中国人膀胱癌BIU-87细胞特异性导向肽-SISSLTH的体内外功能验证

发布时间：2018-11-23 15:03

【摘要】：研究背景：膀胱肿瘤（Bladder carcinoma）是临床上最常见的恶性肿瘤之，其发病率和死亡率在泌尿系肿瘤中均居首位。非肌肉浸润性膀胱癌(non-muscleinvasive bladder cancer）约占全部膀胱肿瘤的75—85%，虽然其恶性程度低，但大约有10%的患者最终可发展为肌肉浸润性膀胱癌。因此如何对膀胱肿瘤进行早期诊断和靶向治疗就成为当今医学领域研究的热点内容。目的：建立中国人膀胱癌BIU-87细胞系的裸鼠皮下移植瘤模型，，对小分子环七肽片段-SISSLTH在细胞和组织不同水平进行验证，鉴定该导向肽对中国人膀胱癌BIU-87细胞结合的特异性和亲和性，为未来临床上对膀胱肿瘤进行早期诊断、术中准确切除和开发靶向药物提供定的理论基础。方法：将Matrigel基质胶作为中国人膀胱癌BIU-87细胞的悬浮剂，用注射器将癌细胞种植于裸鼠腋窝背侧皮下建立裸鼠移植瘤模型。通过化学方法合成小分子环七肽片段-SISSLTH，纯化并标记绿色荧光染料异硫氰酸荧光素FITC，利用免疫荧光技术原理，通过应用流式细胞术和激光共聚焦显微镜技术两种方法，对小分子多肽荧光探针FITC-SISSLTH在细胞和组织不同水平进行验证，鉴定其与中国人膀胱癌细胞结合的特异性和亲和性，以及该小分子探针在荷瘤裸鼠体内的生物分布情况。结果：在细胞水平对小分子多肽荧光探针FITC-SISSLTH进行验证，其结果显示：小分子多肽荧光探针FITC-SISSLTH与中国人膀胱癌BIU-87细胞系的亲和性明显高于人肝癌Hep-G2细胞系和人前列腺癌PC-3细胞系。利用激光共聚焦显微镜技术研究小分子探针在荷瘤裸鼠体内的分布，实验结果表明：该探针可以特异性地在肿瘤组织中富集，而其他脏器中聚集很少。说明该探针对膀胱癌组织有特异性和靶向性。结论：在细胞水平对小分子多肽荧光探针FITC-SISSLTH进行验证，其结果显示：小分子多肽荧光探针FITC-SISSLTH与中国人膀胱癌BIU-87细胞系的亲和性明显高于人肝癌Hep-G2细胞系和人前列腺癌PC-3细胞系。利用激光共聚焦显微镜技术研究小分子探针在荷瘤裸鼠体内的分布，实验结果表明：该探针可以特异性地在肿瘤组织中富集，而其他脏器中聚集很少。说明该探针对膀胱癌组织有特异性和靶向性。
[Abstract]:Background: bladder tumor (Bladder carcinoma) is one of the most common malignant tumors in clinic. Non-muscle invasive bladder cancer (non-muscleinvasive bladder cancer) accounts for 75-85% of all bladder tumors. Although its malignancy is low, about 10% of the patients can eventually develop musculoinvasive bladder cancer. Therefore, how to make early diagnosis and targeted therapy of bladder tumors has become a hot topic in the field of medicine. Objective: to establish a subcutaneous transplanted tumor model of Chinese bladder cancer BIU-87 cell line in nude mice and to verify the different levels of small molecule cyclic heptapeptide fragment (SISSLTH) in cells and tissues. The specificity and affinity of the targeting peptide to BIU-87 cells in Chinese bladder cancer were identified, which provided a theoretical basis for early diagnosis, intraoperative excision and development of targeted drugs for bladder cancer in the future. Methods: Matrigel matrix glue was used as the suspension of Chinese bladder cancer BIU-87 cells. The tumor cells were implanted into the dorsal subcutaneous axillary of nude mice with syringe to establish the transplanted tumor model in nude mice. SISSLTH, was synthesized by chemical method for the purification and labelling of green fluorescent dye fluorescein isothiocyanate (FITC,). Flow cytometry and laser confocal microscopy were used to purify and label the green fluorescent dye fluorescein isothiocyanate (FITC,) by using the principle of immunofluorescence. The specificity and affinity of the small molecular polypeptide fluorescence probe (FITC-SISSLTH) to Chinese bladder cancer cells were confirmed at different levels of cell and tissue, and the biological distribution of the small molecule probe in nude mice was also studied. Results: the fluorescence probe FITC-SISSLTH of small polypeptide was verified at the cell level. The results showed that the affinity of small molecular peptide fluorescent probe FITC-SISSLTH to Chinese bladder cancer BIU-87 cell line was significantly higher than that of human hepatocellular carcinoma Hep-G2 cell line and human prostate cancer PC-3 cell line. The distribution of small molecular probe in nude mice bearing tumor was studied by laser confocal microscopy. The experimental results showed that the probe could be specifically enriched in tumor tissue, but few in other organs. The results showed that the probe was specific and targeted to bladder cancer. Conclusion: the fluorescence probe FITC-SISSLTH of small polypeptide was verified at cell level. The results showed that the affinity of small molecular peptide fluorescent probe FITC-SISSLTH to Chinese bladder cancer BIU-87 cell line was significantly higher than that of human hepatocellular carcinoma Hep-G2 cell line and human prostate cancer PC-3 cell line. The distribution of small molecular probe in nude mice bearing tumor was studied by laser confocal microscopy. The experimental results showed that the probe could be specifically enriched in tumor tissue, but few in other organs. The results showed that the probe was specific and targeted to bladder cancer.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14

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