本文選題：HER-2　切入點(diǎn)：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(yán)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(jìn)行乳腺癌的治療,雖然能夠使患者病情得到改善、有效延長(zhǎng)生存時(shí)間,但是會(huì)對(duì)機(jī)體產(chǎn)生嚴(yán)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(xì)胞,減少對(duì)正常細(xì)胞的毒副作用,成為癌癥領(lǐng)域的研究熱點(diǎn)。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(xì)胞的融合蛋白。它能通過導(dǎo)向載體選擇性地與腫瘤細(xì)胞表面標(biāo)志物結(jié)合,使毒素分子進(jìn)入腫瘤細(xì)胞,提高藥效的同時(shí)減少對(duì)正常組織細(xì)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(duì)免疫毒素的構(gòu)建尤為重要。研究顯示,人表皮生長(zhǎng)因子受體-2(HER-2)在約有25%-30%的乳腺癌患者中高表達(dá),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(duì)HER-2為靶點(diǎn)的研究意義重大。以HER-2為靶點(diǎn)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(dá)病灶的特點(diǎn),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(duì)腫瘤細(xì)胞有良好的細(xì)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(shí)驗(yàn)室通過對(duì)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(shí)驗(yàn)選擇針對(duì)HER-2為靶點(diǎn)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(dá)獲得目的蛋白,并通過體外實(shí)驗(yàn)初步探討sc Fv-Mmut抗乳腺癌活性及作用機(jī)制。具體實(shí)驗(yàn)包括以下幾個(gè)方面:(1)sc Fv-Mmut的畢赤酵母表達(dá)體系建立首先構(gòu)建畢赤酵母表達(dá)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(yáng)性轉(zhuǎn)化子;利用0.5%甲醇誘導(dǎo)表達(dá),表達(dá)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(dá)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(cè)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(cè)利用MTT法檢測(cè)sc Fv-Mmut對(duì)HER-2陽(yáng)性乳腺癌細(xì)胞株BT474和HER-2陰性乳腺癌細(xì)胞MCF-7的抑制作用,結(jié)果顯示sc Fv-Mmut對(duì)于HER-2陽(yáng)性乳腺癌BT474細(xì)胞有明顯的抑制效果,且明顯高于對(duì)HER-2陰性乳腺癌MCF-7細(xì)胞的抑制效果。利用DNA ladder、DAPI染色、流式細(xì)胞儀初步檢測(cè)免疫毒素sc Fv-Mmut對(duì)BT474細(xì)胞的作用機(jī)制;DNA ladder實(shí)驗(yàn)結(jié)果顯示,BT474細(xì)胞染色體DNA發(fā)生斷裂,出現(xiàn)DNA ladder,標(biāo)志細(xì)胞發(fā)生凋亡;DAPI染色后熒光顯微鏡下觀察,與空白對(duì)照組細(xì)胞的細(xì)胞核比較,給藥組細(xì)胞核皺縮,形成顆粒物質(zhì),說明細(xì)胞發(fā)生凋亡;流式細(xì)胞周期檢測(cè)結(jié)果顯示,BT474細(xì)胞被阻滯在S期。利用熒光染料標(biāo)記細(xì)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(xì)胞中的分布以及靶向細(xì)胞的能力,結(jié)果顯示sc Fv-Mmut能夠靶向腫瘤細(xì)胞并進(jìn)入細(xì)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(dá)系統(tǒng)中成功表達(dá)sc Fv-Mmut;sc Fv-Mmut的生物學(xué)活性和作用機(jī)制的研究結(jié)果表明:以HER-2為靶點(diǎn)設(shè)計(jì)的sc Fv-Mmut能特異性靶向HER-2陽(yáng)性乳腺癌細(xì)胞,進(jìn)入細(xì)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，

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