免疫毒素scFv-Mmut的表达及抗乳腺癌作用的初步研究

发布时间：2018-03-23 14:32

  本文选题：HER-2　切入点：乳腺癌　出处：《吉林大学》2017年硕士论文

【摘要】：乳腺癌是目前严重威胁女性健康的恶性肿瘤之一,是全身性、高度异质性疾病。临床上,常采用手术治疗为主、放化疗为辅进行乳腺癌的治疗,虽然能够使患者病情得到改善、有效延长生存时间,但是会对机体产生严重的非特异性损伤。二十世纪初,Ehrlich提出肿瘤的靶向治疗,它能够选择性的杀伤肿瘤细胞,减少对正常细胞的毒副作用,成为癌症领域的研究热点。免疫毒素是一种靶向药物,它是由导向载体和毒素分子(又称为效应分子)构成的能特异性杀伤肿瘤细胞的融合蛋白。它能通过导向载体选择性地与肿瘤细胞表面标志物结合,使毒素分子进入肿瘤细胞,提高药效的同时减少对正常组织细胞的损害。因此,选择肿瘤特异的导向载体和成药性良好的毒素分子对免疫毒素的构建尤为重要。研究显示,人表皮生长因子受体-2(HER-2)在约有25%-30%的乳腺癌患者中高表达,与乳腺癌的发生、转移以及预后密切相关。所以针对HER-2为靶点的研究意义重大。以HER-2为靶点的单链抗体sc Fv既保留了天然抗体的部分亲和力,又具有分子量小、易穿透组织到达病灶的特点,成为免疫毒素导向载体的良好选择。蜂毒肽是蜂毒的主要成分,分子量小,免疫原性低,具有破膜活性和肿瘤凋亡诱导活性,对肿瘤细胞有良好的细胞毒作用,是毒素分子的首选。但蜂毒肽的溶血活性限制了其在临床治疗中的应用,本实验室通过对蜂毒肽分子结构的优化,得到了既有诱导凋亡能力又无溶血活性的蜂毒肽类似物(Mmut),本文即以此蜂毒肽类似物作为效应分子。本实验选择针对HER-2为靶点的单链抗体sc Fv作为导向载体,选用蜂毒肽类似物(Mmut)作为效应分子,构成免疫毒素sc Fv-Mmut,通过毕赤酵母诱导表达获得目的蛋白,并通过体外实验初步探讨sc Fv-Mmut抗乳腺癌活性及作用机制。具体实验包括以下几个方面:(1)sc Fv-Mmut的毕赤酵母表达体系建立首先构建毕赤酵母表达质粒p PIC9K/sc Fv-Mmut,通过限制性内切酶SacⅠ线性化质粒,利用电穿孔法转化至毕赤酵母GS115菌株;通过MM/MD平板、遗传霉素G418抗性筛选阳性转化子;利用0.5%甲醇诱导表达,表达产物经过15%SDS-PAGE电泳分析,获得工程菌株sc Fv-Mmut1。(2)sc Fv-Mmut的纯化首先通过0.5%甲醇诱导表达工程菌株sc Fv-Mmut1;离心收集发酵上清液,经硫酸铵沉淀,Ni-Sepharose 6FF亲和层析纯化;15%SDS-PAGE鉴定,BCA标准蛋白试剂盒检测后,获得浓度为0.42μg·μL-1的目的蛋白溶液。(3)sc Fv-Mmut的活性检测利用MTT法检测sc Fv-Mmut对HER-2阳性乳腺癌细胞株BT474和HER-2阴性乳腺癌细胞MCF-7的抑制作用,结果显示sc Fv-Mmut对于HER-2阳性乳腺癌BT474细胞有明显的抑制效果,且明显高于对HER-2阴性乳腺癌MCF-7细胞的抑制效果。利用DNA ladder、DAPI染色、流式细胞仪初步检测免疫毒素sc Fv-Mmut对BT474细胞的作用机制;DNA ladder实验结果显示,BT474细胞染色体DNA发生断裂,出现DNA ladder,标志细胞发生凋亡;DAPI染色后荧光显微镜下观察,与空白对照组细胞的细胞核比较,给药组细胞核皱缩,形成颗粒物质,说明细胞发生凋亡;流式细胞周期检测结果显示,BT474细胞被阻滞在S期。利用荧光染料标记细胞核(Hochest33342标记)、线粒体(Mito Tracker?Red CMXRos标记)和免疫毒素sc Fv-Mmut(异硫氰酸荧光素FITC标记),通过激光共聚焦扫描显微镜观察sc Fv-Mmut在细胞中的分布以及靶向细胞的能力,结果显示sc Fv-Mmut能够靶向肿瘤细胞并进入细胞质发挥作用。综上所述,本研究成功构建了重组质粒p PIC9K/sc Fv-Mmut,并在毕赤酵母真核表达系统中成功表达sc Fv-Mmut;sc Fv-Mmut的生物学活性和作用机制的研究结果表明:以HER-2为靶点设计的sc Fv-Mmut能特异性靶向HER-2阳性乳腺癌细胞,进入细胞质诱导凋亡。
[Abstract]:Breast cancer is one of the most serious threat to women's health of malignant tumor at present, is a systemic, highly heterogeneous disease. Clinically, often using surgical treatment, radiotherapy and chemotherapy as treatment for breast cancer, although it can make the patient's condition improved, effectively prolong the survival time, but causes non-specific serious damage to the body. At the beginning of twentieth Century, Ehrlich proposed the targeted therapy of tumors, it can selectively kill tumor cells, reduce the side effect on normal cells, has become a hot research field of cancer. Immune toxin is a targeted drug, which is composed of carrier and toxin molecules (also known as effector molecules) which can specific killing tumor cell fusion protein. It can selectively bind carrier and tumor cell surface markers, the toxin molecules into tumor cells, improve the efficacy and reduce to normal The tissue and cell injury. Therefore, selection of tumor specific targeting carrier and good druggability toxin molecule construction of immunotoxins is particularly important. Research shows that human epidermal growth factor receptor -2 (HER-2) is highly expressed in about 25%-30% of patients with breast cancer, and breast cancer, metastasis and prognosis. So for the HER-2 as the research target. The great significance of targeting the HER-2 scFv SC Fv retains some affinity of natural antibodies, with small molecular weight, easy to penetrate the tissue to the characteristics of the lesions, become a good choice to guide the immunotoxin carrier. Melittin is the main component of bee venom, molecular weight a small, low immunogenicity, has broken membrane activity and apoptosis inducing activity, have good cytotoxic effect on tumor cells, is a toxin molecule preferred. But the hemolytic activity of melittin limits its in clinical treatment 鐨勫簲鐢，

本文编号：1653873