CXCR4靶点近红外光学分子成像检测骨肉瘤原位及肺转移肿瘤病灶的研究

发布时间：2018-06-25 19:03

本文选题：骨肉瘤 + 转移　；参考：《第四军医大学》2015年硕士论文

【摘要】：【背景】骨肉瘤是最常见、高表达CXCR4、具有高肺转移率的原发恶性骨肿瘤。目前的解剖学成像手段并不能在分子水平早期诊断病变,如能研发一种新的具有CXCR4靶点特异性的分子成像模式,对骨肉瘤原位及肺转移病灶的早期诊断将大有裨益。【目的】利用CXCR4作为分子靶点研发一种近红外荧光分子成像造影剂,通过验证此造影剂与体内外骨肉瘤细胞的结合特性以及探测体内骨肉瘤肺部转移灶的可行性,为早期检测骨肉瘤及其肺转移灶提供一种新的分子成像工具。【方法】1.合成能够靶向CXCR4的肽段,并用IR-783近红外荧光染料标记的CXCR4靶点近红外造影剂CXCR4-IR-783。利用q RT-PCR、蛋白免疫印记实验以及免疫细胞荧光化学实验验证两种骨肉瘤细胞系(F5M2与F4)CXCR4的表达。通过慢病毒转染(sh RNA)建立CXCR4敲除的稳转F5M2骨肉瘤细胞系用于验证此造影剂特异性实验。利用近红外荧光共聚焦显微镜以及Kodak体内多光谱成像系统进行验证骨肉瘤细胞与此染料的结合实验。2.活体以及解剖后的裸鼠人骨肉瘤异种移植模型成像分别进行CXCR4-IR-783近红外荧光成像和(或)Micro CT成像。同时原位肿瘤以及解剖的器官和组织由HE染色进行验证,并由免疫组织化学检测骨肉瘤CXCR4的表达。3.体内F5M2原位异种移植裸鼠模型的肺部转移灶探测由Micro-CT、CXCR4靶点近红外荧光成像以及18F-FDT PET完成。将动物解剖后,各主要器官以及肺组织再次进行近红外体外荧光成像。骨肉瘤的肺部转移灶最终由HE染色进行验证,并由免疫组织化学检测其CXCR4表达。【结果】1.成功合成了一种CXCR4靶点的近红外荧光造影剂:CXCR4-IR-783。骨肉瘤细胞水平结合实验证实CXCR4-IR-783能够特异结合于表达CXCR4的骨肉瘤细胞。同时,CXCR4-IR-783与高表达CXCR4的骨肉瘤细胞结合表现出时间依赖性、剂量依赖性,且能够探测出数量仅为100个的F5M2细胞团。2.CXCR4-IR-783可被人骨肉瘤异种移植裸鼠模型中表达CXCR4的原位肿瘤细胞吸收及内化。其中高表达CXCR4的F5M2骨肉瘤荷瘤裸鼠获得的靶点背景比(4.87)约为低表达CXCR4的F4骨肉瘤荷瘤裸鼠(2.24)的两倍。同时,CXCR4-IR-783表现出从体内正常器官清除的时间依赖性。3.利用CXCR4-IR-783进行的近红外荧光成像可以探测到裸鼠原位种植F5M2骨肉瘤细胞4周后的自发性肺部转移灶,同时利用18F-FDG PET并不能探测到这些转移灶。【结论】通过利用CXCR4靶点特异性近红外造影剂CXCR4-IR-783进行近红外光学分子成像可以探测到CXCR4表达阳性的骨肉瘤原位病灶以及较早期的骨肉瘤转移灶。其可能成为探测骨肉瘤及其转移的一种具有潜力的分子成像手段。如将此种成像手段结合解剖学成像手段将可提高骨肉瘤的早期检出诊断率。
[Abstract]:Background Osteosarcoma is the most common, highly expressed CXCR 4, with high lung metastasis rate of primary malignant bone tumors. The present anatomical imaging methods can not be used for early diagnosis of lesions at the molecular level, such as the development of a new molecular imaging model with CXCR4 target specificity. It will be helpful for the early diagnosis of osteosarcoma in situ and lung metastases. [objective] to develop a near infrared fluorescence imaging contrast agent using CXCR4 as a molecular target. In order to provide a new molecular imaging tool for early detection of osteosarcoma and its lung metastases, the binding properties of the contrast medium with osteosarcoma cells in vivo and in vitro and the feasibility of detecting pulmonary metastasis in vivo were verified. [methods] 1. The peptide fragment which can target CXCR4 was synthesized and labeled with IR-783 near infrared fluorescent dye. CXCR4-IR-783was used as the target near infrared contrast agent. The expression of CXCR4 in two osteosarcoma cell lines (F5M2 and F4) was confirmed by Q RT-PCR, protein imprinting assay and immunocytofluorescence assay. CXCR4 knockout stably transformed F5M2 osteosarcoma cell line was established by lentivirus transfection (sh RNA) to verify the contrast agent specificity. Near infrared fluorescence confocal microscopy and Kodak multispectral imaging system were used to verify the binding of osteosarcoma cells to this dye. CXCR4-IR-783 near infrared fluorescence imaging and / or micro CT imaging were performed on xenotransplantation models of human osteosarcoma in vivo and after anatomy in nude mice. The expression of CXCR4 in osteosarcoma was detected by immunohistochemistry. The detection of lung metastasis in nude mice model of in vivo F5M2 orthotopic xenotransplantation was performed by micro-CT-CXCR4 target near infrared fluorescence imaging and 18F-FDT PET. After dissecting the animals, the main organs and lung tissues were re-imaged with NIR fluorescence imaging in vitro. The lung metastasis of osteosarcoma was finally verified by HE staining, and the expression of CXCR4 was detected by immunohistochemistry. [results] 1. A CXCR4 target near infrared fluorescent contrast agent: CXCR4-IR-783was successfully synthesized. Osteosarcoma cell level binding assay confirmed that CXCR4-IR-783 could specifically bind to osteosarcoma cells expressing CXCR4. The binding of CXCR4-IR-783 to osteosarcoma cells with high expression of CXCR4 was time-dependent and dose-dependent. Only 100 F5M2 cell clusters could be detected. 2. CXCR4-IR-783 could be absorbed and internalized by in situ tumor cells expressing CXCR4 in xenotransplantation of human osteosarcoma in nude mice. The target background of F5M2 osteosarcoma bearing nude mice with high expression of CXCR4 was about twice as high as that of F4 osteosarcoma bearing nude mice with low expression of CXCR4 (4.87) (2.24). At the same time, CXCR4-IR-783 showed a time dependence on the removal of normal organs from the body. Near-infrared fluorescence imaging using CXCR4-IR-783 could detect spontaneous pulmonary metastasis of F5M2 osteosarcoma cells implanted in situ in nude mice for 4 weeks. At the same time, 18F-FDG PET could not detect these metastatic foci. [conclusion] Near-infrared optical molecular imaging using CXCR4-IR-783 as a target specific contrast agent for CXCR4 can detect in situ lesions of osteosarcoma with CXCR4 positive expression. Early metastases of osteosarcoma. It may be a potential molecular imaging tool for detecting osteosarcoma and its metastasis. If this imaging method is combined with anatomical imaging, the early detection rate of osteosarcoma can be improved.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R738.1

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