【作者】 赵宇；

【导师】 孙曼霁； 王玉霞；

【作者基本信息】 中国人民解放军军事医学科学院 ， 药理学， 2010， 博士

【摘要】 目前,癌症仍然是危害人类健康的主要疾病之一。临床数据表明,超过50%的肿瘤的发生均与肿瘤抑制基因P53的突变有关。P53基因是细胞中重要的肿瘤抑制基因,其编码产物p53蛋白在修复细胞基因组损伤,维持基因组稳定,以及在细胞处于极度应激条件下,发生不可逆损伤后诱导细胞凋亡,防止其由于基因变异发生恶性转化等方面发挥着重要的作用。因此,p53蛋白被称为细胞中的“基因卫士”。p53蛋白极易由于基因的突变发生功能的异常。变异的p53蛋白不仅丧失了阻止正常细胞发生癌变的作用,还由于突变所产生的异常功能(Gain of function)启动一些原癌基因的过度表达,促进细胞的恶性转化,加速肿瘤的发生和发展。因此,p53蛋白已经成为抗肿瘤治疗的热点分子。虽然目前报道了许多以p53为治疗效应分子或以其为作用靶点的生物类制剂和小分子类化合物,可以通过补充野生型p53蛋白或修复肿瘤细胞中失活的p53蛋白功能抑制肿瘤细胞的增殖,清除癌细胞,但是由于其细胞穿透性和体内运输的靶向性较差,使其抗肿瘤效果很不理想,应用也受到了极大限制。在恶性肿瘤中,90%以上属于实体瘤。实体瘤的一个显著病理生理特征是组织内缺氧,即在实体瘤内部由于肿瘤细胞增殖和新生血管形成的速度不平衡,使得其组织内部存在瘤内缺氧区域(Hypoxia)。处于该区域中的肿瘤细胞被称为缺氧肿瘤细胞(Hypoxic tumor cells)。这种细胞的特点在于对于传统的放化疗作用具有极强的抗性,不易被彻底清除,是实体瘤发生晚期转移的主要原因之一,与这些细胞内缺氧诱导因子(Hypoxia-inducible factors, HIFs)的活性特异性增强密切相关。HIFs家族包括三个主要成员HIF-1,HIF-2和HIF-3,其中HIF-1的结构和功能研究得最详细。HIF-1是由α和β两个亚基组成的异源二聚体分子。HIF-1β,在细胞中为组成型表达;HIF-1α由于其分子结构中具有氧依赖性降解结构域(Oxygen-dependent degradation domain, ODD),在常氧条件下,氧依赖性HIF-1多聚羟化酶(HIF-1 prolyhydroxylase,HIF PH)羟化其ODD核心脯氨酸(Pro 564),进而通过Von Hippel Lindau (VHL)蛋白介导的泛素-蛋白酶体途径降解;而在缺氧条件下,由于氧原子的缺失,HIF PH失活,HIF-1α亚基可以稳定存在,其可以与β亚基聚合成二聚体,发挥转录因子的作用,调节一系列下游基因的表达,使细胞适应缺氧环境的应激刺激。因此,如何有效的杀伤缺氧性肿瘤细胞是有效及彻底根治实体瘤的重要环节。此外,有证据表明,具有突变型P53基因的肿瘤细胞更能适应缺氧环境,具有更高的恶性表型,更易发生转移和侵袭。蛋白转导结构域(Protein transduction domain, PTD)是一类可以将外源性生物大分子通过跨膜转导作用导入细胞内的一类多肽,包括来自HIV-1的TAT,单纯疱疹病毒的VP22,果蝇头胸足编码序列编码的ANPT,以及其他以这些天然多肽为模板人工合成的小肽类化合物。这些肽类的共同特点是,其氨基酸序列中富含碱性氨基酸,并且可以通过与细胞膜上的某些表面分子相互作用,将本身难以通过易化扩散进入细胞的外源大分子类物质输入细胞。已有研究表明,许多生物大分子,如DNA,siRNA,大分子蛋白质,甚至纳米颗粒都可以在PTD的介导下进入细胞。这一发现为开发大分子生物治疗性药物提供了技术基础。在众多PTD中,TAT是研究最多、应用最广的一个。通过研究表明,该多肽参与跨膜转导作用的核心结构为47~57残基段。利用该区域构建的应用于研究或具有生物治疗作用的蛋白包括:TAT-EGFP,TAT-HT,TAT-p27等。实验证实,这些偶联了TAT的蛋白可以有效的进入细胞或组织,并发挥其应有的生物学活性,为研发更多的TAT融合蛋白类药物提供了可靠的实验证据。本课题以实体瘤的以上两个显著的生化生理特征为靶标,设计了一种新型的p53融合蛋白,TAT-ODD-p53,一方面可修复肿瘤细胞中突变的p53蛋白,重建正常的p53功能通路,另一方面有效的杀伤缺氧性肿瘤细胞,有效的切断实体瘤复发和转移的根源。我们利用具有将外源大分子物质导入细胞的蛋白转导结构域TAT47-57多肽,可以使蛋白具有缺氧靶向稳定性的结构域ODD557-574与人野生型p53融合,构建了TAT-ODD-p53融合基因。同时我们还构建了p53,TAT-p53和TAT-ODD-EGFP融合蛋白作为该蛋白的对照蛋白。将上述基因克隆到原核表达载体pET28a载体中,在E.coli.(BL21)工程菌中表达。将表达后的蛋白经变性,纯化和复性,得到高纯度的TAT-ODD-p53融合蛋白。我们利用免疫组织化学染色,免疫荧光染色和Western blotting的方法检测了融合蛋白在体外和体内的跨膜转运功能和缺氧靶向稳定性;利用MTT染色法,Annexin V-FITC & PI双染色经过流式细胞仪分析,测定了TAT-ODD-p53蛋白在不同氧分压下对肿瘤细胞生长的抑制活性和诱导细胞凋亡作用;通过PI单染色细胞流式分析测定了其在常氧和缺氧条件下诱导肿瘤细胞发生周期抑制的作用;通过Western blotting在细胞水平检测p53相关下游基因表达水平的变化,对其可能的抑瘤机制进行了探讨。另外,我们利用裸鼠建立了小鼠人源肿瘤荷瘤荷瘤模型,通过腹腔连续给药的方式,测定了TAT-ODD-p53的抑瘤活性,同时检测了蛋白在动物体内的分布和稳定性。应用免疫组化技术,分析了该蛋白对肿瘤组织内p53下游相关基因的表达水平的调节,对其在体内发挥抗肿瘤活性的调节通路进行了研究。实验结果表明:1.通过分子生物学技术,我们构建了TAT-ODD-p53融合基因,并将其克隆到原核表达载体中,在工程菌BL21中成功的进行了表达;2.通过包涵体变性复性,并结合亲和层析法成功的得到了大量的高纯度的目的蛋白;3.通过细胞免疫荧光染色和Western blotting检测显示,TAT-ODD-p53可以有效的进入肿瘤细胞;与在常氧细胞内相比,TAT-ODD-p53在缺氧细胞内的稳定性显著提高,半衰期明显延长;4.细胞生长抑制实验显示,缺氧条件下,TAT-ODD-p53蛋白可以有效的抑制肿瘤细胞的生长,而在常氧条件下,作用不明显;5.通过流式细胞分析,与常氧条件比较,TAT-ODD-p53在缺氧条件下诱导肿瘤细胞发生凋亡的比例明显提高,更易引发肿瘤细胞生长的周期抑制;6. Western blotting结果表明,缺氧状态下TAT-ODD-p53发挥其生物活性的主要机制是上调p21蛋白的表达水平及激活caspase-3通路诱导细胞凋亡,属于p53蛋白的经典作用途径;7.体内实验结构显示,TAT-ODD-p53蛋白可以选择性的积累于实体瘤内的缺氧区域,与瘤体内HIF-1α高表达区域完全一致,而在正常组织,如肝脏内几乎检测不到;8.通过统计的瘤重和瘤体积的数据表明,与其他对照融合蛋白相比,TAT-ODD-p53可以显著的抑制荷瘤鼠肿瘤的生长,没有观察到任何严重的副作用;9.对治疗后肿瘤组织中p53相关基因以及肿瘤特异性标志蛋白表达水平检测表明,TAT-ODD-p53可以显著上调一些p53相关基因的表达,如p21,PUMA,caspase-3,并下调了肿瘤恶性标志性蛋白的表达,如EGFR和VEGF。通过以上结果得出如下结论:TAT-ODD-p53可以有效的穿透细胞膜进入细胞内部,并在实体瘤的缺氧区域内稳定存在;通过上调p53相关基因的表达以及抑制肿瘤恶性标志物蛋白的表达,抑制肿瘤细胞增殖,诱导缺氧性肿瘤细胞凋亡,实现其靶向抗肿瘤的作用。在治疗过程中,通过观察动物日常行为和检测动物的血液生化指标,没有发现TAT-ODD-p53对受试动物有明显的毒副作用。更多还原

【Abstract】 Cancer is the major public problem around the world. Increasing clinical reports suggested that over 50% tumors contain the mutant P53 gene. P53 gene is one of the most important tumor suppression genes and its encoding product, p53, plays a central role to inhibit the tumorigenesis, such as sustaining the genomic stability, repairing the DNA damage, and protecting the malignant transformation via inducing apoptosis of cells which are suffered the irreversible injury. Therefore, p53 is called as“Genome guard”. However, p53 is highly susceptible to various mutations leading to abnormality. Mutant p53 not only fails to protect the normal cells undergoing the tumorigenesis, but also triggers the over expression of a set of oncogenes to promote the cellular carcinogenesis and accelerate the tumor progression. Hence, p53 has been a promising therapeutic target of tumor treatment. Although various reagents have been developed for restoration of inactive p53, their utility extensively limited because of their poor cell permeability and low targeting delivery.Moreover, over 90% tumors are solid tumors. Inadequate oxygenation, known as hypoxia, is the predominant pathophysiological feature in the solid tumors. The unequal balance between extensive cell proliferation and angiogenesis is the leading cause to the insufficient supplement of oxygen in tale. Malignant cells under this microenvironment are hypoxic tumor cells which are extremely resistant to traditional chemo- and radiotherapy and result in the metastasis in the advanced stage of cancers. Hypoxia-inducible factors (HIFs) play a pivotal role in these biological processes. HIFs family consists of three members, including HIF-1, HIF-2 and HIF-3. HIF-1 is well-studied. HIF-1 is a heterodimer, containingαandβsubunits. HIF-1βis constitutive, but HIF-1αis the regulatory subunit. Under normoxia, HIF-1αis unstable. Its oxygen-dependent degradation domain (ODD) could induce its degradation via unbiquitin-proteasome pathway mediated by Von-Hipple Lindau protein (pVHL). In the hypoxia, however, oxygen is deprived and HIF-1 prolyhydroxylase (HIF PH) become inactivated, fail to degrade HIF-1αvia pVHL dependent pathway. The stable HIF-1αcould combine withβsubunit, functioning as transcriptional factor to regulate a set of genes expression for the hypoxic adaption of tumor cells. Therefore, how to thoroughly clean the hypoxic tumor cells is the key step for the regime of solid tumors. Moreover, it is suggested that hypoxia could select the subpopulation of cancer cells harboring the mutant p53, which are more easily to prone to have aggressive phenotype to promote the progression, invasion and metastasis of tumors.Protein transduction domains, shorted as PTDs, are series of polypeptides which are capable to deliver bio-macromolecules into cells via cross-membrane transduction, including TAT peptide of HIV-1, VP22 from HSV, antennapedia homeoprotein of drosophila and a set of synthetic polypeptides with cell permeability. The predominant feature of these peptides is rich of basic amino acid in the sequence, resulting in the interaction with some bio-molecules on the cell surface to induce the internalization of the macromolecules with poor cell permeability, such as DNA, siRNA, proteins with big molecular weight and even nano-particles. This put the promising insight on the development of therapeutic protein drugs. TAT is one of the most well-studied and widely used PTDs. Its minimum functional domain is the residue 47~57. Many repots displayed that various of TAT fused proteins were developed for research and/or therapeutic utilities, such as TAT-EGFP, TAT-HT and TAT-p27, which could play their bio-functions delivered into cells and/or tissues via TAT transportation. These results support the fundamental information for further creation of TAT-based biological reagents.In this study, aiming at restoration of dysfunctional p53 and scavenging the hypoxic cancer cells in solid tumor, a novel p53 fusion protein, TAT-ODD-p53, was designed for targeting therapy of solid tumors. This protein was fused with TAT47-57, protein transduction peptide, the minimum functional motif of ODD (ODD557-574) and human wild-type p53 protein. This protein should have higher cell permeability and selectively localizes in the hypoxic regions of solid tumor to induce hypoxic tumor cells apoptosis. First, the encoding genes of p53, TAT-p53 and TAT-ODD-p53 were constructed and cloned into pET28a prokaryotic expression vector, respectively. Second, these p53 fusion proteins were expressed in E.coli, BL21 (DE3) engineering bacteria. The highly purified proteins were prepared via the procedure of extraction, denaturation, purification and renaturation. The transmembrane delivery and targeting stability of TAT-ODD-p53 were assessed by fluorescent immunocytochemistry and Western blotting in vitro. MTT assay, Annexin V & PI staining combined with cytometry analysis were used for evaluation of the cytotoxicity of this fusion protein; TAT-ODD-p53 induced cell-cycle arrest was analyzed using cytometry with PI staining. To investigate the mechanism of anti-tumor affect of TAT-ODD-p53, a set of p53 downstream genes expression was analyzed by Western blotting in vitro. On the other hand, tumor-bearing mode was established with Balb/c nude mice by subcutaneous injection of H1299, human non-small lung cancer cell line, to investigate the distribution and tumor suppression activity of TAT-ODD-p53. The protein’s accumulation in tumor and normal tissues was determined after the ip injection of p53 fusion protein at the dosage of 5 mg/kg. To assess the tumor suppression activity of TAT-ODD-p53, the proteins were i.p. injected to mice at the dosage of 5 mg/kg/time for 12 days with one day’s intermission. Immunochemical staining was used to analysis the existence of TAT-ODD-p53 in different tissues and its anti-tumor mechanism in vivo. Our experimental results demonstrated that: 1. TAT-ODD-p53 fusion gene was constructed, cloned into pET28a vector, and successfully expressed; 2. The fusion protein was purified via affinity chromatography; 3. TAT-ODD-p53 could be effectively delivered into tumor cells and accumulated in the cytoplasm and nucleus, the protein was more stable in hypoxia than that under normoxia, the half-life time significantly prolonged. 4. MTT assay displayed that TAT-ODD-p53 had predominant growth inhibition on various tumor cell lines under hypoxia, but had not significant function in the normal oxygen tension condition. 5. Cytometry analysis suggested that under hypoxia the apoptosis percentage of tumor cells increased significantly when the cells were treated with TAT-ODD-p53 at the dose of 30μg/ml with the prolonged time and the cell cycle was blocked in the G1 phase. 6. Western blotting displayed that TAT-ODD-p53 could up-regulate the p21 expression and activate the caspase-3 pathway to suppress the tumor cell growth in vitro. 7. In vivo, TAT-ODD-p53 could selectively accumulate in the hypoxic regions of solid tumor of mice, but not be detected in the normal tissue, such as liver. The co-localization assessment showed that TAT-ODD-p53 could scatter the area where HIF-1αwas highly expressed. 8. The weight and volume of tumor in mice treated with TAT-ODD-p53 were lower than those of other treatment groups, and there were no any severe side-effects observed; 9. The immunochemical staining showed that the p53-associated gene expression was up-regulated after treatment with TAT-ODD-p53, and some proteins involved in tumor locaregional spread, distant metastasis and angiogenesis were down-regulated in the tumor tissue.In conclusion, TAT-ODD-p53 has good cell permeability and selectively accumulated in the hypoxic regions of solid tumor. It effectively cleans the hypoxic tumor cells and suppresses the tumor growth via inducing tumor cells undergoing apoptosis via up-regulation of a set of p53 down-stream genes expression.更多还原