【作者】 贾培媛；

【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， 2009， 博士

【摘要】 促性腺激素释放激素(gonadotrophin releasing hormone,GnRH)是下丘脑分泌的十肽激素,其生理功能是调节垂体前叶促性腺激素的分泌。近年来的研究发现在乳腺、卵巢、子宫内膜、前列腺、胰腺、结肠、肺、肝脏的肿瘤细胞上有GnRH受体分布。GnRH及其类似物可抑制这些肿瘤细胞生长,它们的抗肿瘤效应是通过垂体及垂体外的机制实现的。缺氧是实体肿瘤的特征性病理改变。氧依赖性降解区域(oxygen-dependent degradation domain,ODD)是缺氧诱导因子-1α(HIF-1α)上控制其稳定的关键区域,通过其核心脯氨酸的羟化,在常氧状态时使HIF-1α被蛋白酶降解。p53是细胞内重要的肿瘤抑制因子,它可以通过诱导肿瘤细胞凋亡和诱发肿瘤细胞发生细胞周期阻滞而产生抗肿瘤作用。研究表明人类50%以上的肿瘤的发生都与细胞内P53基因突变、缺失有关,因此p53一直是近年来抗肿瘤研究的热点。本课题旨在以p53为肿瘤治疗的效应分子,通过GnRH与其受体特异结合介导蛋白进入相应的肿瘤细胞,应用ODD减轻p53对正常组织的损伤,增强蛋白对实体肿瘤的靶向作用,构建pET28a-GnRH-P53(pET28a-GP)、pET28a-GnRH-ODD-P53(pET28a-GOP)、pET28a-GnRHⅢ-P53(pET28a-G3P)、pET28a-GnRHⅢ-ODD-P53(pET28a-G3OP)、pET28a-GnRH-ODD557-574aa-P53 (pET28a-GSOP)、pET28a-GnRHⅢ-ODD557-574aa-P53(pET28a-G3SOP)原核表达载体,转化大肠杆菌BL21,诱导重组质粒表达相应的融合蛋白,将表达后菌体超声处理后离心,收集包涵体后进行变性、复性、纯化,从而得到具有生物学功能的融合蛋白。在细胞和动物水平研究融合蛋白的抗肿瘤作用。具体方法为:应用MTT法测定融合蛋白对肿瘤细胞生长的抑制作用;细胞免疫化学法、细胞免疫荧光法测定融合蛋白在细胞中的定位;应用流式细胞术测定融合蛋白诱导肿瘤细胞凋亡及其诱发肿瘤细胞周期阻滞;Western blot检测融合蛋白抗肿瘤的可能机制。以H1299细胞建立BALB/c裸鼠荷瘤模型,将实验动物随机分为C (正常对照组),TC (肿瘤对照组),GP(GnRH-p53),GOP(GnRH-ODD-p53),G3P (GnRHⅢ-p53),G3OP(GnRHⅢ-ODD- p53)六组,以5mg/kg/天的剂量腹腔给药(对照组动物给予等量的生理盐水),测定融合蛋白对模型鼠的抗肿瘤作用,同时对模型鼠血浆生化指标进行测定初步判断融合蛋白对动物的毒副作用。本课题的实验结果:①构建了pET28a-GnRH-P53系列重组质粒载体,将其转入大肠杆菌BL21后成功表达了相应的融合蛋白。②经细胞免疫荧光法、细胞免疫化学法、Western blot检测表明融合蛋白可以进入GnRH-R表达阳性的H1299细胞、DU145细胞、T47D细胞、MDA-MB-231细胞以及SW480细胞。③经MTT法测定表明GP、GSOP、G3P、G3SOP在常氧和缺氧条件下均可以抑制H1299细胞的生长,并且这种作用具有剂量依赖性,即随着蛋白浓度的增加其抑制H1299细胞生长作用增强;GOP和G3OP在缺氧条件下对H1299细胞的生长抑制作用比其在常氧条件下的作用明显;融合蛋白对DU145细胞、T47D细胞、MDA-MB-231细胞以及SW480细胞也具有增殖抑制作用;融合蛋白对H1299细胞的增殖抑制作用强于相同剂量的GnRH类似物亮丙瑞林和G662。④流式细胞术测定融合蛋白诱导肿瘤细胞凋亡表明,融合蛋白在常氧状态下可以增加H1299细胞早期凋亡的百分率,其作用在48h最强,而与PBS对照相比,G3OP在给药24h时可显著诱导H1299细胞发生早期凋亡;在缺氧状态下,给药24h,与RPMI1640空白培养基对照相比较,各融合蛋白均可显著诱导H1299细胞发生早期凋亡; GOP和G3OP在缺氧状态下的作用比其在常氧状态下诱导细胞凋亡作用明显增强。⑤在常氧和缺氧状态下,各融合蛋白可以诱导H1299细胞G0/G1期细胞比例增加,而处于S期细胞的百分数减少,DNA合成受到抑制,即发生细胞G1期阻滞。⑥经Western blot检测,融合蛋白可以上调H1299细胞中的Caspase-3蛋白、p21蛋白表达水平,推测融合蛋白抑制H1299细胞生长的机制与活化Caspase-3蛋白诱导细胞凋亡及上调p21蛋白表达促进细胞发生G1期阻滞相关。⑦测量、计算各组实验动物瘤体体积发现,与TC组比较GP抑制瘤体生长作用十分显著(p<0.01),GOP、G3P、G3OP抑瘤作用较明显(p<0.05)。⑧免疫组化方法检测荷瘤模型小鼠肝脏和肿瘤切片,发现p53蛋白主要分布于肿瘤组织中,而且给药组动物肿瘤组织切片中Caspase-3蛋白、p21蛋白、PUMA蛋白的含量较对照组高,表明融合蛋白对荷瘤模型小鼠的抗肿瘤作用主要是通过诱导肿瘤细胞凋亡和发生细胞周期阻滞而实现的。⑨动物血浆生化指标测定结果表明,融合蛋白对动物的肝、肾功能几乎没有影响。由以上结果得出如下结论:在细胞和动物整体水平上,融合蛋白GP、GOP、G3P、G3OP具有抑制肿瘤生长作用,其抗肿瘤作用主要是通过诱导肿瘤细胞凋亡和发生细胞周期阻滞而实现的。GP等融合蛋白有望开发成为新型的抗肿瘤药物。更多还原

【Abstract】 As a 10-amino acid regulator of reproduction, gonadotrophin-releasing hormone (GnRH) is released by neurons in the hypothalamus, and transported via the hypothalamo-hypophyseal portal circulation to the anterior pituitary to trigger gonadotropin release for luteinizing hormone and follicle-stimulating hormone, which in turn stimulate the gonads for steroid production. In the past, GnRH receptor (GnRH-R) was found to be expressed in normal human reproductive tissues (e.g. breast, ovary, endometrial, prostate, pancreas, colon, lung and liver) and tumors derived from these tissues. Numerous studies have provided evidence for the suppress effect of GnRH and GnRH-a (GnRH analogue) in these tumor cells proliferation. The multiple actions of GnRH could be linked to the divergence of signaling pathways that are activated by GnRH-R. The oxygen-dependent degradation domain (ODD) of HIF-1αis a key on protein stability. In normal oxygen level, ODD can accelerate the degration of HIF-1αby the hydroxylation of its proline. p53, one of the most well studied tumor suppressor factor, is responsible to a variety of damage owing the induction of apoptosis and cell cycle arrest in the tumor cells. More than 50% of human tumors contain a mutation or deletion of p53. Therefore p53 has been an appealing target for new anticancer therapeutic strategies.The aim of this project is to investigate the targeted tumor inhibition induced by p53 fusion proteins against tumors with p53 gene mutation or deficient. GnRH, as the ligand of GnRH-R, was used to deliver p53 into tumor cells. ODD was used to control the stability of proteins in tissue under different oxygen stress. GnRH, GnRHⅢ, ODD and p53 gene was constructed and cloned into the pET28a vector. The p53 fusion proteins were expressed and their targeted anti-tumor effects were determined. The viability of tumor cells treated with each protein was measured by the MTT method. Immunocytochemistry analysis and immunofluorescence analysis were also used to detect the location of p53 fusion proteins in tumor cells. The flow cytometry was employed to analyze the apoptosis and cell cycle arrest in the proteins treated H1299 cells. The possible mechanism of proliferation inhibition induced by p53 fusion proteins was measured by Western blot. The mice with xenograft tumor of H1299 cells were used to test the tumor proliferation inhibition induced by fusion proteins (GP, GOP, G3P and G3OP) in vivo, while saline buffer was used as control. The plasma biochemical parameters of mice were also determined.A series of novel fusion proteins, GnRH-p53(GP), GnRH-ODD-p53(GOP),GnRHⅢ-p53 (G3P),GnRHⅢ-ODD-p53 (G3OP), GnRH-ODD557-574aa-p53(GSOP), GnRHⅢ-ODD557-574aa-p53(G3SOP), were expressed in E coli. BL21 (DE3). As the control protein, wild type p53 (p53), was also constructed. These proteins were used to investigate the targeted tumor inhibition induced by p53 fusion proteins. Immunocytochemistry analysis and immunofluorescence analysis showed that all of fusion proteins could permeate into the H1299 cells and located in the cytoplasm and nucleus. Cellular experiments demonstrated that GP, GSOP, G3P and G3SOP could obviously decrease the viability of H1299 cells, whereas GOP and G3OP did weaker anti-proliferation function under normoxia than hypoxia. The fusion proteins also had proliferation inhibition function in DU145 cells, T47D cells, MDA-MB-231 cell and SW480 cells. p53 fusion proteins had stronger cellular toxicity in tumor cells than the same molar concentration of GnRH analogues (Leuprorelin and G662) did. The results of flow cytometry showed that p53 fusion proteins could induce early apoptosis and G1 cell cycle arrest in H1299 cells. The fusion proteins fused with ODD domain exhibit mild apoptosis promoting action on the tumor cells under normoxia. The results of Western blot showed that the proteins in H1299 cells treated with fusion proteins could be well recognized by the mouse anti-p53 monoclonal antibody, rat anti-caspase-3 monoclonal antibody and mouse anti-p21 monoclonal antibody. This result of Western blot also indicated that the fusion proteins could increase the expression of caspase-3 proteins and p21 proteins in H1299 cells, which showed that the proliferation inhibition was related with cell apoptosis and cell cycle arrest induced by p53. Repeated intraperitoneal (i.p.) injection of fusion proteins at 5mg/kg/day resulted in the reduction in mass and volume of xenograft tumor, compared with saline buffer treatment control(GP, p<0.01; GOP, G3P, G3OP, p<0.05). The result of immunohistochemistry indicated that the fusion proteins could increase the expression of caspase-3, p21 and PUMA proteins in tumors. The plasma biochemical parameters of mice determined in this research were almost normal.In conclusion, GnRH mediates its fusion proteins transformation into cancer cells. The intracellular delivery of p53 fusion proteins exerted the inhibition of cancer cells growth in vitro and the reduction of tumor volume in vivo. Their anti-tumor effect was functioned by the apoptosis and cell cycle arrest induced by p53. Hence, the fusion protein could be a novel protein drug for anti-tumor therapy.更多还原