基于甘露糖受体的新型非病毒基因传递载体的制备及评价

发布时间：2018-09-02 06:52

【摘要】：目的：根据甘露糖载体的特点设计合成精胺—甘露聚糖载体(SM),对其及其基因复合物进行表征,并将SM基因传递体系应用到体内基因传递中,实现肝脏靶向转染；将其应用在体外基因传递,实现对甘露糖受体表达的细胞高效转染,并探索其作用机理。方法：1.合成不同接枝率SM载体并以FTIR、DSC、H1-NMR及元素分析测试表征,MTT法筛选其细胞毒性,通过虫荧光素酶报告基因筛选其转染效率,孵育法制备其复合物并进行粒径、电位、形态表征；2.利用SM/FITC-DNA通过活体成像技术观察SM基因复合物的组织分布,筛选其靶向性,测定粒径及转染效率评价血清对其干扰,利用组织切片HE染色评价SM传递体系的肝脏毒性,并通过绿色荧光蛋白报告基因载体转染肝脏评价其转染效率；3.MTT法评价SM基因传递体系在不同细胞的细胞毒性,并转染评价其在不同细胞上的转染效率,与PEI、Lipo、SP在MMR-及MMR+细胞上进行转染及毒性比较,并通过不同的竞争抑制剂、入胞抑制剂及钙离子螯合剂探究SM入胞机理,通过标记SM与DNA探索其在细胞内转运情况,并通过溶酶体染色探索其溶酶体逃逸情况。制备原代的骨髓源树突状细胞并初步鉴定,流式细胞术测定其对SM/FITC-DNA的摄取,利用EGFP报告基因对BMDC进行转染评价。结论：成功设计合成了精胺—甘露聚糖非病毒基因载体材料,并对其进行表征。通过改变CDI的反应比例,成功合成了不同接枝率的SM载体材料,并对其进行了细胞毒性及转染效率的筛选,为后续的应用提供了实验依据。通过对SM与DNA孵育形成复合物的表征,证明SM与DNA在一定N/P可以形成粒径-100nm,电位～45mV的稳定的复合物体系,并具有规整的形态。将SM基因传递体系应用于体内的基因传递中,针对肝脏靶向性研究,SM/DNA复合物可以在一定接枝比例下一定N/P比时靶向于肝脏,相较裸DNA可以延长DNA在肝脏积累。与文献报道的阳性对照组PEI基因复合物相比较,SM基因传递体系具有更强的抗血清干扰能力,且对肝脏产生较小的毒副作用。在体内的转染评价中,SM基因传递体系可以产生与PEI相似及更好的转染效率。将SM基因传递体系应用于体外基因传递中,成功在四种细胞中进行了细胞毒性及转染效率的筛选,而SM在MMR表达的细胞中转染尤为高效。通过对比SM与其他非病毒载体,SM在MMR+细胞中具有高效、低毒的转染优势。通过进一步对SM在MMR+细胞中作用机制的研究,证明SM是通过MMR受体介导,并通过网格蛋白及内陷小窝两条入胞途径进入细胞中,其MMR介导需要钙离子的存在。而对SM/DNA复合物的胞内转运及内涵体逃逸的研究表明,SM/DNA可以成功从细胞内涵体中逃逸,并将DNA释放进入细胞核,而SM本身并不进入细胞核中。还进一步将SM基因传递体系应用在了原代骨髓源树突状细胞中,证明BMDC对SM的摄取率要远高于其他非病毒载体,并成功实现对BMDC的转染。
[Abstract]:Aim: according to the characteristics of mannose vector, we designed and synthesized spermine mannanan vector (SM), to characterize it and its gene complex, and applied SM gene transfer system to gene transfer in vivo to achieve liver targeted transfection. It was applied to gene transfer in vitro to realize the efficient transfection of mannose receptor expression cells and to explore its mechanism. Method 1: 1. SM vectors with different grafting rates were synthesized and characterized by FTIR,DSC,H1-NMR and elemental analysis. The transfection efficiency was screened by luciferase reporter gene, and its complex was prepared by incubating method, and its particle size, potential and morphology were characterized. SM/FITC-DNA was used to observe the tissue distribution of SM gene complex by in vivo imaging, to screen its targeting, to determine the particle size and transfection efficiency to evaluate the interference of serum, and to evaluate the liver toxicity of SM transmission system by HE staining of tissue sections. The transfection efficiency was evaluated by transfection of green fluorescent protein reporter gene vector. 3. MTT assay was used to evaluate the cytotoxicity of SM gene transfer system in different cells and its transfection efficiency in different cells. Compared with PEI,Lipo,SP transfection and toxicity in MMR- and MMR cells, the mechanism of SM entry was explored by different competitive inhibitors, inhibitors and calcium chelators, and the intracellular transport of SM was investigated by labeling SM and DNA. Lysosome escape was investigated by lysosome staining. Primary bone marrow-derived dendritic cells were prepared and identified. SM/FITC-DNA uptake was determined by flow cytometry and BMDC transfection was evaluated by EGFP reporter gene. Conclusion: spermine-mannanan non-viral vector material was successfully designed and synthesized and characterized. By changing the reaction ratio of CDI, the SM carrier materials with different grafting rates were successfully synthesized, and their cytotoxicity and transfection efficiency were screened, which provided the experimental basis for further application. By the characterization of SM and DNA to form complex, it is proved that SM and DNA can form stable complex system with particle size of -100 nm and potential of 45mV at a certain N / P, and have regular morphology. SM gene transfer system was applied to gene transmission in vivo. The aim of this study was to study the liver targeting of SMC / DNA complex at a certain grafting ratio of N / P to the liver, while bare DNA could prolong the accumulation of DNA in the liver. Compared with the PEI gene complex reported in the literature, SM gene delivery system has stronger antiserum interference ability and less toxic side effect on liver. In vivo transfection evaluation, SM gene delivery system can produce similar to PEI and better transfection efficiency. The SM gene transfer system was applied to gene transmission in vitro. The cytotoxicity and transfection efficiency of SM were successfully screened in four kinds of cells, and SM transfection was especially efficient in the cells expressing MMR. SM and other non-viral vectors SM have high efficiency and low toxicity in MMR cells. Through further study on the mechanism of SM in MMR cells, it is proved that SM is mediated by MMR receptor and enters into cells through two pathways of grid protein and invagination fossa. The existence of Ca ~ (2 +) is required by MMR mediated by SM. However, studies on intracellular transport of SM/DNA complex and escape of intension showed that SM-DNA could successfully escape from cell intension and release DNA into nucleus, while SM itself did not enter nucleus. Furthermore, SM gene transfer system was applied to primary bone marrow-derived dendritic cells, which proved that the uptake rate of SM by BMDC was much higher than that of other non-viral vectors, and the transfection of BMDC was successfully realized.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943

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