放射性标记CGRRAGGSC在IL-11Rα阳性表达乳腺癌中的显像及体外干预研究

发布时间：2018-03-07 09:38

  本文选题：多肽　切入点：白细胞介素-11受体α　出处：《南京医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景:乳腺癌是女性发病率最高的恶性肿瘤,且有65%-75%的乳腺癌患者发生骨转移。研究发现IL-11/IL-11Rα信号通路参与乳腺癌骨转移发生发展,IL-11Rα能作为乳腺癌、骨肉瘤等受体显像的潜在靶点,其在乳腺癌显像及治疗的应用值得进一步探索。目的:本实验通过评估IL-11类似物环九肽CGRRAGGSC对IL-11Rα的靶向性,并在此基础上构建放射性核素标记CGRRAGGSC探针进行乳腺癌模型鼠显像及体外对乳腺癌细胞抑制作用的研究。方法:采用蛋白印迹(Western blot)检测乳腺癌细胞(MDA-MB-231、MCF-7和BT549)和正常乳腺细胞MCF-10A中IL-11Rα的表达。体外细胞免疫荧光观察冷CGRRAGGSC或抗IL-11Rα抗体处理前后,CGRRAGGSC、对照环肽CGSPGWVRC和酪氨酸(Tyr)或螯合剂(DTPA)修饰后的CGRRAGGSC对各乳腺癌细胞的结合情况。近红外线显像评估CGRRAGGSC在乳腺癌MCF-7模型鼠体内对IL-11Rα的靶向特异性情况。通过氯化亚锡还原法标记环九肽CGRRAGGSC获得标记产物99mTc-DTPA-CGRRAGGSC,纸层析检测标记率及稳定性,以乳腺癌皮下移植瘤模型鼠进行SPECT成像研究。通过氯氨T法标记CGRRAGGSC获得131I-CGRRAGGSC,薄层层析检测标记率及稳定性,行噻唑蓝(MTT)法、Transwell、划痕实验及 Western blot 检测 131I-CGRRAGGSC 对乳腺癌细胞的抑制作用及处理前后细胞内IL-11Rα和STAT3、p-STAT3蛋白的表达变化。结果:IL-11Rα在本研究的乳腺癌细胞中的表达高于正常乳腺细胞MCF-10A。CGRRAGGSC、Tyr-CGRRAGGSC 和 DTPA-CGRRAGGSC 都能与各乳腺癌发生特异性结合,而对照肽CGSPGWVRC几乎不结合;用冷CGRRAGGSC或抗IL-11Rα抗体处理后,CGRRAGGSC的结合能力明显减弱。近红外显像示注射Cy7-CGRRAGGSC后肿瘤部位的荧光信号明显高于其它器官或组织,而注射Cy7组肿瘤的荧光信号与正常器官或组织相似。成功制备了 131I-CGRRAGGSC和99mTc-DTPA-CGRRAGGSC,两者的标记率都达95%以上,并且在37℃放置12 h后的放射性化学纯度都在90%以上。SPECT显像示瘤体间质内给药后,99mTc-DTPA-CGRRAGGSC在荷瘤鼠瘤体内的滞留时间长且放射性高。131I-CGRRAGGSC处理后,乳腺癌细胞的增殖及迁移能力减弱,其机制可能与降低IL-11Rα阻断STAT3的磷酸化相关。结论:放射性核素标记CGRRAGGSC方法简便,标记率高,并且对IL-11Rα靶向性强,有望用于阳性表达IL-11Rα肿瘤的显像及治疗研究。
[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 伪 signaling pathway is involved in the development of breast cancer bone metastasis and IL-11R 伪 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 伪 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 伪 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# 伪 antibody, CGSPGWVRC and tyrosine tyrosine tyrosine (CGSPGWVRC) or chelating agent to breast cancer cells. The target specificity of CGRRAGGSC to IL-11R 伪 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 伪 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 伪 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 伪 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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