抑癌基因PTEN与细胞凋亡关系的研究进展

PTEN是近期发现的一种新型肿瘤抑制基因,现已证明PTEN基因是第一个具有双特异性磷酸酶活性的抑癌基因,不仅参与细胞周期的调控,而且调节细胞的正常生长和发育,最终促进细胞凋亡。PTEN基因是继p53之后,另一个在肿瘤细胞中突变与缺失较为广泛,与细胞的生长及凋亡关系密切的抑癌基因,现就其与细胞凋亡的研究进展作一综述。     

端粒与寿命

正常细胞是有寿命的,每分裂一次,端粒缩短一截,当没有端粒时,细胞就寿终正寝了。癌基因是引起癌症的基因,抑癌基因是抑制癌症的基因,所有基因都以英文命名。比如癌基因有ras、raf、neu等,抑癌基因有p53、Rb、BRCA1等。抑癌基因是监督其他基因的,又叫分子警察。比如一个广场,平时有几个警察维持秩序就行了,但如果出现了暴乱,则必须增加警力。     

端粒与寿命

正正常细胞是有寿命的,每分裂一次,端粒缩短一截,当没有端粒时,细胞就寿终正寝了。癌基因是引起癌症的基因,抑癌基因是抑制癌症的基因,所有基因都以英文命名。比如癌基因有ras、raf、neu等,抑癌基因有p53、Rb、BRCA1等。抑癌基因是监督其他基因的,又叫分子警察。比如一个广场,平时有几个警察维持秩序就行了,但如果出现了暴乱,则必须增加警力。     

INK4a/ARF基因与乳腺癌的关系

INK4a/ARF是直接调控细胞周期并抑制细胞增殖的抑癌基因,本文就INK4a/ARF基因以及p53与乳腺癌的关系作一综述。     

p53-EGFP表达载体的构建及其对A549的影响

构建具有EGFP标签的抑癌基因p53表达载体,并验证其对肺癌细胞A549的影响。以质粒pcDNA3.1-p53为模板,设计引物并进行PCR扩增获得目的基因p53,将获得的目的基因克隆至载体pcDNA3.1-EGFP上,用琼脂糖凝胶电泳检测目的条带,双酶切以及测序验证。将克隆好的载体,利用脂质体法转染A549细胞,荧光显微镜下观察,同时用MTT法测不同时间段细胞OD值。结果表明,琼脂糖凝胶电泳结果显示,目的基因扩增成功。双酶切及DNA测序结果均证实,成功构建了载体pcDNA3.1-EGFP-p53。转染A549细胞后,荧光显微镜下观察到绿色荧光。MTT法测定结果表明,目的基因转染的A549细胞组所测得的OD值明显低于对照组(p0.01)。因此,具有EGFP标签的抑癌基因p53表达载体pcDNA3.1-EGFP-p53构建成功,转染A549细胞后,对A549细胞具有一定抑制作用。为后续继续研究p53-MDM2生物发光能量共振转移作用奠定了基础。     

突变型抑癌基因p53在鸡马立克氏病肿瘤组织中的表达研究

[目的]探讨抑癌基因p53在鸡马立克氏病的发生与发展中的作用和意义。[方法]采用免疫组织化学和细胞化学技术,观察抑癌基因p53在鸡马立克氏病肿瘤组织及体外培养感染马立克病毒的鸡胚成纤维细胞中的表达。[结果]在体外培养的鸡胚成纤维细胞(CEF)感染马立克氏病毒后,感染的CEF呈阳性。在马立克氏病的发生过程中,突变型抑癌基因p53在病鸡的肝脏、肾脏、肿瘤、心脏、脾脏、肺脏、胸腺及法氏囊中均可检出中等量的表达。p53发生突变时所产生的p53蛋白突变体的半衰期比未发生突变时要长。[结论]突变型p53基因具有直接转化细胞和阻碍野生型p53发挥作用的特点,致使突变的DNA得以积累,最终导致癌变的发生。     

中国肝细胞癌p53基因热点突变R249S的载体构建及其在细胞中的表达

抑癌基因p53突变是人肝细胞癌（Hepatocellular carcinoma，HCC）中常见的现象，中国江苏启东地区的HCC患者中p53基因的第249位密码子突变AGG→AGT/Arg→Ser（R249S）被认为是热点突变。本研究以人肝cDNA为模板，扩增人全长p53基因，将其克隆到pCMV-Myc载体中。利用定点突变引物以pC-MV-p53为模板，构建R249S定点突变体pCMV-R249S，转染p53缺陷型的H1299细胞系。Western Blot检测表明，克隆在pCMV-Myc载体中的p53基因和249位密码子热点突变的p53基因都可以在H1299细胞系中表达。     

p53与结肠直肠癌的发生、转移、治疗与预后及筛查关系的研究现状

p53是目前公认的重要抑癌基因,在大肠癌中表达率较高,与大肠癌的发生发展等各方面关系密切。本文阐述了p53与大肠癌发生转移、治疗、预后和筛查关系的研究现状。     

p16、p53基因蛋白在胃粘膜肠上皮化生与胃癌组织中的异常表达

目的:探讨抑癌基因与胃癌发生的关系及其在胃癌早期诊断中的价值.方法:采用免疫组化s p法,检测p16、p53突变蛋白在不同类型的肠上皮化生和不同组织类型的胃癌组织中异常表达.结果:p16、p53突变蛋白在不同类型的肠上皮化生和胃癌组织中的表达差异显著.结论:p16、p53蛋白突变与肿瘤细胞的增殖、分化有密切关系,可作为胃癌早期诊断的细胞生物学标志.     

p53信号通路在MTB感染肺泡Ⅱ型上皮细胞系A549中的免疫调控作用

【目的】探讨p53通路在结核分枝杆菌(MTB)感染肺泡Ⅱ型上皮细胞(AECⅡ)中的免疫调控作用。【方法】将供试的p53基因过表达和干扰载体转染293T细胞,对其作用效果进行验证。利用脂质体分别转染p53基因过表达和干扰载体到AECⅡ细胞系A549中,建立p53基因过表达和干扰A549系模型细胞。用牛结核分枝杆菌减毒株(BCG)分别感染未经任何处理的A549细胞(感染对照组)及模型细胞,以未感染BCG的各类A549细胞为参照。以β-actin为内参基因,通过实时荧光定量PCR和Western blot来分析信号分子p53、p300、NF-κB、TLR-4、TRAF6在mRNA和蛋白水平的表达,以及炎症细胞因子TNF-α、IFN-γ、IL-6和IL-8的mRNA表达情况。【结果】过表达载体plRES2-EGFP-p53 WT和干扰载体TP53-RNAi(2648)作用效果明显,成功建立了p53基因过表达和干扰的A549细胞模型。BCG感染的对照组、p53基因过表达组和干扰组的A549细胞中,p53、p300、NF-κB、TLR-4、TRAF6在mRNA和蛋白水平的表达显著或极显著高于相应的BCG未感染组,其中p300表达与p53基因表达量呈正相关,NF-κB、TLR-4、TRAF6表达与p53基因表达量呈负相关;BCG感染可引起TNF-α、IFN-γ、IL-6和IL-8的显著表达,且与p53基因表达量呈负相关。【结论】BCG感染A549细胞时,p53信号通路中的p53协同p300来抑制NF-κB、TLR-4和TRAF6的活化及负调控TNF-α、IFN-γ、IL-6和IL-8的分泌,从而抵抗MTB的侵染。     

单细胞中P53和Mdm2蛋白活性的不同振动行为研究

综述了p53基因及Mdm2基因的生物学特征,介绍了P53-Mdm2网络。     

果蝇p53基因的克隆及序列分析

从紫外刺激的果蝇(Drosophila melanogaster)抽提总RNA,通过RT-PCR方法扩增得到有完整读码框的p53基因,并且通过对p53基因的序列分析表明,所得到的序列与网上已经登陆的果蝇p53序列有99.4%的相似系数,而与其他生物的p53序列存在一定的分歧。系统进化树的分析表明,7个物种来源的p53基因起源于同一个祖先分子,且同属于一个有相似功能作用的蛋白家族。但由于长久以来的进化发展,p53基因在不同的物种内发生了一定的突变,并延续了这种变异。     

斑马鱼p53基因原核表达载体的构建及其在大肠杆菌中的表达

利用原核表达系统克隆表达斑马鱼p53基因。RT-PCR法从斑马鱼胚胎中扩增获得p53基因编码区,并将其克隆至原核表达载体pET28a上,构建重组质粒pET28a/z-p53,将重组质粒转化E.coli BL21(DE3)受体菌,IPTG诱导表达,表达产物经镍柱纯化、尿素透析复性,SDS-PAGE电泳分析,结果表明,p53基因在大肠杆菌中成功表达,表达的p53融合蛋白分子量大约为53kD,透析复性后获得了高纯度可溶性的p53蛋白。     

海岛棉GbWRKY53基因的克隆与表达分析

黄萎病被称为棉花的"癌症",挖掘和鉴定抗病相关基因是棉花抗黄萎病分子育种的基础。WRKY转录因子在植物对生物和非生物胁迫应答过程中起重要调控作用。从高抗黄萎病品种海岛棉Pima90-53中克隆了WRKY转录因子Gb WRKY53,其开放阅读框(open reading frame,ORF)为999 bp,编码332个氨基酸,含有1个WRKY结构域及HXC型锌指结构,属于WRKY转录因子家族的第Ⅲ组。基因结构分析表明,Gb WRKY53基因有3个外显子和2个内含子。系统进化分析表明,Gb WRKY53与雷蒙德氏棉(Gossypiumr aimindii)Gr WRKY53的亲缘关系最近。当黄萎病菌诱导后,Gb WRKY53基因表达量呈先增加后降低的变化趋势,处理后12 h其表达量达到最大;水杨酸(SA)诱导后,Gb WRKY53表达量在8 h明显增加,且一直持续增高到24 h;而茉莉酸甲酯(MeJA)诱导后,Gb WRKY53表达量在12 h达到最高值随后下降,且其表达水平明显低于同时间水杨酸诱导后的表达量。1-氨基环丙烷-1-羧酸(ACC)诱导后,Gb WRKY53基因表达量仅微量上调。由此推断,Gb WRKY53基因可能参与了复杂的植物信号途径并在棉花抗黄萎病菌胁迫应答中起重要作用。     

Genetic mechanisms of tumor suppression by the human p53 gene

Mutations of the gene encoding p53, a 53-kilodalton cellular protein, are found frequently in human tumor cells, suggesting a crucial role for this gene in human oncogenesis. To model the stepwise mutation or loss of both p53 alleles during tumorigenesis, a human osteosarcoma cell line, Saos-2, was used that completely lacked endogenous p53. Single copies of exogenous p53 genes were then introduced by infecting cells with recombinant retroviruses containing either point-mutated or wild-type versions of the p53 cDNA sequence. Expression of wild-type p53 suppressed the neoplastic phenotype of Saos-2 cells, whereas expression of mutated p53 conferred a limited growth advantage to cells in the absence of wild-type p53. Wild-type p53 was phenotypically dominant to mutated p53 in a two-allele configuration. These results suggest that, as with the retinoblastoma gene, mutation of both alleles of the p53 gene is essential for its role in oncogenesis.     

Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas

Previous studies have demonstrated that allelic deletions of the short arm of chromosome 17 occur in over 75% of colorectal carcinomas. Twenty chromosome 17p markers were used to localize the common region of deletion in these tumors to a region contained within bands 17p12 to 17p13.3. This region contains the gene for the transformation-associated protein p53. Southern and Northern blot hybridization experiments provided no evidence for gross alterations of the p53 gene or surrounding sequences. As a more rigorous test of the possibility that p53 was a target of the deletions, the p53 coding regions from two tumors were analyzed; these two tumors, like most colorectal carcinomas, had allelic deletions of chromosome 17p and expressed considerable amounts of p53 messenger RNA from the remaining allele. The remaining p53 allele was mutated in both tumors, with an alanine substituted for valine at codon 143 of one tumor and a histidine substituted for arginine at codon 175 of the second tumor. Both mutations occurred in a highly conserved region of the p53 gene that was previously found to be mutated in murine p53 oncogenes. The data suggest that p53 gene mutations may be involved in colorectal neoplasia, perhaps through inactivation of a tumor suppressor function of the wild-type p53 gene.     

Identification of p53 gene mutations in bladder cancers and urine samples

Although bladder cancers are very common, little is known about their molecular pathogenesis. In this study, invasive bladder cancers were evaluated for the presence of gene mutations in the p53 suppressor gene. Of 18 tumors evaluated, 11 (61 percent) were found to have genetic alterations of p53. The alterations included ten point mutations resulting in single amino acid substitutions, and one 24-base pair deletion. In all but one case, the mutations were associated with chromosome 17p allelic deletions, leaving the cells with only mutant forms of the p53 gene products. Through the use of the polymerase chain reaction and oligomer-specific hybridization, p53 mutations were identified in 1 to 7 percent of the cells within the urine sediment of each of three patients tested. The p53 mutations are the first genetic alterations demonstrated to occur in a high proportion of primary invasive bladder cancers. Detection of such mutations ex vivo has clinical implications for monitoring individuals whose tumor cells are shed extracorporeally.