ND2SmoA1转基因荷瘤小鼠的培育、鉴定和脑血管成像研究

Study on cultivation, identification and cerebrovascular imaging of transgenic tumor-bearing ND2:SmoA1 mice

杨毅诚

11849028

硕士

生物医学工程学

吴长锋

2020-06-08

2020-07-20

哈尔滨工业大学

深圳

恶性颅内肿瘤是致死率最高的恶性肿瘤之一，其中髓母细胞瘤是儿童群体中最常见的神经系统恶性肿瘤。对于癌症研究来说，在小鼠上创建可靠的疾病模型对疾病诊断以及治疗手段的开发与完善有着重要作用。目前对于恶性颅内肿瘤，外科手术介入依旧是预防和治疗癌症症状的关键手段。研究表明，广泛，精准的手术切除可以大大延长脑瘤病人尤其是高级别恶性胶质瘤病人的生存期。近年来，利用肿瘤荧光标记物实现肿瘤诊断以及实时成像指导手术切除成为一项提高肿瘤清除精度与患者术后生存期的重要技术。而半导体聚合物纳米颗粒(semiconducting polymer nanoparticles, SPN)由于其作为荧光探针的多种突出优点引起了相当大的关注，在该领域拥有巨大的潜能。本文的主要内容是对The Jackson Laboratory公司的髓母细胞瘤模型，ND2:SmoA1转基因小鼠进行繁育以及基因与表型鉴定，证明了该模型产生脑瘤的能力。通过静脉注射，对荷瘤小鼠脑部血管进行基于合成的半导体共轭聚合物纳米材料的近红外二区荧光成像实验，证明了该材料的脑部血管成像能力以及作为原发性颅内恶性肿瘤模型在开发脑瘤实时成像系统方面的应用潜能。本文通过纳米沉淀法合成了半导体共轭聚合物纳米材料，通过对其吸收发射以及生物兼容性的表征实验证明其具有高量子效率以及优秀的近红外二区成像能力。在繁育该品系小鼠时使用了纯合ND2:SmoA1公鼠与C57野生型母鼠交配，再通过子代自交与定量PCR基因鉴定技术筛选出纯合ND2:SmoA1小鼠。与直接使用纯合ND2:SmoA1小鼠交配繁衍后代的方法相比，大大增加小鼠扩群速度的同时，规避了ND2:SmoA1纯合母鼠在怀孕以及哺育后代期间因脑瘤发病死亡的风险。在此基础上，本文完成了对早期与晚期荷瘤小鼠的脑部近红外二区荧光成像实验，与对照组正常小鼠数据对比可明显分辨小鼠是否荷瘤，并通过病理切片进行了验证。未来通过此模型与先进的半导体纳米成像材料配合，有望进一步开发出精准可靠的颅内肿瘤实时成像系统。

Malignant intracranial tumor is one of the malignant tumors with the highest lethality rate, among which medulloblastoma is the most common malignant tumor of nervous system in children. For cancer research, the creation of reliable disease models on mice plays an important role in the development and improvement of disease diagnosis and treatment. At present, for malignant intracranial tumors, surgical intervention is still the key method for preventing and treating cancer symptoms. Studies have shown that extensive and precise surgical resection can greatly extend the survival of patients with brain tumors, especially those with high-grade malignant gliomas. In recent years, the use of tumor fluorescent markers to achieve tumor diagnosis and real-time imaging-guided surgical resection has become an important technology to improve tumor removal accuracy and patient survival. Semiconductor semiconducting nanoparticles(SPN) have attracted considerable attention because of their many outstanding advantages as fluorescent probes, and have great potential in this field. The main content of this article is the breeding of the Jackson Laboratory medulloblastoma model, ND2:SmoA1 transgenic mice and the identification of genes and phenotypes, which proves the model's ability to produce brain tumors. Afterwards, an intravenous two-region infrared imaging experiment based on synthetic semiconductor conjugated polymer nanomaterials was performed on the brain blood vessels of tumor-bearing mice, which proved the brain blood vessel imaging ability of the material and the primary intracranial malignancy The application potential of tumor models in developing real-time imaging systems for brain tumors. In this paper, the semiconductor conjugated polymer nanomaterials were synthesized by nanoprecipitation method[2], and their absorption and emission and biocompatibility characterization experiments proved that they have high quantum efficiency and excellent infrared two-region imaging capability. When breeding mice of this strain, homozygous ND2:SmoA1 male mice were mated with C57 wild-type female mice, and homozygous ND2:SmoA1 mice were screened by progeny self-crossing and quantitative PCR gene identification technology. Compared with the method of directly mating and breeding offspring using homozygous ND2:SmoA1 mice, it greatly increases the expansion rate of mice while avoiding the risk of ND2:SmoA1 homozygous female mice due to brain tumors during pregnancy and feeding offspring. On this basis, this paper completed the infrared two-region fluorescence imaging experiment on the brain of early and late tumor-bearing mice. Comparison with the data of normal mice in the control group can clearly distinguish whether the mice are tumor-bearing and verified by pathological section. In the future, with this model and advanced semiconductor nano-imaging materials, it is expected to further develop an accurate and reliable intracranial tumor real-time imaging system.

颅内肿瘤模型 模式动物培育与鉴定 半导体聚合物 近红外二区荧光成像

intracranial tumor model model animal breeding and identification semiconductor nanomaterial NIR-II fluorescence imaging

中文

联合培养

南方科技大学

杨毅诚. ND2SmoA1转基因荷瘤小鼠的培育、鉴定和脑血管成像研究[D]. 深圳. 哈尔滨工业大学,2020.