转移外源性野生型p53基因治疗人类恶性肿瘤的实验研究

野生型ｐ５３基因是一个重要的肿瘤抑制基因，人类多种恶性肿瘤的发生都与ｐ５３基因的突变有关。近年来国外不少实验室试图应用基因转移技术将外源性野生型ｐ５３基因转移到人类恶性肿瘤细胞系中以抑制它们的肿瘤生物学特性。已的研究结果显示转移外源性野生型ｐ５３基因能对多种恶性肿瘤细胞系产生增殖抑制或诱导细胞编程死亡的作用。其中虽有许多确切的机理尚未完全明了，但该项工作的前景将是令人乐观的。     

野生型p53基因脂质体转染细胞疫苗的制备

目的：制备具有治疗活性的野生型Ｐ５３基因真核表达载体的细胞疫苗。方法：利用ＲＴ－ＰＣＲ从人外周血淋总ｍＲＮＡ中扩增出全长的野生型Ｐ５３基因,将其克隆入ＴＡ克隆载体的经扩增后,将ｐ５３ｃＤＮＡ连入ＰＣＭＶ真核表达载体中,利用ｌｉｐｏｆｅｃｔａｍｉｎｅ将其转染ＤＵ１４５细胞并通过免疫组化的方法检测其表达情况。结果：Ｐ５３基因可在ＤＵ１４５细胞中稳定表达。结论：制备了可稳定表达可ＤＵ１４５细胞中的野生型     

p53基因研究新进展

肿瘤抑制基因在肿瘤发生过程中有重要作用,其中P~(53)基因最引现人注目。P~(53)基因的突变与人类一半以上的肿瘤发生相关。P~(53)基因参与细胞生长的负调控。研究发生P~(53)基因编码的P~(53)蛋白可通过调控Cipl基因表达而调控细胞生长。即P~(53)蛋白可刺激Cipl基因产生分子量为21kD的蛋白,这种蛋白能够有效抑制某些促使细胞通过细胞周期进入有丝分裂的酶活性,从而抑制细胞生长。这就解释了为什么正常的P~(53)基因的缺失可导致细胞的无限生长。此外,P~(53)基因与DNA损伤药物诱导的细胞凋亡有关,与非DNA损伤药物诱导的细胞凋亡无关。     

野生型p53对肺癌细胞生长的抑制作用

目的　探讨外源性野生型p5 3对肺癌细胞生长的抑制作用 .方法　通过向肺癌细胞中分别转染携带有荧光蛋白基因的野生型P5 3基因和突变型P5 3基因 ,而后用荧光显微镜观察荧光蛋白表达 ;中性红和MTT染料摄入法测定细胞生长活性 .结果　p5 3蛋白与绿色荧光蛋白之融合蛋白表达定位在胞浆和胞膜 ;经中性红和MTT两种方法测定 ,外源性野生型p5 3对肺癌细胞生长具有抑制作用 ;OD值分别为 0 .71± 0 .0 81(OD540 )和 0 .6 5± 0 .0 5 9(OD490 ) ,均低于对照组 (p <0 .0 5 ) .结论　p5 3外显子 5～ 8可在肺癌细胞中获得表达并对细胞生长活性有抑制作用 .     

p53研究的新进展

肿瘤抑制基因p53是目前研究最为广泛和系统的抑癌基因之一。野生型p53(wt-p53)参与了DNA损伤修复、细胞周期调控、细胞凋亡及抑制血管生成等过程。p53基因的突变使上述功能丧失,从而导致肿瘤的形成。在大约50％人类肿瘤中可发现p53基因的突变,且几乎可见于各种类型的肿瘤细胞中。本文就目前p53研究及相关基冈治疗的进展作一综述。     

野生型p53基因在人肝癌细胞的表达及诱导其凋亡

目的:探讨外源性野生型p53基因(wt-p53)对人源性肝癌细胞系生物学的影响。方法:用脂质体转染法将真核表达质粒p53-pcDNA3转染人源性肝癌细胞系HepG2中,用G418筛选细胞;用生物素标记p53的cDNA探针,通过RNA原位杂交方法,检测p53-pcDNA3在细胞中的表达;用流式细胞术(FCM)检测细胞的凋亡指数。结果:G418筛选出了阳性转染细胞;RNA原位杂交显示HepG2的胞质成棕黄色,证明了p53的表达;FCM检测表明,转染p53-pcDNA3的HepG2细胞,其增殖能力下降,细胞凋亡指数由转染前的10.03%上升为54.17%。结论:外源性wt-p53可通过脂质体转染法在HepG2细胞中成功表达,并诱导其发生凋亡,有较好的临床应用前景。     

乳腺癌中p53基因缺失及p53蛋白的异常表达

乳腺癌中ｐ５３基因缺失及ｐ５３蛋白的异常表达郭文斌张嘉庆杨德启乔新民张庆广阚秀虞有智一、材料与方法１．材料来源：４７例乳腺癌及正常腺体均来自我院乳腺中心１９９４～１９９６年间手术切除标本。全部病例均有病理学诊断。２．Ｓｏｕｔｈｅｒｎ杂交：用酚、氯仿／...     

p53 肿瘤抑制基因研究进展

肿瘤抑制基因（又称抑癌基因或抗癌基因）的失活与癌基因的激活参与人类各种肿瘤的形成和发展。肿瘤是细胞的遗传突变，连续的多步遗传变化是细胞癌变的中心问题，原癌基因突变、重排、移位和扩增成为细胞癌基因，具有转化致瘤潜能；抑癌基因阻遏转化致瘤，是能够抑制细胞...     

p53基因结构与功能研究的新进展

ｐ５３蛋白的信号区有１个ＳＨ－３区结合部位,非专一ＤＮＡ结合区可促进顺序专一的ＤＮＡ结合区与ＤＮＡ损伤部位结合。ｐ５３蛋白可整合细胞对各种危急情况的反应,其信息传递途径上游是各种危急状况,有ＡＴＭ类的基因起作用。ｐ５３蛋白通过靶基因ｐ２１ＷＡＦ－１及ＧＡＤＤ４５调控Ｇ１关卡,也参加了Ｇ２／Ｍ关卡的调控;通过上调靶基因Ｂａｘ、ＩＧＦ－ＢＰ－３,抑制ＢｃＬ－２基因表达,调控凋亡;维持基因组的遗传学稳定性,行使抑瘤基因功能。     

p53基因网络王国

抑癌基因p53作为多种细胞应激与细胞应答的中间环节,与其上下游的各种调控因子及相 关基因共同构成了一个错综复杂的信号传导通路。上游:DNA损伤、缺氧、原癌基因激活、核苷酸衰竭、 端粒缩短等因素均可激活p53的功能网络;中游:p53的功能活性受mdm2(mouse double minus 2)、 p14ARF(alternative reading frame)、共价修饰、蛋白质与蛋白质的相互作用等多种相互联系又相互制约 的因素的调控;下游:p53肩负着介导细胞周期阻滞、促进细胞凋亡、维持基因组稳定、抑制肿瘤血管发 生等多种功能使命,在整个机体组织细胞的生长、发育、分化过程中起着举足轻重的作用。因其功能的 多样性及与肿瘤发生发展的高度相关性,p53已成为肿瘤发生机制研究及基因治疗的-个重要靶点。因 此,作为细胞内信号传递网络的核心之一,深入理解p53基因网络的各个层面将对细胞生长代谢调控 研究及肿瘤遗传学研究大有裨益。     

Cancer gene therapy by adenovirus-mediated gene transfer

Cancer arises as a direct result of genetic mutations. It therefore stands to reason that cancer should be well suited for the correction through gene therapy. Recent advances in the understanding of the molecular pathogenesis of cancer and the rapid development of recombinant DNA technology have made cancer gene therapy feasible in the clinical setting. The current efforts for cancer gene therapy mainly focus on immunogene therapy, chemogene therapy, restoration of tumor suppressor gene function, and oncolytic virus therapy. Central to all these therapies is the development of efficient vectors for gene delivery--this remains a work in progress. These vectors can be classified as viral and non-viral vectors. This paper will concentrate on viral vectors because of their practical advantages over non-viral vectors. Of the viral vectors, by far the most important are the human adenoviruses as is reflected by the enormous data and literature accumulated by studies relating to animal tumor models and clinical trials. In this review, we examine the recent progress in adenovirus-mediated cancer gene therapy with regard to cytokine gene, tumor suppressor gene, chemogene, and oncolytic adenovirus. We also discuss the current limitations of the adenoviral vector system and how they may be circumvented in future developments relating to targeted gene delivery.     

In vivo recombinant adenovirus-mediated p53 gene therapy in a syngeneic rat model for colorectal cancer

The p53 gene has a significant role in controlling genomic stability of cancer. The purpose of this study was to evaluate the tumor response of allograft colorectal tumor treated with Ad5CMV-p53 in a syngeneic rat model. Two weeks after the inoculation of WB-2054-M5 tumor cells in the flank of rats, rats were randomly assigned by tumor size to one of three groups (n=18 in each): phosphate buffered saline (PBS), Ad5CMV, and Ad5CMV-p53. Recombinant adenovirus or PBS was administered through intratumoral injection at three divided doses every other day for 4 weeks. Apoptosis of the tumors was evaluated using TUNEL assay. After 2 and 4 weeks of treatment, the volume (cm(3)) of tumors in PBS, Ad5CMV, and Ad5CMV-p53 was as follows: 2 week: 1.66 +/-0.43, 1.40 +/-0.47, 0.75 +/-0.26 (p<0.001), 4 week: 4.41 +/-0.88, 3.93 +/-1.86, 2.33 +/-0.51 (p<0.001). Tumor growth showed no statistically significant difference between the PBS and Ad5CMV groups (6-week vol. p=0.32). The TUNEL assay results revealed more apparent apoptotic cells in Ad5CMV-p53-treated tumors than in other groups. Growth of allograft colorectal cancer in the syngeneic rat model was significantly suppressed by intratumoral Ad5CMV-p53 gene therapy. These results demonstrate that gene replacement therapy with p53 may provide a novel modality of treatment in conjunction with other present treatments for metastatic colorectal cancer.     

重组腺病毒介导的p^16与p^53基因联合转移对人肺癌细胞系 …

目的 探讨ｐ＾１６和ｐ＾５３基因对肺癌细胞的协同抑制效应及凋亡诱导作用。方法 首先用同源重组技术构建重组ｐ＾１６和ｐ＾５３腺病毒载体,然后单独或联合感染人肺癌细胞系Ｈ３５８,用免疫组织法及Ｗｅｓｔｅｒｎ ｂｌｏｔ检测腺病毒介导的基因转移效率与表达水平,用克隆形成实验、原位末端标记及流式术观察它们对Ｈ３５８生长特性及凋亡的影响,结果 免疫组织化学染色结果表明重组腺病毒载体可高效地将外源基因ｐ＾５３转     

p53 protein expression in non-Hodgkin's lymphomas is infrequently related to p53 gene mutations

Accumulation of the p53 protein has been found In several types of lymphomas. However, p53 gene mutations have been infrequently demonstrated in some specific types of lymphomas. In the present study, a correlation between p53 immunoreactivity and p53 gene mutations in a large panel of non-Hodgkin's lymphoma (NHL) cases is attempted. A panel of 202 cases of NHL was evaluated by immuno-hlstochemical staining for p53 protein. All cases that were immunohistochemically positive for p53 protein were analyzed by the polymerase chain reaction (PCR) single strand conformation polymorphism (SSCP) method to identify mutations within the pS3 gene. In order to confirm the mutation, sequencing of PCR-amplified p53 gene segments was performed. Overexpression of p53 protein was found in 59 of the 202 cases of NHL, but only four of these 59 cases showed a shift on SSCP analysis, and point mutations were detected in three of them by the subsequent sequencing. p53 Immunoreactivity was generally greater in high-grade lymphoma. The results of this study suggest that Immunohistochemlcal reactivity for p53 protein is not a reliable Indicator of the presence of their structural alterations of p53 gene exons 4–9 in NHL.     

Receptor-independent augmentation of adenovirus-mediated gene transfer with chitosan in vitro

Recombinant adenovirus is one of the most widely used viral vectors for gene delivery. This study was designed to evaluate the ability of chitosan, a cationic, linear polysaccharide composed of beta(1,4) linked glucosamine partly containing N-acetyl-glucosamine, to enhance the in vitro infectivity of adenovirus to mammalian cells. Wild type and a fiber-mutant replication-defective recombinant adenoviruses expressing beta-galactosidase were used. In the latter, an RGD peptide, the binding site for alpha(v)beta3 and alpha(v)beta5 integrin, was introduced in the fiber knob enabling adenovirus receptor-independent viral infection. Enhanced effect of chitosan on the infectivity of both adenoviruses was observed in Chinese hamster ovary cells that do not express the receptor for adenovirus with beta-galactosidase activity assay and x-gal staining. These data indicate the receptor-independent mechanism(s) for this enhancement effect. In addition, we found that pH of the culture medium, and molecular mass and concentration of chitosan are also critical factors. Thus, the highest effect was obtained with 0.1-1 microg/ml of chitosan with molecular mass of 19K and 40K in the culture medium of pH 6.4; on the other hand, the effect was negligible with the higher chitosan concentrations (10 microg/ml or more), lower or higher molecular mass (11K and 110K) of chitosan, or at pH of 7.4. Studies using several cell lines with variable levels of adenoviral infectivity revealed that this enhanced effect is evident in the cells with poor infectivity to adenovirus. Since chitosan is biocompatible and inexpensive, these data indicate that chitosan may be a potential candidate for a non-viral vector to safely increase adenoviral infectivity to mammalian cells, particularly those with poor susceptibility to adenoviral infection.     

重组腺病毒介导的野生型p53对人胰腺癌细胞凋亡的诱导作用

目的研究恢复外源性野生型（ｗｔｐ）ｐ５３的表达对人胰腺癌细胞生长和凋亡的作用。方法构建了一个复制缺陷型５型腺病毒ｗｔｐ５３表达载体Ａｄ５ＣＭＶｗｔｐ５３，含有人ＣＭＶ启动子，人野生型ｐ５３和ＳＶ４０ｐｏｌｙＡ信号，经腺病毒包装细胞２９３细胞包装、扩增后，转染胰腺癌ＰＣ２细胞。ＰＣＲ和免疫沉淀技术证实转染的细胞有外源ｗｔｐ５３基因的存在和表达。结果转染的ＰＣ２细胞的生长率和３Ｈ掺入率降低。原位凋亡检测、流式细胞术和ＤＮＡ凝胶电泳显示转染细胞凋亡明显增多。而ＰＣ２细胞和用Ａｄ５ｐＸＪ转染的细胞没有这些改变。结论复制缺陷型腺病毒是一种安全高效的基因转移载体，恢复ｗｔｐ５３的表达可以有效地诱导凋亡和抑制胰腺癌细胞生长     

Transplantation tolerance by donor MHC gene transfer

Replacing the function of diseased organs by transplantation has proved to be highly successful in the past four decades. The immune system poses the most significant barrier to the long term survival of the transplanted organs. Without lifelong treatment with powerful immunosuppressive agents to keep the immune response at bay, organ grafts will invariably be rejected. However, current immunosuppressive agents are non-specific and leave transplant recipients more susceptible to opportunistic infections and tumour development. Achieving donor specific tolerance would eliminate the need for lifelong treatment with these agents and thereby, avoid the associated side effects. There have been many exciting new developments in immunopharmacology and in the understanding of the mechanisms of rejection and tolerance. These developments on their own, or in combination with the use of gene therapy techniques may allow the induction of transplantation tolerance in human recipients of allografts in the future. Pretransplant exposure to donor MHC antigens has been highly successful strategy for tolerance induction in experimental models. Pretransplant blood transfusion, and more recently administration of donor bone marrow, has been used in an attempt to prolong graft survival in human. However, using fully allogeneic donor bone marrow carries the risk of graft versus host disease. Gene therapy may allow this approach to be used without this risk. Here, we review efforts to induce transplantation tolerance by gene transfer of donor MHC genes to recipient derived cells and show that this may be a potential strategy to achieve the holy grail of organ transplantation: donor specific tolerance.     

Adenoviral-mediated transfer of p53 gene enhances TRAIL-induced apoptosis in human hepatocellular carcinoma cells

p53 is a tumor suppressor protein with numerous biological functions including transformation, regulation of cell growth, differentiation and apoptosis. The TNF-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in various transformed cell lines. We investigated the effects of combining wild-type p53 gene transduction by adenoviral infection (Ad-p53) with addition of TRAIL on cell death, expression levels of TRAIL receptors (TRAIL-R1, TRAIL-R2), FLICE inhibitory protein (FLIP) and X-linked inhibitor of apoptosis protein (XIAP) on human hepatocellular carcinoma (HCC) cell lines. HCC cell death was increased by combination of Ad-p53 infection and addition of TRAIL compared to either alone. Western blotting demonstrated decreased TRAIL-R1 and TRAIL-R2 levels after infection with Ad-p53. FLIP levels decreased in Huh7 cells and Hep3B cells, and XIAP levels decreased in all three HCC cell lines after infection with Ad-p53. Thus, death of HCC cells due to combined p53 gene transduction and exogenous TRAIL may be due to down regulation of FLIP or XIAP.