恶性肿瘤严重威胁人们的生活质量，是我国最常见的致死因素之一。因此，发展新的诊断技术和诊疗方法防治癌症具有重要的社会意义。近年，活体成像技术的蓬勃发展对疾病诊疗领域做出了意义深远的贡献。体内成像系统能够直接监测机体的细胞活性和基因行为，应用前景广阔，为癌症的诊断提供了新的思路。随着对分子成像技术的深入研究，多模态成像技术现今已成为一个研究热点。该技术通过构建两种或两种以上成像模式相结合的方法，应用各自的优势，互补各自的劣势，进而为疾病的诊断提供更加全面的信息。生物发光成像（Bioluminescence Imaging, BLI）是基于荧光素酶-荧光素对反应产生的生物发光，具有特异性强、灵敏度高、极低的背景和极高的信噪比等优点。萤火虫荧光素酶（Firefly Luciferase, Fluc）是众多荧光素酶的一种，与其他天然荧光素酶相比，发光波长较长（560 nm），是一种常用的信号分子。光声成像（Photoacoustic Imaging, PAI）是一种基于不同生物组织发色团对光吸收差异的新型无损成像方法，将高对比度的光学成像和高穿透深度的超声成像相结合，克服了光学成像组织穿透深度的不足，并能提供多尺度、多维度的光声图像信息。金纳米粒子（Au NPs）可以有效地增强组织的特异性吸收，与常规的光吸收造影剂（如ICG）相比高出约10⁵倍。金纳米棒（Au NRs）具备独特的光学特性、良好的生物相容性和可控的表面修饰等优点，使其有望在生物成像、癌症治疗等多种领域得到应用。上述两种成像模式都可以对活体动物不同组织部位的各种生化过程进行实时、连续的监测，且不会对组织造成损伤。

基于此，本论文开发了一种双模态成像探针。该探针可以同时进行生物发光成像和光声成像，并在此基础上修饰靶向分子叶酸（Folic Acid, FA）和耐久霉素（Duramycin），实现双模态探针特异性靶向肿瘤细胞的功能，达到对肿瘤细胞精准成像的目的。首先构建Fluc的基因重组表达菌株，对目标蛋白表达纯化，使其可用于生物发光成像研究。其次制备Au NRs，以实现光声成像的研究。然后通过化学合成的方法制备靶向叶酸受体阳性细胞的双模态成像探针，从而提升对特定肿瘤的靶向效果。最后通过化学合成的方法制备双模态细胞凋亡检测探针，进一步对凋亡细胞进行可视化检测。通过上述研究，本论文的工作将有望为以细胞凋亡为主要特征的相关疾病的诊断提供新思路。具体工作内容如下：

（1）采用克隆载体pRSETA，宿主细胞E.coli BL21（DE3）构建了Fluc的基因重组表达菌株E.coli BL21（DE3）（pRSETA-Fluc）。异丙基-β-D-硫代半乳糖苷（IPTG）诱导Fluc过表达，镍柱亲和层析法初步纯化Fluc蛋白。SDS-PAGE分析得出61 kDa左右出现目的条带，借助具有相对截留分子量的超滤管超滤浓缩得到后续实验所需的蛋白。

（2）晶种法合成Au NRs。首先，将CTAB与HAuCl₄快速混匀，再加入冰冷的NaBH₄，在27℃恒温培养箱中静置2 h。随后依次添加CTAB，HAuCl₄，AgNO₃，H₂SO₄制备生长液，再滴加L-抗坏血酸。最后，加入金种子溶液并将其置于27°C恒温培养箱中静置过夜。离心后用去离子水洗涤以除去多余的CTAB，并在去离子水中重新分散以获得后续实验所需要的Au NRs。

（3）制备靶向叶酸受体阳性细胞的双模态成像探针（FA-Fluc-Au, FFA），实现叶酸受体阳性细胞的生物发光和光声成像。叶酸受体（FR）集中表达在许多恶性肿瘤细胞表面，几乎不表达在正常细胞表面。FFA探针通过FR的特异性内吞作用选择性靶向到特定肿瘤细胞，后续可以配合治疗药物实时监测肿瘤细胞的变化。体内外实验表明，FFA没有毒副作用，生物相容性较好。与FA阻断组、非靶向组和正常细胞对照组相比，实验组具有更高的信号强度，表明该探针具有特异性靶向FR阳性细胞的能力。

（4）为了提高双模态成像探针的应用性，构建了基于耐久霉素（Duramycin）的双模态细胞凋亡检测探针（Duramycin-Fluc-Au, DFA）。细胞凋亡是生物体内生理性和程序性细胞死亡的过程。在细胞凋亡早期，细胞膜内的磷脂酰乙醇胺（Phosphatidylethanolamine, PE）翻转到膜外，Duramycin可以与PE特异性结合，实现对细胞凋亡的无创、实时监测，精准判断肿瘤细胞对治疗药物的反应。体内外实验表明，DFA没有明显的细胞毒性，生物相容性较好。更重要的是，阿霉素（DOX）诱导细胞凋亡后，将DFA与肿瘤细胞孵育，可以明显看出随着DOX诱导时间和浓度的增加，探针信号增强，表明探针可以特异性靶向凋亡细胞。进一步从体内动物实验中得出，与DOX未诱导组、非靶向组相比，实验组具有更高的信号强度，再次验证了探针特异性靶向凋亡细胞的能力，为后续监测肿瘤细胞凋亡奠定了基础。

Malignant tumors severely threaten people's quality of life and are one of the most common causes of death in China. Therefore, it is socially important to develop new theranostic methods to prevent and treat cancer. Recently, the flourishing development of in vivo imaging technology has made significant contributions to the field of medical imaging. In vivo imaging system can directly monitor the cellular activity and genetic behavior of the body, which has a good and broad application prospect and provides a new way of thinking for the diagnosis and treatment of cancer. With the increasing development and further investigation of molecular imaging technology, multimodal imaging has become a hot spot, which provides more comprehensive information for disease diagnosis by constructing a method combining two or more imaging modalities, applying their respective advantages and complementing their respective disadvantages. Bioluminescence imaging (BLI) is based on bioluminescence generated by luciferase-luciferin pair reaction, which has high specificity and sensitivity, low background and high signal-to-noise ratio. Firefly luciferase (Fluc) is one of the many luciferases with wavelengths up to 560 nm compared to other natural luciferases and is a commonly used signaling molecule. Photoacoustic imaging (PAI) is a new nondestructive imaging method based on the difference in light absorption by chromophores of biological tissues, with high contrast of pure optical imaging and high penetration depth of pure ultrasound imaging, overcoming the inadequate methods of traditional medical imaging and giving multi-scale and multi-dimensional photoacoustic image information. Gold nanoparticles (Au NPs) can effectively enhance tissue-specific absorption, which is about 10⁵ times higher compared to conventional light-absorbing contrast agents (e.g. ICG). Gold nanorods (Au NRs) possess exceptional optical characteristics, excellent biocompatibility, and accessible surface modifications, which make them promising for a variety of applications in bioimaging, cancer therapy, and other fields. Both imaging modalities allow real-time, continuous monitoring experiments of various biochemical processes in different tissue sites of living animals without causing damage to the tissues, and are nondestructive imaging modalities.

 

Based on the above considerations, this thesis explores the use of bioluminescence imaging and photoacoustic imaging as two imaging modes of dual-mode imaging probes, based on which folic acid (FA) and Duramycin are further added as targeting molecules to realize the function of dual-mode probes to specifically target tumor cells for precise targeting and imaging studies. Firstly, we constructed a recombinant expression strain of firefly luciferase and purified the expression of the target protein, which can be used for bioluminescence imaging studies. Secondly, gold nanorods were prepared to enable photoacoustic imaging studies. Then dual-mode imaging probes targeting malignant tumor cells with high expression of folate receptors were prepared by chemical synthesis to improve the targeting effect on specific tumors. Finally, a dual-mode apoptosis detection probe was prepared by chemical synthesis to further visualize apoptotic cells. With the above studies, the work in this thesis will hopefully provide new ideas for the diagnosis of related diseases with apoptosis as the main feature. Details of the work are as follows:

 

(1) A recombinant expression strain of the gene for firefly luciferase, E.coli BL21 (DE3) (pRSETA-Fluc), was constructed using the cloning vector pRSETA, host cell E.coli BL21 (DE3). Isopropyl-β-D-thiogalactoside (IPTG) overexpression of Fluc was followed by Ni-NTA affinity chromatography purification. SDS-PAGE analysis revealed the target band to be approximately 61 kDa, and the protein needed for further experiments was determined by the ultrafiltration concentration in relation to the ultrafiltration tube's interception molecular weight.

 

(2) Gold nanorods were synthesized by seed method. First, CTAB and HAuCl₄ were mixed quickly, then ice-cold NaBH₄ was added and placed in a constant temperature incubator at 27°C for 2 h. Subsequently, CTAB, HAuCl₄, AgNO₃, H₂SO₄ were added sequentially to prepare the growth solution, and then L-ascorbic acid was added dropwise. Finally, the gold seed solution was added and placed in a constant temperature incubator at 27°C and left overnight. After centrifugation, they were washed with ultrapure water to remove excess CTAB and redispersed in ultrapure water to obtain the Au NRs required for subsequent experiments.

 

(3) A dual-mode imaging probe (FA-Fluc-Au, FFA) targeting folate receptor highly expressing malignant tumor cells was prepared by chemical synthesis to achieve bioluminescence and photoacoustic imaging of folate receptor highly expressing malignant tumor cells. The FFA probe selectively targets to specific tumor cells through the specific endocytosis of FR and can be subsequently used with therapeutic drugs to monitor tumor cell changes in real time. In vitro and in vivo experiments have shown that FFA has no toxic side effects and is biocompatible. More importantly, the experimental group had higher signal intensity compared with the FA blocking group, non-targeting group and normal cell control group, indicating that the probe has the ability to specifically target folate receptor high expression malignant tumor cells.

 

(4) To improve the applicability of the bimodal imaging probe, a dual-mode apoptosis detection probe (Duramycin-Fluc-Au, DFA) based on Duramycin was constructed. Apoptosis is a process of physiological and programmed cell death in living organisms. At the early stage of apoptosis, phosphatidylethanolamine (PE) flips from inside to outside the cell membrane. Duramycin can specifically bind to PE to achieve non-invasive, real-time monitoring of apoptosis and accurately determine the response of tumor cells to therapeutic drugs. In vivo and in vitro experiments have shown that DFA has no significant cytotoxicity and good biocompatibility. More importantly, after DOX induced apoptosis, incubation of the DFA probe with tumor cells clearly showed enhanced probe signal with increasing DOX induction time and concentration, indicating that the probe could specifically target apoptotic cells. Further from in vivo animal experiments, it was concluded that the experimental group had higher signal intensity compared with the DOX uninduced group and the non-targeted group, which again verified the ability of the probe to specifically target apoptotic cells and laid the foundation for subsequent monitoring of tumor cell apoptosis.

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