.In the present study we have investigated the expression ofthis isoenzyme in three small cell lung cancer cell lines inorder to determine whether or not this isoenzyme can be usedas a general marker of carcinogenesis. GST activity towards1-chloro-2,4-dinitrobenzene in two of the cell lines (NES andNOC-361) was moderately higher than that in normal human lung,but this activity was markedly suppressed in one of the celllines (NCI-H69). Quantitation of the GST isoenzymes in the tumorsgrown in nude mice by injecting these cell lines also revealeda moderate increase of GST--type isoenzyme in NES and NOC-361and its suppression in NCI-H69. Immunocytochemical localizationstudies with these tumors using antibodies raised against GST-also indicated a drastic decrease of GST--type isoenzyme inNCI-H69 and this finding was confirmed by Western biot studies.These results suggest that GST- or the isoenzyme(s) having similarimmunological nature to GST-, cannot be used as the generalmarkers of neoplastic states.     

Expression of an estramustine-binding associated protein in human lung cancer cell lines

Estramustine-binding protein has previously been demonstrated in normal rat prostatic tissue, in normal human prostate epithelium, and in prostatic carcinomas. It binds specifically estramustine and estromustine, the cytotoxic metabolites of estramustine phosphate (Estracyt), a drug which is used in the treatment of prostatic carcinoma. In this study we have examined the presence of an estramustine-binding associated protein in a panel of human cell lines, representing the major histopathological types of lung cancer. A mouse (murine) monoclonal antiserum developed against rat estramustine-binding protein was used for immunohistochemical detection. Fast protein liquid chromatography was used for biochemical characterization. As judged from the immunohistochemical investigation, estramustine-binding protein was present in large amounts in five of six non-small cell carcinoma cell lines, while seven of eight small cell carcinoma cell lines were essentially negative. Fast protein liquid chromatography analyses of lysated cells from the lung cancer cell lines, incubated with [3H]estromustine, concurred with the results from the immunohistochemical stainings. These data strongly indicate a convincing connection between the immunoreactivity and ligand-binding properties of estramustine-binding protein in the cell lines examined. The presence of an estramustine-binding associated protein in human lung cancer cell lines has implications for further investigations into the biological relevance and the potential for eventual therapeutic applications.     

Expression of cytokeratin 19 mRNA in human lung cancer cell lines.

The present study was designed to clarify the mechanism by which some lung cancer cell lines can produce cytokeratin 19 (CK19) fragment and others cannot. We hypothesized that some lung cancer cell lines which cannot release CK19 express an incomplete sequence of CK19 mRNA. Expression of mRNA was evaluated by RT-PCR using several primer pairs for CK19. CK19 in the culture supernatant was measured by an immuno-radiometric assay. CK19 protein synthesis was evaluated by Western immunoblot and immunohistochemistry. Among 16 lung cancer cell lines, 7 released significant amounts of CK19 in the supernatant. In some cell lines, expression of CK19 mRNA was observed only in some combinations of primers, suggesting that incomplete mRNA was expressed. 3'-RACE analysis detected amplified products of a shorter size compared with normal amplified products in cell lines which expressed incomplete CK19 mRNA, suggesting that 3'-ends of mRNA for CK19 were deleted. Results of Western immunoblot and immuno-histochemical staining using anti-human CK19 monoclonal antibody completely correlated with the results on CK19 levels in culture supernatants as well as with complete expression of mRNA. We conclude that levels of CK19 closely relate to the expression of complete mRNA for CK19.     

Expression of cadherin and NCAM in human small cell lung cancer cell lines and xenografts.

Tumour cell adhesion, detachment and aggregation seem to play an important part in tumour invasion and metastasis, and numerous cell adhesion molecules are expressed by tumour cells. Several families of cell-cell adhesion molecules have been described, of which two groups are particularly well characterised, the cadherin family and the Ig superfamily member, neural cell adhesion molecule (NCAM). We investigated expression of these two adhesion molecule families in small cell lung cancer (SCLC) cell lines and xenografts by immunoblotting. Nineteen tumours established from 15 patients with SCLC were examined. All tumours but one expressed both cadherin and NCAM. The tumours expressed one, two or rarely three cadherin bands, and different combinations of two major isoforms of NCAM with M(r)'s of approximately 190,000 and 135,000. Polysialylation of NCAM, a feature characteristic of NCAM during embryonic development, which may play a role in connection with tumour invasion and metastasis, was found in 14/18 NCAM expressing SCLC tumours. Individual tumours grown as cell lines and as nude mouse xenografts showed no qualitative differences in cadherin or NCAM expression.     

DNA mismatch binding in human lung tumor cell lines

Defects in mismatch repair (MMR) genes have been involved in several types of sporadic and hereditary cancers. In order to elucidate the role of MMR in human lung carcinogenesis we examined DNA mismatch binding in cell-free extracts of seven lung tumor cell lines and five corresponding lymphoblastoid cell lines from lung cancer patients. Using the technique of bandshift assay we have demonstrated that 2/7 of the tumor cell lines are aberrant in binding to specific DNA mismatches while all lymphoblastoid cell lines were proficient in binding to all tested mismatches. Both extracts were aberrant in binding to G/T mismatch whereas one of the cell lines showed deficiency in binding to the C:A mismatches as well. Immunoblotting analysis showed that all known DNA mismatch repair (MMR) proteins were present in these extracts. The cell line deficient in binding to both G:T and C:A mismatches showed microsatellite instability (MSI) in tumor DNA and higher resistance to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This report indicates that DNA mismatch binding deficiencies may be implicated in at least a subgroup of human lung cancer.     

人肺癌细胞株中hnRNP A2/B1表达的研究

目的 研究人肺癌细胞株中核内不均一核糖核蛋白(hnRNP)A2/B1及hnRNP B1的表达。方法 采用特异性hnRNP A2／B2及hnRNP B1引物,应用RT-PCR方法研究肺癌细胞株hnRNP A2／B1 mRNA的表达,以抗hnRNP B1单克隆抗体免疫细胞化学染色方法研究肺癌细胞hnRNP A2/B1蛋白的亚细胞定位。结果 RT—PCR显示人肺腺癌细胞株SPC-A1及Y-90,小细胞肺癌细胞株NCI-H446及鳞癌细胞株A431均表达hnRNP A2／B1及hnRNP B1,其PCR产物分子量分别在661及1039bp左右,与预期大小的hnRNP A2/B1 cDNA片断相符。免疫细胞化学研究发现,肺腺癌细胞株SPC—A1及Y-90．小细胞肺癌细胞株NCI—H446及鳞癌A431的细胞浆及细胞核内均可见特异的hnRNP B1染色,鳞癌A43l及小细胞肺癌NCI—H446 hnRNP B1染色较肺腺癌细胞株SPC—A1及Y-90明显。结论 肺癌细胞株hnRNP A2／B1及hnRNP B1表达明显增高。     

Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines.

Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinity labeling, and Northern blotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data demonstrated that the cells bound between 3 and 52 fmol/mg protein with a KD ranging from 0.5 x 10(-10) to 2.7 x 10(-10) M. EGF binding to the receptor was confirmed by affinity-labeling EGF to the EGF receptor. The cross-linked complex had a M(r) of 170,000-180,000. Northern blotting showed the expression of EGF receptor mRNA in all 10 cell lines that were found to be EGF receptor-positive and in one cell line that was found to be EGF receptor-negative in the radioreceptor assay and affinity labeling. Our results provide, for the first time, evidence that a large proportion of a broad panel of small cell lung cancer cell lines express the EGF receptor.     

Peripheral airway cell differentiation in human lung cancer cell lines

Clara cells and type II pneumocytes are the progenitor cells of the bronchioles and alveoli, respectively. These peripheral airway cells (PAC) contain characteristic cytoplasmic structures and express surfactant associated proteins. PAC cell markers are expressed by many pulmonary adenocarcinomas having papillary and/or lepidic growth patterns, which are characteristics of the bronchioloalveolar and papillary subtypes. We investigated the expression of PAC markers in a panel of 41 lung cancer cell lines. Ultrastructural studies demonstrated the presence of cytoplasmic structures characteristic of Clara cells or of type II pneumocytes in 9 of 34 (26%) non-small cell lung cancer cell lines, including 7 of 17 (41%) adenocarcinomas, one squamous cell carcinoma, and one large cell carcinoma. Of interest, the cytoplasmic structures were present in 5 of 6 (83%) cell lines initiated from papillolepidic adenocarcinomas. In addition, we examined the lines for expression of the surfactant associated proteins SP-A, SP-B, and SP-C. Eight of the nine cell lines containing cytoplasmic inclusions characteristic of PAC cells also expressed protein and/or RNA of SP-A, the major surfactant associated protein. Five of these lines expressed SP-B RNA (either constitutively or after dexamethasone induction), while a single line expressed SP-C only after dexamethasone induction. None of six small cell lung cancer cell lines examined expressed any of the PAC markers. Thus, PAC markers are expressed frequently (but not exclusively) in pulmonary adenocarcinoma cell lines, especially in those initiated from tumors having papillolepidic growth patterns. The establishment and identification of multiple cell lines expressing PAC features provide an important new resource for biological and preclinical therapeutic studies.