高低转移肺腺癌细胞系中肿瘤抑制基因p16、p21和p53表达研究

目的 探讨肿瘤抑制基因对肺腺癌细胞生长的抑制作用。方法 利用FuGene转染方式分别将p21和p16基因的表达质粒转入－对肺腺癌细胞系Anip973和AGZY83－a中,同时用含野生型p53 基因的腺病毒感染p16基因转染前后的这一对细胞系。对P16和P21蛋白过表达的细胞系进行了细胞生长曲线、克隆形成率、原位末端标记分析和流式细胞仪分析。结果 p16基因的过表达只能使细胞系的G1期细胞比例提高,但细胞生长曲线,克隆形成率均未出现改变,未检测到凋亡信号。P21蛋白过表达的一对细胞系细胞生长曲线斜率降低,克隆形成能力下降,并出现明显的G1期阻滞,但未检测到凋亡信号。p53基因感染AGZY83－a,Anip973及经过p16基因转染的细胞AGZY83－ap16和Anip973p16后呈现时间依赖性表达,细胞生长曲线和四唑盐比色法分析提高,野生型p53基因的大量表达明显抑制以上4种细胞的生长,Anip973和Anip973p16的生长抑制率高于AGZY83－a和AGZY83－ap16;Anip973p16和AGZY83-ap16的生长抑制率高于Anip973和AAGZY83－a。这4种细胞在感染p53后出现典型的凋亡信号。结论p16基因的过表达并不能抑制细胞系的生长,而p21基因的过表达通过G1期阻滞抑制这1对肺腺癌细胞的生长;野生型p53基因在AGZY83－a和Anip973中高效表达可产生明显的细胞生长抑制效应;野生型p53基因对肺腺癌高转移细胞系Anip973抑制作用更为明显。     

BRI基因在一对转移能力不同的肺腺癌细胞系中的序列分析与表达研究

目的 确认淀粉样纤维蛋白基因(amyloid fibrils，BRI)基因在l对同源但转移能力不同的肺腺癌细胞系AGZY83-a和Anip973中的序列并分析其表达。方法 采用测序技术，Northern印迹杂交。G显带后荧光原位杂交分析BRI基因在肺腺癌细胞系的序列与表达。结果 BRI基因在高转移肺腺癌细胞系Anip973中高表达，在其低转移母系AGZY83-a中低表达，两细胞系BRI基因染色体定位区均存在断裂重排。该基因染色体定位区在Anip973中出现扩增。已知BRI基因的-116bp～-5bp处碱基序列和-115bp～-5bp处碱基序列在AGZY83-a和Anip973中分别突变为CTCAGCAGCCCGC和TCAGCCGC。结论 BRI基因在转移能力不同的肺腺癌细胞系差异表达与该基因的染色体定位区域的断裂重排无关，与该基因染色体定位区拷贝数增加及5′非翻译区存在不同的突变可能相关。     

应用mRNA差异显示技术克隆人肺腺癌转移相关基因

肿瘤转移是恶性肿瘤最基本的生物学特征。为了探讨恶性肿瘤的侵袭和转移的机理，选择两株具有相同的细胞来源、但具有不同转移能力的人肺腺癌细胞系ＡＧＺＹ８３ａ和Ａｎｉｐ９７３作为研究材料，采用ｍＲＮＡ差异显示（ｍＲＮＡｄｉｆｅｒｅｎｔｉａｌｄｉｓｐｌａｙ）技术，对这两个细胞系的基因表达差异情况进行了分析。结果显示，在这两个细胞系之间存在明显的基因表达差异，说明在低转移的ＡＧＺＹ８３ａ向高转移的Ａｎｉｐ９７３演变过程中涉及多个基因的激活与失活。研究结果还提示，在高转移的Ａｎｉｐ９７３细胞系中，某些基因的表达或高表达与人类逆转录病毒基因ＬＴＲ的整合有关。另一个克隆与人类线粒体基因ＮＤ４具有高度同源性，其高度表达与Ａｎｉｐ９７３的转移表型有关。表明肿瘤的转移是一个多基因参与的过程，由于这些基因的相互作用及细胞内外环境因素的影响，最终决定了肿瘤细胞的转移表型。     

Inhibition of Cyclooxygenase‐2 Suppresses Lymph Node Metastasis via VEGF‐C

Most experimental work addressing cyclooxygenase‐2 (COX‐2) inhibitor has focused on suppressing hematogenic spread. Little is known about the mechanism by which this inhibitor can also block lymphatic metastasis. Here, the effects of COX‐2 inhibitor on vascular endothelial growth factor‐C (VEGF‐C) expression, lymphangiogenesis and lymph node metastasis were investigated. Utilizing the highly metastatic human lung adenocarcinoma cell line Anip973 and its parental line AGZY83‐a, which has a low metastatic capacity, we found elevated VEGF‐C and COX‐2 immunoreactivity in Anip973 cells compared with AGZY83‐a cells. Celecoxib down‐regulated expression of VEGF‐C mRNA and protein in Anip973 cells while PGE₂ up‐regulated expression of VEGF‐C mRNA and protein in AGZY83‐a cells in a concentration‐dependent manner. The expression of COX‐2 and VEGF‐C was significantly increased in xenografted Anip973 tumors compared with AGZY83‐a tumors. The Anip973 tumors showed more lymphatic vessels and lymph node metastasis than the AGZY83‐a tumors. In vivo, celecoxib decreased VEGF‐C expression in Anip973 tumor‐treated mice to a similar level to that in the AGZY83‐a tumor‐treated mice. Consistent with this decrease in VEGF‐C expression, the density of lymphatic vessels and lymph node metastasis in Anip973 tumor‐treated mice were suppressed to approximately that found in the AGZY83‐a tumor‐treated ones. Taken together, our results suggest that the differential expression of COX‐2 and VEGF‐C might help explain the different metastasis phenotype of lung adenocarcinoma cancer, and that COX‐2 inhibitor mediates VEGF‐C to block lymphangiogenesis and lymph node metastasis. Thus, COX‐2 may be a potential therapeutic target for blocking lymph node metastasis in lung adenocarcinoma. Anat Rec, 2009. © 2009 Wiley‐Liss, Inc.     

COX‐2‐Mediated Regulation of VEGF‐C in Association With Lymphangiogenesis and Lymph Node Metastasis in Lung Cancer

The mechanisms underlying the effects of COX‐2 on tumor lymphangiogenesis remain largely undefined. Here, the human lung cancer cell lines A549, 95D, Anip973, and AGZY83‐a with different metastatic capacities were investigated by immunostaining, western blotting, and real‐time RT‐PCR. We observed increased expressions of COX‐2 and VEGF‐C in the three highly metastatic cell lines compared with the less metastatic AGZY83‐a cell line. The COX‐2‐specific inhibitor Celecoxib suppressed VEGF‐C expression whereas the main COX‐2 metabolite PGE₂ elevated VEGF‐C expression in Anip973 and AGZY83‐a cells in positive and negative experiments. To determine the functional link to COX‐2 more specifically and elucidate the mechanistic pathway, we used a siRNA to knock down the high COX‐2 expression in Anip973 cells and transfected a COX‐2 cDNA to enhance the low COX‐2 expression in AGZY83‐a cells, and then treated the cells with EP1/EP4 agonists or antagonists, respectively. The results revealed that the EP1/EP4 agonists significantly increased VEGF‐C production in the COX‐2‐knockdown Anip973 cells. In contrast, the EP1/EP4 antagonists diminished VEGF‐C production in the COX‐2‐overexpressing AGZY83‐a cells. Furthermore, animal models provided evidence that Celecoxib decreased VEGF‐C expression, lymphangiogenesis, and lymph node metastases in Anip973 cells, whereas PGE₂ treatment increased the same factors in the parental AGZY83‐a cells. A positive correlation between COX‐2 and VEGF‐C was also confirmed in vivo. The present data suggest that COX‐2 regulates VEGF‐C expression via the PGE₂ pathway, and that EP1/EP4 receptors are involved in PGE₂‐mediated VEGF‐C production. Thus, COX‐2 may represent a candidate gene for blocking tumor lymphangiogenesis and lymph node metastasis. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

抗人肺癌细胞转移相关抗原单克隆抗体的制备及初步鉴定

用转移能力及其它生物学特性不同的2个人肺腺癌细胞系免疫BALB/c小鼠,制备出一组可与人肺腺癌细胞发生特异性反应,而与其它脏器肿瘤组织无交叉反应的单克隆抗体。经初步鉴定,其中的3个单抗分别识别低转移或高转移肺腺癌细胞系表面抗原。     

BRI基因的表达与非小细胞肺癌发生及转移潜能的关系

目的 探讨BRI基因在人非小细胞肺癌中的差异表达情况及其与肺癌发生及转移能力形成的相关性。方法 应用逆转录-聚合酶链反应(RT-PCR)、Northern印迹杂交方法分析BRI基因在一对遗传背景相同、转移能力不同的人肺腺癌细胞系AGZY83-a和Anip-073中的差异表达,并进一步检测BRI基因在另外6个非小细胞肺癌细胞系(SPC-A-1,A549,95D,TKB-18,GLC-82,PAa)及30例临床肺癌标本中的表达情况。结果 BRI基因在2细胞系中存在明显的差异表达,在具有高转移潜能的Anip973中高表达。在其他6个细胞系中BRI的表达与肺癌转移潜能可能有关。同时发现与同一病例的正常肺组织相比,BRI基因在肺癌组织中高表达,其过表达在有淋巴结转移的肺癌为6／8,无转移者为45．5％(10／22),并且BRI基因在肺癌与正常肺组织之间存在转录本的差异。结论 BRI基因在肺癌中表达上调,其1．6kb转录本表达的增加可能与肺癌的发生和转移能力的形成有关。     

异丙酚下调PKM2抑制人肺腺癌细胞系增殖、迁移和侵袭

目的探讨异丙酚对肺腺癌细胞增殖、迁移和侵袭的影响。方法 MTT法检测异丙酚(60、100、120μmol/L)处理的人肺腺癌细胞系A549、Anip973的抑制率或增殖;将si-NC组(转染si-NC)、si-PKM2(丙酮酸激酶M2)组(转染si-PKM2)、pc DNA3. 1组(转染pc DNA3. 1)、pc DNA3. 1-PKM2组(转染pc DNA3. 1-PKM2)用脂质体法转染至Anip973细胞,部分组用120μmol/L异丙酚处理;Transwell小室法检测细胞迁移和侵袭;RT-qPCR检测细胞中PKM2的mRNA的表达;Western blot检测细胞中PKM2、E-cadherin、MMP-2的蛋白表达。结果异丙酚(60、100和120μmol/L)呈浓度依赖性抑制人肺腺癌细胞A549、Anip973增殖(P <0. 05),Anip973细胞对异丙酚的敏感性较强,最适浓度为120μmol/L;异丙酚可抑制Anip973细胞迁移和侵袭,并下调PKM2、MMP-2蛋白表达,上调E-cadherin蛋白表达;敲减PKM2具有与异丙酚相同的抑制Anip973细胞的增殖、迁移和侵袭作用,下调MMP-2蛋白表达,上调E-cadherin蛋白表达的作用;过表达PKM2可减轻异丙酚对Anip973细胞增殖、迁移和侵袭及E-cadherin、MMP-2蛋白表达。结论异丙酚可抑制肺腺癌细胞系的增殖、迁移和侵袭,其机制与下调PKM2表达相关。     

组织型纤溶酶原激活剂基因转染AGZY83-a细胞移植入小鼠体内的表达研究

目的 探讨人组织型纤溶酶原激活剂（tissue-plasminogen activator,t-PA)基因转染AGZY83－a细胞（AGZY83－a/tPA)在动物体内表达的稳定性，以及通过细胞移植方法进行t-PA基因治疗的可行性。方法 将转染t-PA cDNA的AGZY83－a细胞在G418的严格筛选下体外培养，收集细胞制成悬液，注射入小鼠体内不同部位，定期检测小鼠血浆中t-PA的含量。结果 移植后，小鼠血浆中t-PA的含量与移植前及对照组比较差异有显著性（P＜0．01），并且至少可持续105天。其中腹腔注射组表达水平最高，皮下注射组次之，两者均明显高于股四头肌注射组。结论 转t-PA基因AGZY83－a细胞移植于小鼠体内可稳定表达t-PA，并且通过腹腔和皮下植入效果好于骨骼肌植入，为临床通过细胞移植进行t-PA基因治疗提供了初步的实验依据。     

Multidrug resistance-associated protein 3 and Bcl-2 contribute to multidrug resistance by vinorelbine in lung adenocarcinoma

Although cancer cells initially respond to vinorelbine (NVB), the acquisition of resistance to the treatment is the main cause of chemotherapeutic failure in lung cancer. The intrinsic mechanism of drug resistance induced by NVB in lung cancer is not clear and tumor cell models to study NVB resistance have not been widely studied. We previously established a NVB resistant cell line, Anip973/NVB, derived from the Anip973 lung adenocarcinoma cell line. The aim of this study was to investigate the molecular mechanisms involved in the resistance to NVB in lung adenocarcinoma. Genetic profiles of Anip973/NVB and Anip973 cells were compared by microarray analysis and qRT-PCR. Tumor xenografts were obtained by grafting Anip973/NVB and Anip973 cells into nude mice and the xenograft response to NVB or control treatment was evaluated. Morphological assessment of xenograft tissues was performed by transmission electron microscopy (TEM). Immunohistochemistry (IHC) was used to compare Bcl-2 and MRP3 protein expression in xenografts. Fifty-five up-regulated genes and 88 down-regulated genes in Anip973/NVB cells compared with Anip973 cells were identified by cDNA microarray analysis. Up-regulation of MRP3 and Bcl-2 was confirmed by qRT-PCR. NVB inhibits xenografts of Anip973 growth but did not affect xenografts of Anip973/NVB growth. Ultrastructural changes observed by TEM showed that NVB induces apoptosis in the Anip973-treated group but not in the Anip973/NVB-treated group. Higher expression rates of Bcl-2 and MRP3 were observed in Anip973/NVB xenograft cells compared with Anip973 xenograft cells by IHC. In conclusion, in the present study, we identified a set of genes responsible for multidrug resistance in Anip973/NVB cells. Among them, MRP3 and Bcl-2 may participate in lung adenocarcinoma multidrug resistance induced by NVB.     

RAB5A反义RNA对人肺腺癌细胞系侵袭转移影响的体外研究

目的　已知RAB5A基因过表达与人非小细胞肺癌恶性表型形成相关,本研究在此基础上进一步探 讨该基因过表达在人肺腺癌恶性演进中的作用。　方法　将RAB5A反义表达载体稳定转染入高浸润人肺腺癌细 胞系L18和高转移人肺腺癌细胞系95D中,采用重组基底膜侵袭、与基底膜成分黏附能力测定、趋化运动能力测 定、明胶酶分泌与活性检测等体外实验方法观察转染前后细胞生物学特性的改变。　结果　RAB5A反义RNA稳 定转染后L18细胞趋化运动和侵袭基底膜能力显著降低(P<0.05),明胶酶活性检测发现转染后细胞MMP 2的分 泌能力明显减弱;稳定转染后95D细胞趋化运动和侵袭基膜能力显著降低(P<0.05)。　结论　体外实验显示, RAB5A基因过表达对肿瘤细胞的趋化运动能力和侵袭重组基底膜能力起重要作用,进一步提示RAB5A基因过表 达在人肺腺癌的侵袭转移过程中发挥一定作用。     

肺腺癌细胞微环境及转移与黏附分子表达的关系

目的研究肺腺癌细胞生长环境及转移性与黏附分子CD44v6和CD29的表达关系。方法将起源相同、转移性不同的两个肺腺癌细胞系AGZY和Anip分别用简便肿瘤多细胞球体(MTS)培养法培养，并设常规单层贴壁细胞培养对照。通过倒置显微镜、扫描及透射电镜观察MTS形成情况，并用免疫组化法分别对MTS及贴壁细胞上CD44v6和CD29表达进行检测。结果MTS培养成功，贴壁细胞与MTS在细胞结构及细胞连接结构上相似，两种MTS在形态及结构上差异无显著性。免疫组化结果显示，CD29在高转移性的Anip细胞及其MTS上呈阳性表达；在低转移性的AGZY细胞及其MTS上阴性表达。CD44v6在Anip和AGZY细胞及MTS上均呈阳性表达，差异无显著性。贴壁细胞与MTS上两种黏附分子表达均无差异。结论成功建立了一种简易制备MTS的方法。细胞生长方式(单层贴壁与MTS)可能不影响CD44v6和CD29的表达。CD29表达可能与肺腺痛转移性相关；CD44v6表达可能与肺腺癌转移无关。     

Cytogenetic studies of four human lung adenocarcinoma cell lines

G-banded karyotypes were analyzed on four human lung adenocarcinoma cell lines, AGZY83-a, LTEP-a1, LTEP-a2, and GLC-82. More than 50 cells were counted and 20-40 cells were karyotyped for each cell line at different passages. The chromosome numbers of cell line AGZY83-a, LTEP-a1, LTEP-a2, and GLC-82 were 53-69, 100-110, 51-56, and 61-63, respectively. Each cell line had a number of markers involving complex chromosome rearrangements. Chromosomal analysis at different passages showed the stability of these cell lines in vitro. It was revealed that the marker chromosomes involving deletions or rearrangements of the short arm of chromosome #1 were present in all the four cell lines with breakpoints at 1p12-1p22.     

野生型p53基因诱导凋亡相关基因ANNEXIN A2的表达研究

目的探讨胡基因过表达介导不同转移潜能肿瘤细胞的凋亡分子机理，寻找与细胞凋亡和肿瘤转移的相关基因。方法应用mRNA差异显示方法分析腺病毒介导的野生型,053基因感染不同转移潜能的肺癌细胞系前后存在的表达差异基因，并用逆转录一聚合酶链反应、Northern印迹和Western印迹方法加以验证。结果分析发现，在柳基因诱导后ANNEXIN A2基因的表达显著差异有统计学意义，经加基因诱导后ANNEXIN A2基因的表达有明显下调。并且以Arfip 973细胞的表达缺失最为显著。经逆转录一聚合酶链反应、Northern印迹和Western印迹方法也验证了这种差异的存在。结论表明ANNEXIN A2基因的表达与p53基因诱导的肿瘤细胞凋亡存在密切的相关性。     

Metastatic patterns of lung cancer visualized live and in process by green fluorescence protein expression

We demonstrate here the visualization of human lung cancer metastasis live and in process in nude mice by green fluorescent protein (GFP) expression. The human lung adenocarcinoma cell line Anip 973 stably transfected with the humanized GFP-S65T cDNA was selected for very bright green fluorescence. GFP-transfected lung cancer cells were initially inoculated subcutaneously in nude mice. Five weeks after transplantation, the resulting tumor had reached over 1 cm in diameter and had very bright GFP fluorescence. Fragments of subcutaneous tumor were implanted onto the visceral pleura of the left lung of nude mice by surgical orthotopic implantation (SOI) of histologically-intact tissue via transverse thoracotomy. The ipsilateral resulting tumor was highly fluorescent due to GFP expression. GFP expression allowed the visualization of the advancing margin of the ipsilateral tumor into the fresh normal lung tissue. Lymphogenous and direct-seeding metastases in the pulmonary hilum, cervical lymph nodes, the mediastinum and contralateral pleural cavity and contralateral lung in the SOI-treated mice were brightly visualized by GFP expression in fresh tissue. GFP-transfected and untransfected tumor had similar metastatic characteristics suggesting that GFP expression had no effect on metastasis itself. The results with the GFP-transfected tumor cells, combined with the use of SOI, demonstrate a fundamental advance in the visualization and study of lung cancer metastasis in process.     

p21活化激酶4在非小细胞肺癌中的表达及其临床意义

目的:探讨p21活化激酶4(PAK4)在人非小细胞肺癌细胞系及人非小细胞肺癌组织中的表达及其临床意义。方法:采用Western blot及实时荧光定量PCR检测人支气管上皮(HBE)细胞、非小细胞肺癌细胞(A549、NCI-H520、NCI-H460和NCI-H596细胞)、20例新鲜非小细胞肺癌组织及相应的癌旁组织中PAK4的表达情况;采用免疫组化检测210例非小细胞肺癌组织PAK4的表达情况;采用Kaplan-Meier法评估非小细胞肺癌患者术后5年生存率;用Cox比例风险模型分析PAK4对患者预后的影响。结果:非小细胞肺癌细胞(A549、NCI-H520、NCI-H460和NCI-H596细胞)PAK4蛋白及mRNA表达均显著高于HBE细胞(P0.05);20例非小细胞肺癌组织中PAK4蛋白及mRNA表达高于癌旁组织PAK4蛋白及mRNA表达(P0.05);10例转移性非小细胞肺癌组织PAK4 mRNA表达显著高于10例原发性非小细胞肺癌组织(P0.05);免疫组化染色结果示210例非小细胞肺癌PAK4评分显著高于对应的癌旁组织。临床资料分析显示PAK4蛋白表达与非小细胞肺癌的分化程度、淋巴结转移、远处转移及临床分期有关(P0.05);PAK4高表达组患者5年生存率显著低于PAK4蛋白低表达组患者(logrank检验,P0.05),PAK4蛋白表达是非小细胞肺癌患者的独立预后因素。结论:PAK4蛋白高表达是非小细胞肺癌患者死亡的独立危险因素。     

cDNA cloning of a new member of the Ras superfamily, RAB9-like, on the human chromosome Xq22.1–q22.3 region

Members of the RAB protein family regulate vesicular trafficking and reside in specific intercellular compartments. A new member of the RAB family was identified through a public database search, and its full-length cDNA was isolated from a human fetal brain cDNA library. The predicted protein product of the gene consists of 201 amino acid residues, and the protein has 86% similarity to human RAB9 at the amino acid level. We designated the new gene RAB9-like. Northern blot analysis showed that the gene was transcribed ubiquitously in various human tissues. A database search revealed that the gene is divided into three exons and spans approximately 7.2 kb of the genome DNA of chromosome Xq22.1–q22.3 region. Received: March 10, 2000 / Accepted: August 4, 2000     

人肺癌中OPB7-1 cDNA片段的克隆定位

目的：寻找肺腺癌发生及转移的相关基因，探讨肺癌发生发展的分子机理。方法：应用细胞培养、逆转录－PCR、RH基因定位及RNA原位杂交方法研究确定肺癌相关基因。结果：OPB7－1 cDNA片段，经RH基因定位方法定位于1p31-1p34。在正常人肺cDNA文库、低转移能力肺腺癌细胞系AGZY83－a cDNA中存在的片段大小一致，但序列中存在个别碱基的差别。OPB7－1 cDNA片段（974bp）与GenBank中已知序列同源性差，但与其有同源性的3个毗连群克隆均位于1号染色体上（1p31-1p34）。24例临床病例分析发现，该片段在肺癌组织中均有不同程度的表达，在正常对照组织中未见明显表达，且在有淋巴结转移病例的肺癌组织中表达程度呈明显增高的趋势。结论：提示OPB7－1可能为新的基因，与肺癌的发生、转移可能有一定的相关性。