本文選題：卵巢癌 + 腫瘤內(nèi)皮細(xì)胞標(biāo)記物1�。� 參考：《吉林大學(xué)》2014年博士論文

【摘要】：腫瘤內(nèi)皮細(xì)胞標(biāo)記物1(TEM1)作為腫瘤血管標(biāo)記物被認(rèn)為能夠觀察腫瘤血管的再生。我們的前期工作顯示：與正常卵巢組織比較,卵巢癌組織的腫瘤血管內(nèi)皮細(xì)胞過表達(dá)TEM1,因此,推測TEM1可能是一個腫瘤血管治療的標(biāo)記物。同時,我們使用酵母抗體庫篩選分離出人的TEM1特異性單鏈抗體(scFv78),并發(fā)現(xiàn)TEM1能結(jié)合到人和鼠的TEM1的胞外區(qū)(aa.324-390),其親和指數(shù)Kd值約2nM,表明其親和力較差。另外,scFv78分子量小,容易經(jīng)腎臟排泄,血漿半衰期短,限制了其在早期特異性診斷和治療等方面的應(yīng)用。 本實驗為了提高TEM1特異性抗體的親和力與延長其在血液中的半衰期。我們構(gòu)建了一系列新的scFv78多價抗體衍生物,包括二聚體Fc融合蛋白(來自hu IgG1)(78Fc),四聚體CH2融合蛋白(78CH2),二聚體CH3融合蛋白(78mb)和二聚體的CH1-鉸鏈區(qū)融合蛋白(78F(ab')2),以便從中發(fā)現(xiàn)溫度穩(wěn)定和血清穩(wěn)定性更好以及親和力更高的抗體；通過瞬時轉(zhuǎn)染293F細(xì)胞并且對培養(yǎng)上清進(jìn)行親和純化獲得這些蛋白；利用活細(xì)胞ELISA對這些蛋白進(jìn)行親和力的分析；同時,在動物體內(nèi)對上述5個蛋白的血液代謝動力學(xué)進(jìn)行檢測；并利用近紅外熒光成像技術(shù)對篩選出的最佳候選蛋白進(jìn)行了體內(nèi)成像檢測,利用細(xì)胞殺傷實驗和成管實驗,觀察其體外選擇性地殺傷TEM1陽性表達(dá)細(xì)胞的效果。實驗結(jié)果顯示：與我們前期工作發(fā)現(xiàn)的scFv78相比,本實驗構(gòu)建的78Fc親和力(Kd=0.14±0.01nM)增加了約15倍；動物體內(nèi)代謝動力學(xué)結(jié)果表明78Fc的慢相半衰期約5.1小時；體內(nèi)功能分析表明78Fc不針對Naive鼠的正常器官,而只是針對體內(nèi)TEM1+腫瘤；近紅外熒光成像技術(shù)結(jié)果顯示78Fc在鼠體內(nèi)只靶向TEM1陽性表達(dá)的細(xì)胞；最后78Fc-MMAE可以在體外選擇性地殺傷TEM1陽性表達(dá)細(xì)胞。 綜上所述,我們可以初步認(rèn)為,通過將scFv78和Fc段融合后,提高了該蛋白的穩(wěn)定性及親和力,血液動力學(xué)結(jié)果表明78Fc有相對長的血清半衰期,可以用來做成像的探針。體內(nèi)近紅外熒光成像技術(shù),體外細(xì)胞殺傷實驗與成管實驗與也證實其具有較高的特異性。因此,我們認(rèn)為TEM1是一個成像與治療的候選基因,可能為卵巢癌和其它癌癥的靶向診斷與治療提供新的思路；而78Fc可以為卵巢癌的早期診斷和治療提供可能。
[Abstract]:Tumor endothelial cell marker (Tem 1) is considered to be able to observe tumor vascular regeneration. Our previous work showed that tumor vascular endothelial cells in ovarian cancer tissues overexpressed TEM1 compared with normal ovarian tissues. Therefore, we speculated that TEM1 might be a marker of tumor vascular therapy. At the same time, we used yeast antibody library to screen and isolate human TEM1 specific scFv78, and found that TEM1 could bind to the extracellular region of TEM1 in human and mouse, and its affinity index (KD) was about 2 nm, which indicated that the affinity was poor. In addition, scFv78 has small molecular weight, easy excretion through kidney and short plasma half-life, which limits its application in early specific diagnosis and treatment. The purpose of this study was to improve the affinity of TEM1 specific antibodies and prolong their half-life in blood. We have constructed a series of new scFv78 polyvalent antibody derivatives. It includes dimer FC fusion protein (from Hu IgG1, CH2 fusion protein, tetramer CH2 fusion protein, dimer CH3 fusion protein, and dimer CH1-hinge fusion protein) and dimer CH1-hinge region fusion protein, so as to find antibodies with better temperature stability, better serum stability and higher affinity between the two fusion proteins, including the fusion protein Fc from Hu IgG1, the fusion protein from tetramer CH2, the fusion protein from dimer CH3, and the fusion protein from the CH1-hinge region of dimer. These proteins were obtained by transient transfection of 293F cells and affinity purification of the culture supernatant. The affinity of these proteins was analyzed by living cell ELISA. The best candidate protein was detected by near infrared fluorescence imaging in vivo. The effect of selective killing of TEM1 positive cells in vitro was observed by cell killing test and tube forming experiment. The results showed that the affinity of 78Fc constructed in this experiment was increased by about 15 times compared with the scFv78 found in our previous work, and the metabolic kinetics of 78Fc in vivo showed that the slow phase half-life of 78Fc was about 5.1 hours. In vivo functional analysis showed that 78Fc was not directed at normal organs of Naive mice, but only on TEM1 tumors in vivo, near infrared fluorescence imaging showed that 78Fc only targeted TEM1 positive cells in mice. Finally, 78Fc-MMAE can selectively kill TEM1 positive cells in vitro. In conclusion, we can preliminarily conclude that by fusion of scFv78 and FC, the stability and affinity of the protein are improved, and the hemodynamic results show that 78Fc has a relatively long serum half-life and can be used as an image probe. In vivo near infrared fluorescence imaging, in vitro and in vitro cell killing experiments and tube-forming experiments and also confirmed that it has a high specificity. Therefore, we believe that TEM1 is a candidate gene for imaging and treatment, which may provide a new idea for the targeted diagnosis and treatment of ovarian cancer and other cancers, while 78Fc can provide the possibility for early diagnosis and treatment of ovarian cancer.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R737.31

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

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本文編號：1786998