本文選題：多肽　切入點(diǎn)：白細(xì)胞介素-11受體α　出處：《南京醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景:乳腺癌是女性發(fā)病率最高的惡性腫瘤,且有65%-75%的乳腺癌患者發(fā)生骨轉(zhuǎn)移。研究發(fā)現(xiàn)IL-11/IL-11Rα信號(hào)通路參與乳腺癌骨轉(zhuǎn)移發(fā)生發(fā)展,IL-11Rα能作為乳腺癌、骨肉瘤等受體顯像的潛在靶點(diǎn),其在乳腺癌顯像及治療的應(yīng)用值得進(jìn)一步探索。目的:本實(shí)驗(yàn)通過(guò)評(píng)估IL-11類似物環(huán)九肽CGRRAGGSC對(duì)IL-11Rα的靶向性,并在此基礎(chǔ)上構(gòu)建放射性核素標(biāo)記CGRRAGGSC探針進(jìn)行乳腺癌模型鼠顯像及體外對(duì)乳腺癌細(xì)胞抑制作用的研究。方法:采用蛋白印跡(Western blot)檢測(cè)乳腺癌細(xì)胞(MDA-MB-231、MCF-7和BT549)和正常乳腺細(xì)胞MCF-10A中IL-11Rα的表達(dá)。體外細(xì)胞免疫熒光觀察冷CGRRAGGSC或抗IL-11Rα抗體處理前后,CGRRAGGSC、對(duì)照環(huán)肽CGSPGWVRC和酪氨酸(Tyr)或螯合劑(DTPA)修飾后的CGRRAGGSC對(duì)各乳腺癌細(xì)胞的結(jié)合情況。近紅外線顯像評(píng)估CGRRAGGSC在乳腺癌MCF-7模型鼠體內(nèi)對(duì)IL-11Rα的靶向特異性情況。通過(guò)氯化亞錫還原法標(biāo)記環(huán)九肽CGRRAGGSC獲得標(biāo)記產(chǎn)物99mTc-DTPA-CGRRAGGSC,紙層析檢測(cè)標(biāo)記率及穩(wěn)定性,以乳腺癌皮下移植瘤模型鼠進(jìn)行SPECT成像研究。通過(guò)氯氨T法標(biāo)記CGRRAGGSC獲得131I-CGRRAGGSC,薄層層析檢測(cè)標(biāo)記率及穩(wěn)定性,行噻唑藍(lán)(MTT)法、Transwell、劃痕實(shí)驗(yàn)及 Western blot 檢測(cè) 131I-CGRRAGGSC 對(duì)乳腺癌細(xì)胞的抑制作用及處理前后細(xì)胞內(nèi)IL-11Rα和STAT3、p-STAT3蛋白的表達(dá)變化。結(jié)果:IL-11Rα在本研究的乳腺癌細(xì)胞中的表達(dá)高于正常乳腺細(xì)胞MCF-10A。CGRRAGGSC、Tyr-CGRRAGGSC 和 DTPA-CGRRAGGSC 都能與各乳腺癌發(fā)生特異性結(jié)合,而對(duì)照肽CGSPGWVRC幾乎不結(jié)合;用冷CGRRAGGSC或抗IL-11Rα抗體處理后,CGRRAGGSC的結(jié)合能力明顯減弱。近紅外顯像示注射Cy7-CGRRAGGSC后腫瘤部位的熒光信號(hào)明顯高于其它器官或組織,而注射Cy7組腫瘤的熒光信號(hào)與正常器官或組織相似。成功制備了 131I-CGRRAGGSC和99mTc-DTPA-CGRRAGGSC,兩者的標(biāo)記率都達(dá)95%以上,并且在37℃放置12 h后的放射性化學(xué)純度都在90%以上。SPECT顯像示瘤體間質(zhì)內(nèi)給藥后,99mTc-DTPA-CGRRAGGSC在荷瘤鼠瘤體內(nèi)的滯留時(shí)間長(zhǎng)且放射性高。131I-CGRRAGGSC處理后,乳腺癌細(xì)胞的增殖及遷移能力減弱,其機(jī)制可能與降低IL-11Rα阻斷STAT3的磷酸化相關(guān)。結(jié)論:放射性核素標(biāo)記CGRRAGGSC方法簡(jiǎn)便,標(biāo)記率高,并且對(duì)IL-11Rα靶向性強(qiáng),有望用于陽(yáng)性表達(dá)IL-11Rα腫瘤的顯像及治療研究。
[Abstract]:Background: breast cancer has the highest incidence of malignant tumors in women, and 65% to 75% of breast cancer patients have bone metastases. It has been found that IL-11/IL-11R 偽 signaling pathway is involved in the development of breast cancer bone metastasis and IL-11R 偽 can be used as breast cancer. The potential targets of osteosarcoma and other receptor imaging, such as osteosarcoma, are worthy of further exploration in breast cancer imaging and treatment. Objective: to evaluate the targeting of IL-11R 偽 by IL-11 analogues CGRRAGGSC. A radionuclide labeled CGRRAGGSC probe was constructed for the imaging of breast cancer model mice and its inhibitory effect on breast cancer cells in vitro. Methods: breast cancer cells MDA-MB-231 MCF-7 and BT549) and normal breast were detected by Western blot. Expression of IL-11R 偽 in MCF-10A of glandular cells. In vitro, cellular immunofluorescence was used to observe the binding of CGRRAGGSC modified with cold CGRRAGGSC or anti-#en3# 偽 antibody, CGSPGWVRC and tyrosine tyrosine tyrosine (CGSPGWVRC) or chelating agent to breast cancer cells. The target specificity of CGRRAGGSC to IL-11R 偽 was evaluated in MCF-7 mice with breast cancer. The labelled product 99mTc-DTPA-CGRRAGSCC was obtained by stannous chloride reduction method, and the labeling rate and stability were detected by paper chromatography. The SPECT imaging of breast cancer subcutaneous transplanted tumor model mice was studied. 131I-CGRRAGSCs were obtained by using chloramine-T labeling CGRRAGGSC, and the labeling rate and stability were detected by thin layer chromatography. The inhibitory effect of 131I-CGRRAGSC on breast cancer cells and the expression of IL-11R 偽 and STAT3- STAT3-STAT3 protein in breast cancer cells before and after treatment were detected by Western blot and scratching assay. Results the expression of 10% IL-11R 偽 in breast cancer cells was higher than that in normal breast cancer cells. Breast cell MCF-10A.CGRRAGSCC Tyr-CGRRAGGSC and DTPA-CGRRAGGSC can bind to each breast cancer. The binding ability of CGRRAGSC treated with cold CGRRAGGSC or anti IL-11R 偽 antibody was significantly decreased. Near infrared imaging showed that the fluorescence signal of tumor site after Cy7-CGRRAGGSC injection was significantly higher than that of other organs or tissues. The fluorescence signals of tumor in Cy7 group were similar to those in normal organs or tissues. 131I-CGRRAGSC and 99mTc-DTPA-CGRRAGSCC were successfully prepared, and the labeling rates of both groups were above 95%. After being placed at 37 鈩，

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