Cy5标记寡脱氧核苷酸MT01在大鼠重要脏器组织中的分布

发布时间：2018-04-15 00:24

本文选题：寡脱氧核苷酸 + MT01　；参考：《吉林大学》2014年硕士论文

【摘要】：MT01是本课题组成员依据人线粒体DNA序列设计的单链寡脱氧核苷酸，可以靶向阻断TLR9的激活，减轻组织的炎症性损伤，抑制由TLR9激活引起的机体固有免疫应答，其功能和结构已获得国家专利保护[一种具有免疫抑制功能的寡核苷酸，国家发明专利，专利号CN101643496]。本课题组在前期工作中发现，MT01能够促进骨髓间充质干细胞向成骨细胞分化，增强成骨细胞成骨功能；同时，体内实验证实，在大鼠牙周炎模型中局部应用MT01，可以有效抑制牙周炎大鼠牙槽骨吸收。以上研究成果提示，MT01有可能开发为一种减缓牙槽骨吸收的新型制剂。因此，有必要对局部应用时，MT01在体内重要脏器内的分布、代谢、清除率以及毒理反应等进行系统的研究，从而为进一步深入研究开发MT01奠定实验基础。目的：通过局部注射Cy5标记寡脱氧核苷酸MT01，采用激光共聚焦显微镜观察MT01在大鼠体内重要脏器组织中不同时间点的分布情况，初步探讨其代谢分布的规律性。方法：选取60只SPF级健康雄性Wistar大鼠，体重200g±10g，随机数字表法分为实验组和对照组，每组30只。实验组于右侧上颌第一恒磨牙颊侧牙龈粘膜注射Cy5标记的MT012μg，对照组注射未经标记的MT012μg，于注射后15min、1h、4h、8h、16h、1d、2d、3d、4d和5d全麻下取大鼠肺、肝、脾、肾、心和脑组织，制备快速冰冻切片并染色。激光共聚焦显微镜下观测MT01荧光分布，以荧光阳性细胞率表示各脏器组织内MT01进入的量。结果：在对照组中未观察到荧光阳性细胞。在实验组中除心、脑组织外，肺、肝、脾和肾组织细胞中均可观察到荧光阳性细胞；肾组织中荧光阳性细胞呈片状分布，荧光主要集中于肾小管上皮细胞的胞质中；肺、肝、脾组织中荧光阳性细胞呈点状散在分布；肝、脾和肾组织的荧光阳性细胞率随时间呈规律性变化，峰值分别出现在注射后4、3和4d时，而肺组织的荧光阳性细胞率随时间未见明显规律性。肾组织的荧光阳性细胞率明显高于其他组织。结论：MT01经大鼠牙龈局部注射后可进入肺、肝、脾和肾组织，分布具有一定规律性，，MT01主要集中在肾脏组织中，提示肾脏可能为MT01主要代谢器官。
[Abstract]:MT01 is a single-stranded oligodeoxynucleotide designed according to the sequence of human mitochondrial DNA. It can block the activation of TLR9, alleviate the inflammatory injury of tissues and inhibit the innate immune response induced by TLR9 activation.Its function and structure have been protected by national patent [an immunosuppressive oligonucleotide, national invention patent, patent number CN101643496].In our previous work, we found that MT01 could promote the differentiation of bone marrow mesenchymal stem cells into osteoblasts and enhance the osteogenic function of bone marrow mesenchymal stem cells.Local application of MT01 in rat periodontitis model can effectively inhibit alveolar bone resorption in rats with periodontitis.These results suggest that MT01 may be a novel preparation for reducing alveolar bone resorption.Therefore, it is necessary to systematically study the distribution, metabolism, clearance rate and toxicological reaction of TMT 01 in important organs during local application, so as to lay an experimental foundation for further research and development of MT01.Aim: to observe the distribution of MT01 at different time points in important organs of rats by local injection of Cy5 labeled oligodeoxynucleotides (MT01) and to explore the regularity of metabolic distribution of MT01 at different time points.Methods: 60 SPF grade healthy male Wistar rats, weighing 200 g 卤10 g, were randomly divided into experimental group and control group with 30 rats in each group.Cy5 labeled MT012 渭 g was injected into the buccal mucosa of the right maxillary first permanent molar in the experimental group, and the unlabeled MT012 渭 g was injected into the control group. The lung, liver, spleen, kidney, heart and brain tissues of the rats were harvested under general anesthesia for 4 days, liver, spleen, kidney, heart and brain under general anesthesia for 4 days and 5 days.The fluorescence distribution of MT01 was observed under laser confocal microscope, and the amount of MT01 entering into the tissues was expressed by fluorescence positive cell rate.Results: no fluorescent positive cells were observed in the control group.Fluorescence positive cells were observed in the lung, liver, spleen and kidney except heart and brain tissues in the experimental group, and the fluorescent positive cells in the renal tissue were flake distribution, fluorescence mainly concentrated in the cytoplasm of renal tubular epithelial cells, lung, liver, liver, lung, liver, kidney, liver, lung, liver, lung, liver, lung, liver, lung, liver, lung, liver and liver.The percentage of fluorescent positive cells in liver, spleen and kidney showed regular changes with time, and the peak value appeared at 4d and 4d after injection, respectively.However, the rate of fluorescent positive cells in lung tissues did not show obvious regularity with time.The rate of fluorescent positive cells in renal tissue was significantly higher than that in other tissues.Conclusion the lung, liver, spleen and kidney can be injected into the lung, liver, spleen and kidney by local injection of the rat gingival. The distribution of MT01 is mainly concentrated in the kidney tissue, suggesting that the kidney may be the main metabolic organ of MT01.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R783.1

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