MicroRNA (miRNA) 是一类内生的、长度约为20~24个核苷酸的小RNA，其在细胞内具有多种重要的调节作用。每个miRNA可以有多个靶基因，而几个miRNA也可以调节同一个基因。最近的研究表明，microRNA-9（miR-9）作为miRNA的一种，在细胞生长、分化、免疫和凋亡等多个过程中起着重要作用。在很多癌细胞中，miR-9的表达受到抑制，如多种乳腺癌细胞系。传统检测microRNAs表达的方法主要包括Northern杂交、实时定量PCR和基因芯片等技术。然而，这些检测手段常常耗时耗力，并且需要重复制备样本和进行侵入性检测。近年来，分子影像技术的快速发展使得在体、无创、实时研究microRNAs成为可能。光声成像是近年来发展起来的一种非入侵式和非电离式的新型生物医学成像方法。生物组织产生的光声信号携带了组织的光吸收特征信息，通过探测光声信号能重建出组织中的光吸收分布图像。光声成像结合了纯光学组织成像中高选择特性和纯超声组织成像中深穿透特性的优点，可得到高分辨率和高对比度的组织图像。因此，无损光声成像作为一种新兴的医学影像技术，能够在一定的深度下获得足够高的分辨率和图像对比度，图像传递的信息量大，可以提供形态及功能信息，在生物医学应用领域具有广阔的应用前景。酪氨酸酶是黑色素合成的关键酶，而黑色素是一种非常适合于光声成像的介质，无毒、稳定且生物亲和性好。利用酪氨酸酶报告基因光声成像系统来检测miR-9的表达变化，在癌症治疗及其临床应用领域具有重要意义。 本研究主要是利用酪氨酸酶的产物黑色素吸光能力强且生物相容性好的性质，开发监测miR-9表达的酪氨酸酶新型分子探针，利用光声成像分别在细胞水平和动物水平非侵入性监测内源性miR-9的表达变化及甲基化药物5-aza-2’-deoxycytidine刺激下miR-9的表达变化，从而实现miR-9的无创监测，为miRNA在活体表达水平的无创性监测提供了新的成像工具。 一、利用基因克隆技术构建酪氨酸酶报告基因TYR及含有三段miR-9靶序列的酪氨酸酶报告基因TYR-3×PT，通过单独将两种报告基因质粒转染进细胞，以及报告基因质粒与miR-9共转染，检测酪氨酸酶活性和黑色素含量变化，以验证报告基因对内源性和外源性miR-9表达的响应；二、在细胞水平和小鼠体内分别进行光声成像：对于转染了不同浓度质粒的293T细胞、共转同等浓度质粒和不同浓度的miR-9的293T细胞、加药前后处理的A549细胞分别进行3D手持扫描和仿体断层扫描的光声成像；将加药前后处理的A549细胞分别注射在小鼠大腿根部进行光声断层成像。 成功构建了miR-9调控的酪氨酸酶报告基因探针TYR-3×PT，能够监测miR-9的表达水平变化。在一定范围内，酪氨酸酶活性和黑色素含量随报告基因转染浓度的增加而增大，miR-9的抑制作用也随其转染浓度的增大而增强。去甲基化药物5-Aza-2’-deoxycytidine促进了A549细胞内miR-9的表达。光声成像能够检测出由质粒和miR-9的不同转染浓度导致的信号差异，以及药物刺激前后，在小鼠体内和体外的信号差异。 酪氨酸酶报告基因TYR-3×PT适合作为做光声成像的介质，利用靶向miR-9的酪氨酸酶报告基因和光声设备，能够在细胞水平和动物水平检测miR-9的表达变化，为miRNA在活体表达水平的无创性监测提供了新的成像手段。

MicroRNAs (miRNAs), a class of small RNAs including 22~24 nucleotides, are significant regulators of many cellular functions. One miRNA can have many target genes, also different miRNAs can target a same gene. Recent studies have identified microRNA-9 (miR-9) as one such miRNA plays an important role in cell growth, differentiation, neurogenesis, immunity and apoptosis. MiR-9 is decreased in many types of cancer, such as many breast cancer cell lines. Conventional detection methods, such as northern blot, Real-time PCR or microarray, have been used to assess miRNA expression. However, these techniques are labor-intensive and time-consuming, and require the fixation or lysis of cells. Recent remarkable advances in molecular imaging techniques have provided the capability of noninvasive repeated quantitative imaging of miRNAs in living animals. Photoacoustic imaging (PAI), a novel nonionizing and noninvasive modality on ac of optical absorption excitation and ultrasonic detection, provides high-resolution optical contrast information in deep tissues and breaking through resolution restrictions of pure optical imaging techniques, which is gaining attention in the field of medical imaging. It is highly sensitive to melanin, one of the principal absorbers in skin, and tyrosinase is the key enzyme in ge of generation of melanin. Melanin is a very suitable contrast agent for PAI, which is non-toxic, stable and has good biological compatibility. Using tyrosinase report gene and photoacoustic imaging as a system to detect the expression of miR-9, is of great significance of cancer treatment and its clinical application.

 

The goal of this research is to monitor the change of miR-9 expression noninvasively in vivo using photoacoustic imaging and new molecular probes. 5-aza-2'-deoxycytidine can influence the expression of miR-9 in A549 cells. As the product of tyrosinase, melanin has strong absorption ability and good biocompatibility properties, the tyrosinase gene and miR-9 target sequence were synthesized into a new photoacoustic molecular probes which expression is regulated by miR-9. Combine the probes with light sound equipment to monitor the change of miR-9 expression.

 

First, build tyrosinase report gene, detect melanin expression in vitro and validate report gene targeting to miR-9: using gene synthesis plas TYR, TYR-3×PT; Two plass were transfected into 293T cells at a certain concentration gradient, respectively, then detect tyrosinase activity and melanin content; miR-9 and TYR-3×PT were simultaneously transfected into 293T cells at a certain concentration gradient, then detect tyrosinase activity and melanin content. Second, photoacoustic imaging is performed with EP tubes, phantom and mice, respectively: 293T cells transfected with different concentrations of plas, 293T cells transfected with the same concentration of plas and different concentrations of miR-9, and A549 cells transfected by plas before and after dosing were uted 3D photoacoustic handheld scanning with EP tube and photoacoustic tomography imaging with phantom; The mices which thighs were rapidly injected with A549 cells transfected with plas before and after dosing implemented 3D photoacoustic handheld scan and photoacoustic tomography imaging.

 

Successful build the tyrosinase report gene targeting miR-9, miR-9 can inhibit tyrosinase report gene expression. Within a certain range, the report gene expression increases with the increased concentration of its transfection, miR-9 inhibition also along with the increase of its transfection concentration increased. To methylation drug 5-Aza-2'-deoxycytidine promoted the expression of A549 cells miR-9. Photoacoustic imaging to detect the plas and miR-9 different transfection concentration signal caused by the differences, and drugs before, during and after the stimulus signal differences in mice in vivo and in vitro.

 

We proved that tyrosinase report gene is suitable for photoacoustic imaging. Using tyrosinase report gene targeted miR-9 and photoacoustic equipment, change of miR-9 expression can be detected in A549 cells before and after 5-Aza-2'-deoxycytidine stimulus.

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