NP9基因的克隆及对细胞周期素D1转录活性的影响

背景与目的：NP9基因是我们在鼻咽癌中克隆到的一个新的表达下调的基因(GenBank登录号：BF718797),为了进一步研究该基因的功能,我们构建了人NP9基因编码区序列的真核表达质粒,并研究其表达对细胞周期素D1(cyclinD D1)的影响。方法：通过核苷酸同源序列比较(BlastN)得到NP9 EST的同源cDNA,RT-PCR扩增其编码区序列并构建真核表达重组质粒pRe／CMV2-NP9,脂质体转染后G418筛选出稳定、高效转录NP9基因的细胞克隆;再通过脂质体转染cyclinD D1 promoter Luc和NF-kB—Luc质粒到稳定表达NP9基因的细胞克隆,测定荧光素酶的活性以确定NP9基因对cyclinD D1和NF-kB转录活性的影响;同时亚克隆：NP9基因编码序列到载体pEGFP—C1中,脂质体转染细胞以确定NP9蛋白在细胞中的定位。结果：融合的绿色荧光蛋白出现于细胞核。G418抗性克隆中,RT-PCR扩增证实部分克隆表达NP9基因,且强弱不同。表达NP9基因的细胞克隆在瞬间转染cyclinD D1 promoter Luc和NF-kB-Luc 48h后,荧光素酶的活性较其对照组分别下降46％和63％。结论：NP9蛋白在细胞核内表达;NP9基因通过抑制核转录因子NF-kB的转录活性下调cyclinD D1的表达。     

Cyclin D1及PCNA与鼻咽癌放射敏感性相关性研究

[目的]探讨鼻咽癌的细胞周期素(cyclin)D1和增殖细胞核抗原(PCNA)表达与放射敏感性的关系。[方法]65例初治行放疗的鼻咽癌患者,免疫组化测定治疗前活检标本的cyclinD1和PC鄄NA表达,并与放射敏感性(放疗后的近期疗效)进行单因素和多因素的分析研究。[结果]cyclinD1阳性表达率为60%,PCNA表达率为52.3%。放射敏感者(CR)40例(61.5%),放射低敏者(PR和MR)25例(38.5%)。cyclinD1阳性表达和PCNA高表达的CR率分别为74.4%和82.4%,明显高于阴性表达及低表达者(CR率分别为42.3%和38.7%)(P<0.01)。各期中cyclinD1阳性与阴性表达及PCNA高与低表达者疗效为CR的差异有显著性(P<0.01)。Logistic多因素回归分析显示明显影响放疗效果的因素依次为:PCNA,临床分期,原发肿瘤大小,病人年龄和性别。[结论]cyclinD1阳性表达及PCNA高表达的鼻咽癌放射敏感性高,在作为预测放疗敏感性的指标上,PCNA可能较cyclinD1更有意义,为个体化的治疗方案增添了重要参考价值的生物学指标。     

鼻咽癌相关基因NAG7对鼻咽癌细胞周期及凋亡的影响

背景与目的NAG7基因是我室克隆的鼻咽癌相关的肿瘤抑制候选基因,其功能与作用机制目前尚不清楚.研究鼻咽癌相关的潜在抑瘤基因NAG7对鼻咽癌细胞系(HNE1)细胞周期和细胞凋亡的影响,并探讨其作用机制.方法采用脂质体转染技术将NAG7基因导入HNE1细胞,建立稳定表达NAG7的细胞株,Northernblot分析转染细胞NAG7基因的表达,并采用流式细胞技术检测细胞周期、细胞周期素及细胞凋亡的改变,并用westernblot验证.结果NAG7基因重表达的HNE1细胞与空载体转染的HNE1和HNE1细胞相比G0/G1期细胞数增加(P<0.05),S期细胞数减少;细胞凋亡数目增加(P<0.05);细胞周期素A、D1、E表达明显降低(P<0.05),细胞周期素B1表达降低,Westernblot检测亦证实cyclinD1和cyclinE表达明显下调.结论NAG7的重表达导致细胞周期素表达下调,从而延缓细胞经G1期进入S周期及诱导细胞凋亡增加进而抑制NPC细胞的过度增殖.     

肺癌中p16及pRB与cyclinD1和PCNA基因蛋白的表达及预后意义的评估

目的:研究抑癌基因蛋白p16、pRB、细胞周期素D1(cyclinD1)及PCNA在肺癌中的表达及与预后的关系.方法:应用免疫组织化学方法检测p16、pRB、cyclinD1及PCNA的表达,结果由COX回归模型与Kaplan-Meier单因素分析处理.结果:1)65例肺癌标本均示PCNA阳性,但表达程度不同,非小细胞肺癌(NSCLC)＞小细胞肺癌(SCLC),P＜0.05.PCNA高表达与p16-、pRB 、PT状况、远处转移呈正相关.增殖指数(PI)Ⅰ～Ⅱ级的生存时间≥24个月为81.82%,Ⅲ～Ⅳ级为18.18%,P＜0.01.2)p16阳性率为50.77%,p16的表达与腺癌分化程度有关,高分化＞中分化＞低分化,P＜0.01,与鳞癌分化无关.p16 与p16-患者生存期≥24个月的比率分别为72.73%和27.27%,P＜0.05.pRB阳性表达率为58.46%,鳞癌、腺癌较高,SCLC较低,与生存期无关.cyclinD1阳性表达率为60.00%,其中磷癌72.41%,腺癌58.33%,SCLC 25.00%,P=0.05.3)p16-/pRB 细胞增殖活性较强,生存期较短;cyclinD1 /p16-/pRB 生存期较短,增殖活性较强.结论:PCNA及p16分别是肺癌进展及预后判断的独立指标;PCNA、p16、pRB、cyclinD1联合检测对肺癌的预后评估更有价值和更客观;小细胞肺癌pRB表达的缺失具有鉴别诊断意义.     

鼻咽癌细胞中大蒜素抑制MAPK信号通路的作用机制研究

目的:探讨大蒜素导致鼻咽癌细胞G1/S期阻滞的分子机制。方法:采用MTT法测定细胞增殖活性;激酶活性分析测定MAPK激酶的活性;报道基因分析检测转录因子AP-1的转录活性;Western blot法检测细胞周期蛋白CyclinD1的表达水平。结果:大蒜素呈浓度依赖性抑制鼻咽癌细胞增殖,细胞的存活率从100%降为52.8%(50μg/mL),P=0.0068;检测50μg/mL大蒜素处理时MAPK激酶的活性,发现其活性仅为未处理组的42.92%,说明大蒜素抑制了MAPK激酶活性;基因分析显示在大蒜素作用下,转录因子AP-1的转录活性也呈浓度依赖性的降低,从6.3×103U降低到3.8×103U(50μg/mL),P=0.0089,受AP-1调节的G1/S期调节蛋白CyclinD1的相对表达水平也从100%下降为32.2%。结论:大蒜素通过抑制MAPK信号通路而抑制转录因子AP-1的活性,从而下调CyclinD1表达,导致细胞G1/S期阻滞。     

PCNA与AgNORs枯鼻咽癌组织中表达的定量研究

目的：为了增加鼻咽癌的诊断和预后判定指标。方法：应用抗增殖细胞核抗原（抗ＰＣＮＡ）单克隆抗体ＰＣ１０，采用ＳＰ免疫组化方法和ＡｇＮＯＲｓ图像定量分析技术，对有确切随访结果的７８例鼻咽鳞状细胞癌和２２例鼻咽粘膜组织标本进行了定量研究。结果：经ＰＣＮＡ阳性反应计算出的增殖指数（ＰＩ）和图像分析的ＡｇＮＯＲｓ计数及其颗粒总面积均与ＮＰＣ分化程度高度相关，随ＮＰＣ分化程度的增高而减少；ＰＣＮＡ阳性反应增殖     

EB病毒BHRF1基因表达对鼻咽癌细胞周期分布影响的研究

ＥＢ病毒感染与鼻咽癌的发生和发展明显相关。近来研究发现,ＥＢ病毒编码基因的表达与病毒介导的细胞转化与恶变有关。ＢＨＲＦ１为ＥＢ病毒编码的一个早期基因,其与原癌基因Ｂｃｌ－２具有部分相同的序列和共同的超微结构定位,提示其具有相似的功能和机理,即有抑制细...     

抑制ATM表达致鼻咽癌细胞CNE1辐射增敏的细胞周期阻滞研究

背景与目的:细胞周期调控是决定细胞辐射敏感性的决定性因素之一。共济失调毛细血管扩张症突变基因(ataxia-telangiectasia mutant,ATM)功能与细胞DNA损伤修复、细胞周期检查点调控密切相关。我们前期研究通过反义RNA抑制ATM基因表达可增加鼻咽癌细胞系CNE1辐射敏感性,本研究拟探讨其辐射增敏的细胞周期阻滞调控机制。方法:ATM反义组细胞CNE1/pDOR-atm及对照组细胞CNE1/pDOR经2Gy、5GyX线照射后不同时间点(1h、4h、8h、24h、48h)收获,应用流式细胞仪(flowcytometer,FCM)检测各细胞周期百分比及凋亡率。结果:两组细胞X线照射后均未出现明显G1期阻滞和细胞凋亡,但分别在照射后1h、4h、8h出现明显S期阻滞,24h、48h出现明显G2期阻滞,其中反义组S期细胞百分率总均数水平低于对照组(P<0.05),而G2/M期细胞百分率总均数水平高于对照组(P<0.05)。结论:反义RNA抑制ATM表达致CNE1辐射增敏的细胞周期调控机制可能与减少S期细胞比例,增加G2/M期细胞比例有关,与G1期阻滞和细胞凋亡的调控无关。     

细胞周期基因芯片筛选不同增殖状态下CNE-2细胞的差异表达基因

目的：了解不同增殖状态鼻咽癌细胞（CNE-2）细胞周期基因表达差异，探讨鼻咽癌放射治疗中加速再增殖的机理。方法：对不同增殖状态的CNE-2细胞，采用基因芯片技术、实时荧光定量PCR检测其细胞周期相关基因表达差异。结果：细胞增殖活性最高时相（2Gy连续照射第3天）与最低时相（2Gy连续照射第5天）表达有差异的基因有3条，为检测基因总数的3％（3／100），其中细胞增殖活性增高时上调基因为CCNE1、CUL-5，下调基因为CDKN1A。应用相对定量荧光实时PCR技术对部分差异表达在2倍以上的基因（CCNE1、CDKN1A））进行了mRNA水平的验证，结果显示实时PCR实验结果与芯片结果具有良好的一致性。结论：某些细胞周期基因如CCNE1、CUL-5、CDKN1A等可能参与了鼻咽癌放射治疗过程中发生的加速再增殖过程，有必要对这些基因功能作进一步研究，以探明照射过程中肿瘤细胞加速再增殖发生的分子机制。     

应用间期FISH技术探讨cyclinD1基因在鼻咽癌中的扩增

目的：检测ｃｙｃｌｉｎ Ｄ１基因在鼻咽癌（ｎａｓｏｐｈａｒｙｎｇａｌ ｃａｒｃｉｎｏｍａ,ＮＰＣ）细胞中的扩增情况,探讨ｃｙｃｌｉｎＤ１基因在ＮＰＣ发生及发展中的作用。方法：应用间期Ｍｉｃｒｏ－ＦＩＳＨ技术。结果：８３例ＮＰＣ病例可见２３例（占２７．７１％）出现ｃｙｃｌｉｎＤ１扩增;其中Ⅰ、Ⅱ期病例扩增的有５例,Ⅲ、Ⅳ期病例扩增的有１８例;２３例扩增中１６例（６９．５６％）伴有颈淋巴结转移。结论：     

The tetraspanin CD9 inhibits the proliferation and tumorigenicity of human colon carcinoma cells

The implication of the tetraspanin CD9 in cancer has received much recent attention and an inverse correlation between CD9 expression and the metastatic potential and cancer survival rate has been established for different tumor types. In contrast to the well-established role of CD9 in metastasis, very little is known about the involvement of this tetraspanin in the process of development of primary tumors. In the present study, we present evidence on the implication of CD9 in colon carcinoma tumorigenesis. We report here that ectopic expression of CD9 in colon carcinoma cells results in enhanced integrin-dependent adhesion and inhibition of cell growth. Consistently with these effects, treatment of these cells with anti-CD9-specific antibodies resulted in (i) increased beta1 integrin-mediated cell adhesion through a mechanism involving clustering of integrin molecules rather than altered affinity; (ii) induction of morphological changes characterized by the acquisition of an elongated cell phenotype; (iii) inhibition of cell proliferation with no significant effect on cell survival; (iv) increased expression of membrane TNF-alpha, and finally (v) inhibition of the in vivo tumorigenic capacity in nude mice. In addition, through the use of selective blockers of TNF-alpha, we have demonstrated that this cytokine partly mediates the antiproliferative effects of CD9. These results clearly establish for the first time a role for CD9 in the tumorigenic process.     

Overproduction of Cyclin D1 is dependent on activated mTORC1 signal in nasopharyngeal carcinoma: Implication for therapy

Activated mTOR was implicated to play a role in the carcinogenesis of nasopharyngeal carcinoma (NPC). However, the mechanism of activated mTOR/Complex1(mTORC1) signaling pathway in NPC development has not been well established. In this study, we correlated the expression of mTORC1 signal molecules and Cyclin D1 in NPC. We also investigated the effect of blocking mTORC1 signal with rapamycin and mTOR siRNA on Cyclin D1 expression in CNE-2 cells, as well as cell apoptosis and viability. We found a positive association of mTORC1 signal molecules and Cyclin D1 in NPC. Also, we found blockage mTORC1 inhibited Cyclin D1 expression in CNE-2 cells and enhanced cell apoptosis. Our results suggested that mTORC1 signal pathway might be a potential target for NPC therapy.     

鼻咽癌组织中RASSF1A基因甲基化的研究

目的观察RASSF1A基因在鼻咽癌组织和慢性鼻咽炎的甲基化情况。方法采用甲基化特异性PCR技术检测16例鼻咽低分化未角化癌和10例鼻咽黏膜慢性炎组织中RASSF1A基因的甲基化。结果RASSF1A基因的高甲基化率在鼻咽癌组织中为93．75％（15／16）,在慢性鼻咽炎组织中为0,两组病例的RASSF1A基因高甲基化率差别显著,P〈0．005。结论RASSF1A基因在鼻咽癌组织中呈高甲基化,可能是影响鼻咽癌发生发展的抑癌基因之一。     

EFFECT OF PROCYANIDIN ON CELL CYCLE IN MURINE LEWIS LUNG CARCINOMA

Fagopyrumcymosum(Trev.)Meisnisaspeciesofbuckwheat.IthasbeenusedinChinaformedicinalpurposes.Themedicaleffectcomesfromitsroots.Chineseliteraturereportsthatthisplantcan揷learlungheat?and揹isseminatepoison?implyingthatitmayhaveanti-inflammatoryandanti-tumoreffects.ItwasreportedthattheFagopyrumcymosumextractcontainsprocyanidinedimmers(B2,B4andC2),catechins,epi-catechins(EC),(-)epigallocatechin(EGCG),hecogenin,quercetin,bete-sitosterol,P-coumaricacidandferulicacid[1].SomeactiveingredientsofFa…     

Characterization of a novel epigenetically-silenced, growth-suppressive gene, ADAMTS9, and its association with lymph node metastases in nasopharyngeal carcinoma

By using a functional complementation approach, suppression of tumorigenicity was observed after transfer of intact or truncated copies of chromosome 3 into a nasopharyngeal carcinoma (NPC) HONE1 cell line. The extra exogenous chromosome 3 in the microcell hybrids (MCHs) significantly extended the lag period of tumor formation, which may be associated with loss or inactivation of wild type alleles from the normal donor chromosome 3. Representative tumors, which grew in nude mice were reconstituted into culture and expanded as tumor segregants (TSs). In our study, a disintegrin-like and metalloprotease with thrombospondin type 1 motif 9 (ADAMTS9), a gene mapping to 3p14.2, was identified to be critically associated with tumor suppression in NPC. Gene expression analysis showed that ADAMTS9 was either not expressed or was downregulated in HONE1 cells, TSs and NPC cell lines. The mechanism of ADAMTS9 gene inactivation in the NPC cell lines and tissues was attributed to promoter hypermethylation. Using a tissue microarray and immunohistochemical staining, 31 of 66 (47%) of the NPC cases showed downregulated or absence of ADAMTS9 expression. ADAMTS9 expression was downregulated or lost in 17 of 23 (73.9%) lymph node metastatic NPC specimens, which was significantly higher than in 14 of 43 (32.6%) primary tumors. After transfection of the ADAMTS9 gene into 7 NPC cell lines, a dramatic reduction of colony forming ability was observed. These findings support ADAMTS9 as a putative tumor suppressor gene in vivo in NPC that is significantly associated with lymph node metastases.     

Adenovirus-mediated intra-tumoral delivery of the human endostatin gene inhibits tumor growth in nasopharyngeal carcinoma

The growth and metastasis of nasopharyngeal carcinoma (NPC), one of the most common cancers in southern China, is closely related to neovascularization. Here, we examined whether intra-tumoral delivery of endostatin gene could lead to long-term local expression of bioactive endostatin at therapeutic levels. We constructed a recombinant adenoviral vector carrying the human endostatin gene (Ad/hEndo), which expressed high-level endostatin protein in NPC CNE-2 cells, and significantly inhibited the proliferation and migration of vascular endothelial cells in vitro. Tumor growth and angiogenesis in NPC CNE-2 xenografted tumors were significantly inhibited after 5 courses of intra-tumoral treatment with Ad/hEndo in vivo. Endostatin mRNA in tumor tissues peaked at 1-2 days after intra-tumoral administration and disappeared within 1 week, whereas the plasma endostatin protein levels peaked at 3 days after administration and lasted 2-3 weeks. The therapeutically relevant endostatin transgene expression was achieved during the course of multiple intra-tumoral administrations with Ad/hEndo. Multiple injections with adenoviral vectors did not lead to continuous increases of adenovirus neutralizing antibodies in serum. Thus, adenovirus-mediated intra-tumoral introduction of the human endostatin gene may form a viable new treatment for NPC, although readministration every 2-3 weeks may be necessary for the best effect.     

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.     

EB病毒基因BARF1在鼻咽癌细胞中的表达及意义

目的：探讨EB(Epstein—Barr)病毒新基因BARF1(BamHI A rightward open reading frame1)在人鼻咽癌组织中的表达及意义，为深入阐明EB病毒致癌机制提供实验依据。方法：提取RNA后，采用RT—PCR方法扩增标本中的EBNA1(EB virus associated nuclear antigen 1)和BARF1 mRNA，PCR产物经2％琼脂糖凝胶电泳观察并照相。结果：11例RNA合格的标本均表达EBNA1，提示病例均为EB病毒阳性病例；其中9例表达BARF1，占82％：而且9例中的7例为强阳性。结论：EB病毒新基因BARF1 mRNA在鼻咽癌细胞中高表达，这提示除了已经明确的潜伏性膜蛋白1(LMP1)以外，BARF1可能在鼻咽癌细胞恶性增殖中发挥重要作用，具体机制有待深入研究。