【作者】 王小婷；

【导师】 屈伸；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 鸡贫血病毒(chicken anemia virus,CAV)是一种类型独特的环状单链(负链)DNA病毒。研究证实VP3蛋白(Apoptin,亦称凋亡素)是CAV的功能蛋白,可广泛诱导肿瘤细胞凋亡,而且仅诱导具有致瘤表型细胞或转化表型细胞的凋亡,而对正常二倍体细胞无凋亡效应、无细胞毒性。并且,VP3诱导凋亡途径为P53非依赖型,不受Bcl-2高表达的抑制。传统化疗药物常常由于P53突变导致肿瘤细胞对化疗药物敏感性降低,因此,VP3蛋白是一种更具应用前景的抗肿瘤生物制剂。细胞透膜肽(Cell-Penetrating Peptide,CPP)是一类能携带生物大分子进入细胞的短肽,它不依赖经典的细胞内吞。大量的研究表明细胞透膜肽具有强大的运载潜能,不仅可以携带多肽、GFP、RNaseA而且可以携带DNA、反义核酸、有机分子、脂质体等。与其他的蛋白质形成的融合蛋白也能透过细胞膜,并发挥这些蛋白质的生物学功能。因此,本研究正是将两者结合在一起,为肿瘤等疾病的治疗提供新思路。本课题组在前期研究中已证明PTD4能通过生物合成而实现其融合蛋白PTD4-GFP-VP3对体外培养的多种细胞的透膜作用。本研究分别从体内外实验观察PTD4-VP3融合蛋白诱导宫颈癌细胞凋亡的效应。利用PTD4-VP3融合蛋白的原核表达载体,经IPTG诱导融合蛋白表达,镍金属鳌合层析柱纯化获得融合蛋白。利用流式细胞仪检测融合蛋白体外诱导人宫颈癌Hela细胞的凋亡效率。在此基础上,建立人宫颈癌Hela细胞异种移植裸鼠模型,将融合蛋白PTD4-VP3,涂抹在宫颈癌皮下移植瘤的皮肤表面,治疗7天后,比较皮下移植瘤大小并利用TUNEL法观察PTD4-VP3融合蛋白诱导宫颈癌细胞的凋亡效应及对正常组织的毒性。结果显示:随着融合蛋白PTD4-VP3孵育Hela细胞时间的延长,细胞总凋亡率升高,在孵育48小时后细胞总凋亡率最高,达59.73%;裸鼠宫颈癌皮下移植瘤生长曲线显示,治疗组和对照组在涂抹前瘤块体积不存在明显差异(P=0.9533,P>0.05),经融合蛋白涂抹治疗7天后,治疗组裸鼠的瘤块生长速度低于对照组瘤块,治疗结束时两组间瘤块体积有差异,具有统计学意义(P=0.0115,P<0.05)。根据第7天测量的两组瘤块的体积,经计算肿瘤生长抑制率为66.36%。;TUNEL-DAB染色法检测到治疗组宫颈癌细胞的大范围凋亡,对照组宫颈癌细胞少见凋亡;HE染色未见治疗组裸鼠主要脏器发生凋亡。结论:PTD4-VP3融合蛋白可以透过皮肤,并通过特异性诱导肿瘤细胞凋亡的方式抑制肿瘤生长,为进一步VP3治疗肿瘤的研究奠定了基础。更多还原

【Abstract】 Chicken anemia virus is a distinct type of single strand DNA virus. VP3 (apoptin) has shown to induce apoptosis in various tumor cells, but not in normal human cells and murine diploid cells. Apoptin-induced apoptosis does not dependent on functional P53, and cannot be inhibited by overexpression of Bcl-2, but caspase-3 activation is necessary for apoptin-induced rapid apoptosis. So VP3 is a very promising anti-tumor biological agents.Cell-Penetrating Peptide is a short peptide that can take along biological macromolecules into cells. Many research have showed that cell-penetrating peptide can successfully mediate the introduction of biological macromolecules, such as polypeptide , GFP, RNaseA, DNA, polar molecule and lipid body. So it has the potential to deliver both existing and novel anticancer therapeutics. This research combined the two aspect to discuss the anti-tumor effect of the fusion protein.This research is based on the constructing of the prokaryotic expression vector of PTD4-VP3. The fusion protein is reduced by IPTG, and the purification of the fusion protein. The PTD4-VP3 induced apoptosis of tumor cells in vitro was tested by FACS. We construct the nude mice models of cervical cancer by Hela cells. The anti-tumor effect of PTD4-VP3 fusion protein in vivo was tested by TUNEL assay and comparison of the tumor size after treatment. The result showed that PTD4 can take along fusion protein VP3 into cells, and induce apoptosis. The rate of apoptosis is the highest at 48h. The fusion protein can inhibit tumor growth by inducing apoptosis. This research will be the base for the study of the anti-tumor therapy.更多还原