| 【Background】Lung cancer is a common disease which seriously threaten the health and life of human beings, the morbidity and mortality of which in the first place in common malignant tumor. A majority of lung cancer patients were diagnosed with advanced cancer for the clinical symptoms of the initial stage were unapparent, the treatment effect of the traditional means like chemotherapy or radiotherapy is poor, the emerging therapeutic methods as tumor targeted therapy drugs and immune check-point blockers showed sustained survival benefit in the treatment of lung cancer patients, both of the two therapy approach have limitation, a novel targeting treatment strategy were required to make breakthrough in the field of diagnosing and curing lung cancer. Gold nanorods have unique physical properties and biological properties like biocompatibility, surface modification and functionalization, enhanced permeability and retention, Surface Plasmon Resonance, solar-thermal conversion effect, which showed tremendous application foreground in the field of tumor diagnosis and treatment. Generally, gold nanoparticle composite which constructed by attaching target molecule to surface-modified gold nanorods will acquire active targeting function, it was widely applied in the field of tumor diagnosis and treatment such as molecule imaging, targeted drug delivery, tumor radiosensitivity, photothermal therapy. Aptamer is a small fragment DNA or RNA which was screened step by step and then amplified from the random single nucleic acid sequence libraries with the Systematic Evolution of Ligands by Exponential Enrichment technology, and the acquired aptamer could combine with target matter. To compared with traditional antibody, aptamer is a more ideal target molecule for its characteristics, it has high affinity and weak immunogenicity, easy to synthesis on large scale, easy to modified in vivo, easy to penetrate tumor tissue. MUC1 protein is a highly glycosylated Mucin which distributed on the surface of epithelial cell of respiratory tract, gland ducts, gastrointestinal tract, research has revealed the relation in the process of tumor occurrence, growth of tumor cell and metastasis of tumor cell, it will over express on the surface as the cell become cancerous, moreover, epitope on the core peptides of MUC1 was exposed, which making MUC1 become a broad spectrum target molecule. To bring out the potential efficiency of AuNRs and aptamer, we may acquire MUC1-aptamer which can specific binding to MUC1 protein on the surface of lung cancer cells with the SELEX technology, and then construct a novel compound with active targeting function through attaching the MUC1 aptamer to a surface-modified AuNRs. The novel AuNRs compound was applied to the field of early diagnosis and treatment in lung cancer, to explore the probably mechanism of the lung cancer cell uptake the AuNRs compound, and for further study, research were conducted on the targeting killing effect such as targeting drug delivery system, to aim at providing theoretic foundation of the new targeting diagnosis platform in lung cancer, provide a new route of the early diagnosis and therapeutic in lung cancer.【Objective】1. How to acquire AuNRs with two SPR peaks on 525 nm and near 800 nm through controlling the experimental conditions strictly and regulating the aspect ratio.2. Screening and synthesis MUC1-aptamer which have highly affinity with the MUC1 protein on the surface of A549 cells line in lung cancer.3. Based on the succeeded synthesis of the materials as the first steps, constructing AuNRs/MUC1-aptamer compound with active targeting function.4. To observe the characters of the constructed AuNRs/MUC1-aptamer compound as it binding MUC1 protein over-expressed on the surface of A549 cell line, to find the superiority compared with AuNRs. 5. To investigate the specific killing effect when A549 cells combined with AuNRs/MUC1-aptamer compound exposure with near infrared reflection laser which in human tissue projection window.【Method】1. Utilize the Seed-mediated growth methods to synthesis AuNRs, making the AuNRs have dual EPR peaks at 525 nm and near 800 nm by controlling the concentration of AgNO3, CTAB and ascorbic acid, verifying the dual SPR peaks with the method of ultraviolet spectrophotometry, the AuNRs acquired was served at 4℃ standby;2.Choose the S2.2 as the aim MUC1-aptemer which has highly affinity with MUC1 core peptide, connect the thiol at 5’ of the single stranded DNA to combine AuNRs, connect the 6’-FAM fluorophore as a molecular marker when specific binding target cells,correctness of the sequence and purity of the aim aptamer was detected with mass spectrometric detection; 3. Modify gold nanorods with sulfhydrylation MUC1-aptamer both efficiently and quickly by the method of rapid modification at low pH within a short time, the colorimetric method of gold nanoparticles and ultraviolet spectrophotometry were used to characterized and verified the physical property of the constructed AuNRs/MUC1-aptamer compound;4. The constructed AuNRs/MUC1-aptamer compound was incubated with subculture of the lung cancer cell line A549 and its control group human hepatocarcinoma cell line HepG2, respectively, to detect the targeted combination and intake of the constructed compound and target cells, fluorescence intensity on the surface of target cells represent the binding efficiency of targeting compound and cells;5. To utilize the near ultraviolet absorption peak and photo-thermal conversion properties of gold nanorods, radiate the target cells which incubated with AuNRs/MUC1-aptamer compound and gold nanorods with near infrared laser,respectively,then detect the killing effectiveness towards cancer cells with MTT assay, the cell viability was related to the compound it incubated.【Results】Gold nanorods that synthesized with the method of seed-mediated growth methods were detected ultraviolet absorption spectrum by the ultraviolet-spectrophotometer assay, the dual EPR peaks at 525 nm and near 800 nm,making the material to be the aim gold nanorods; modify gold nanorods with sulfhydrylation MUC1-aptamer by the method of rapid modification at low pH, to acquire the AuNRs/MUC1-aptamer compound, the colorimetric method of gold nanoparticles and ultraviolet spectrophotometry verified the succeeded construction of the target compound, and the target compound still has dual EPR peaks at 525 nm and near 800nm; After the constructed AuNRs/MUC1-aptamer was incubated with A549 cell lines,the fluorescence intensity was higher than other groups which observed with flurensence microscope, it was also detected quantitative with BD flow cytometry, fluorescence intensity of the group that A549 cells treated with AuNRs/MUC1-aptamer was 5 fold than the group treated with gold nanorods which has significant difference, while in the HepG2 liver cancer cell line showed no difference, indicated that the constructed target compound can specific bind with A549 lung cancer cell lines; after irradiating the cells incubated with AuNRs/MUC1-aptamer and AuNRs with near infrared laser, calculated the optical density value with MTT assay and the calculated the cell viability, cell viability of the target compound group significantly lower than the negative control group and the HepG-2 liver cell line control group(P<0.05).【Conclusion】Comprehensive analysis all parts of this subject we can draw the following conclusion: our current research have succeed constructed the AuNRs/MUC1-aptamer compound based on the combination of gold nanorods and aptamer, the target compound has the physical and chemical properties of gold nanorods as well as the target ability of aptamer, it can target identification with lung cancer cell line A549 whose surface were over-expressed with MUC1 protein, A549 cells that specific combined with AuNRs/MUC1-aptamer compound can be target killed by near infrared laser, the constructed targeting compound may provide new insight for the targeting diagnosis and therapy of lung cancer. |
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