發(fā)布時間：2018-03-23 14:32

  本文選題：HER-2　切入點：乳腺癌　出處：《吉林大學(xué)》2017年碩士論文

【摘要】：乳腺癌是目前嚴重威脅女性健康的惡性腫瘤之一,是全身性、高度異質(zhì)性疾病。臨床上,常采用手術(shù)治療為主、放化療為輔進行乳腺癌的治療,雖然能夠使患者病情得到改善、有效延長生存時間,但是會對機體產(chǎn)生嚴重的非特異性損傷。二十世紀(jì)初,Ehrlich提出腫瘤的靶向治療,它能夠選擇性的殺傷腫瘤細胞,減少對正常細胞的毒副作用,成為癌癥領(lǐng)域的研究熱點。免疫毒素是一種靶向藥物,它是由導(dǎo)向載體和毒素分子(又稱為效應(yīng)分子)構(gòu)成的能特異性殺傷腫瘤細胞的融合蛋白。它能通過導(dǎo)向載體選擇性地與腫瘤細胞表面標(biāo)志物結(jié)合,使毒素分子進入腫瘤細胞,提高藥效的同時減少對正常組織細胞的損害。因此,選擇腫瘤特異的導(dǎo)向載體和成藥性良好的毒素分子對免疫毒素的構(gòu)建尤為重要。研究顯示,人表皮生長因子受體-2(HER-2)在約有25%-30%的乳腺癌患者中高表達,與乳腺癌的發(fā)生、轉(zhuǎn)移以及預(yù)后密切相關(guān)。所以針對HER-2為靶點的研究意義重大。以HER-2為靶點的單鏈抗體sc Fv既保留了天然抗體的部分親和力,又具有分子量小、易穿透組織到達病灶的特點,成為免疫毒素導(dǎo)向載體的良好選擇。蜂毒肽是蜂毒的主要成分,分子量小,免疫原性低,具有破膜活性和腫瘤凋亡誘導(dǎo)活性,對腫瘤細胞有良好的細胞毒作用,是毒素分子的首選。但蜂毒肽的溶血活性限制了其在臨床治療中的應(yīng)用,本實驗室通過對蜂毒肽分子結(jié)構(gòu)的優(yōu)化,得到了既有誘導(dǎo)凋亡能力又無溶血活性的蜂毒肽類似物(Mmut),本文即以此蜂毒肽類似物作為效應(yīng)分子。本實驗選擇針對HER-2為靶點的單鏈抗體sc Fv作為導(dǎo)向載體,選用蜂毒肽類似物(Mmut)作為效應(yīng)分子,構(gòu)成免疫毒素sc Fv-Mmut,通過畢赤酵母誘導(dǎo)表達獲得目的蛋白,并通過體外實驗初步探討sc Fv-Mmut抗乳腺癌活性及作用機制。具體實驗包括以下幾個方面:(1)sc Fv-Mmut的畢赤酵母表達體系建立首先構(gòu)建畢赤酵母表達質(zhì)粒p PIC9K/sc Fv-Mmut,通過限制性內(nèi)切酶SacⅠ線性化質(zhì)粒,利用電穿孔法轉(zhuǎn)化至畢赤酵母GS115菌株;通過MM/MD平板、遺傳霉素G418抗性篩選陽性轉(zhuǎn)化子;利用0.5%甲醇誘導(dǎo)表達,表達產(chǎn)物經(jīng)過15%SDS-PAGE電泳分析,獲得工程菌株sc Fv-Mmut1。(2)sc Fv-Mmut的純化首先通過0.5%甲醇誘導(dǎo)表達工程菌株sc Fv-Mmut1;離心收集發(fā)酵上清液,經(jīng)硫酸銨沉淀,Ni-Sepharose 6FF親和層析純化;15%SDS-PAGE鑒定,BCA標(biāo)準(zhǔn)蛋白試劑盒檢測后,獲得濃度為0.42μg·μL-1的目的蛋白溶液。(3)sc Fv-Mmut的活性檢測利用MTT法檢測sc Fv-Mmut對HER-2陽性乳腺癌細胞株BT474和HER-2陰性乳腺癌細胞MCF-7的抑制作用,結(jié)果顯示sc Fv-Mmut對于HER-2陽性乳腺癌BT474細胞有明顯的抑制效果,且明顯高于對HER-2陰性乳腺癌MCF-7細胞的抑制效果。利用DNA ladder、DAPI染色、流式細胞儀初步檢測免疫毒素sc Fv-Mmut對BT474細胞的作用機制;DNA ladder實驗結(jié)果顯示,BT474細胞染色體DNA發(fā)生斷裂,出現(xiàn)DNA ladder,標(biāo)志細胞發(fā)生凋亡;DAPI染色后熒光顯微鏡下觀察,與空白對照組細胞的細胞核比較,給藥組細胞核皺縮,形成顆粒物質(zhì),說明細胞發(fā)生凋亡;流式細胞周期檢測結(jié)果顯示,BT474細胞被阻滯在S期。利用熒光染料標(biāo)記細胞核(Hochest33342標(biāo)記)、線粒體(Mito Tracker?Red CMXRos標(biāo)記)和免疫毒素sc Fv-Mmut(異硫氰酸熒光素FITC標(biāo)記),通過激光共聚焦掃描顯微鏡觀察sc Fv-Mmut在細胞中的分布以及靶向細胞的能力,結(jié)果顯示sc Fv-Mmut能夠靶向腫瘤細胞并進入細胞質(zhì)發(fā)揮作用。綜上所述,本研究成功構(gòu)建了重組質(zhì)粒p PIC9K/sc Fv-Mmut,并在畢赤酵母真核表達系統(tǒng)中成功表達sc Fv-Mmut;sc Fv-Mmut的生物學(xué)活性和作用機制的研究結(jié)果表明:以HER-2為靶點設(shè)計的sc Fv-Mmut能特異性靶向HER-2陽性乳腺癌細胞,進入細胞質(zhì)誘導(dǎo)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，

本文編號：1653873