基于甘露糖受體的新型非病毒基因傳遞載體的制備及評價
發(fā)布時間：2018-09-02 06:52
【摘要】：目的：根據(jù)甘露糖載體的特點設計合成精胺—甘露聚糖載體(SM),對其及其基因復合物進行表征,并將SM基因傳遞體系應用到體內(nèi)基因傳遞中,實現(xiàn)肝臟靶向轉(zhuǎn)染；將其應用在體外基因傳遞,實現(xiàn)對甘露糖受體表達的細胞高效轉(zhuǎn)染,并探索其作用機理。 方法：1.合成不同接枝率SM載體并以FTIR、DSC、H1-NMR及元素分析測試表征,MTT法篩選其細胞毒性,通過蟲熒光素酶報告基因篩選其轉(zhuǎn)染效率,孵育法制備其復合物并進行粒徑、電位、形態(tài)表征；2.利用SM/FITC-DNA通過活體成像技術觀察SM基因復合物的組織分布,篩選其靶向性,測定粒徑及轉(zhuǎn)染效率評價血清對其干擾,利用組織切片HE染色評價SM傳遞體系的肝臟毒性,并通過綠色熒光蛋白報告基因載體轉(zhuǎn)染肝臟評價其轉(zhuǎn)染效率；3.MTT法評價SM基因傳遞體系在不同細胞的細胞毒性,并轉(zhuǎn)染評價其在不同細胞上的轉(zhuǎn)染效率,與PEI、Lipo、SP在MMR-及MMR+細胞上進行轉(zhuǎn)染及毒性比較,并通過不同的競爭抑制劑、入胞抑制劑及鈣離子螯合劑探究SM入胞機理,通過標記SM與DNA探索其在細胞內(nèi)轉(zhuǎn)運情況,并通過溶酶體染色探索其溶酶體逃逸情況。制備原代的骨髓源樹突狀細胞并初步鑒定,流式細胞術測定其對SM/FITC-DNA的攝取,利用EGFP報告基因?qū)MDC進行轉(zhuǎn)染評價。 結論：成功設計合成了精胺—甘露聚糖非病毒基因載體材料,并對其進行表征。通過改變CDI的反應比例,成功合成了不同接枝率的SM載體材料,并對其進行了細胞毒性及轉(zhuǎn)染效率的篩選,為后續(xù)的應用提供了實驗依據(jù)。通過對SM與DNA孵育形成復合物的表征,證明SM與DNA在一定N/P可以形成粒徑-100nm,電位～45mV的穩(wěn)定的復合物體系,并具有規(guī)整的形態(tài)。將SM基因傳遞體系應用于體內(nèi)的基因傳遞中,針對肝臟靶向性研究,SM/DNA復合物可以在一定接枝比例下一定N/P比時靶向于肝臟,相較裸DNA可以延長DNA在肝臟積累。與文獻報道的陽性對照組PEI基因復合物相比較,SM基因傳遞體系具有更強的抗血清干擾能力,且對肝臟產(chǎn)生較小的毒副作用。在體內(nèi)的轉(zhuǎn)染評價中,SM基因傳遞體系可以產(chǎn)生與PEI相似及更好的轉(zhuǎn)染效率。將SM基因傳遞體系應用于體外基因傳遞中,成功在四種細胞中進行了細胞毒性及轉(zhuǎn)染效率的篩選,而SM在MMR表達的細胞中轉(zhuǎn)染尤為高效。通過對比SM與其他非病毒載體,SM在MMR+細胞中具有高效、低毒的轉(zhuǎn)染優(yōu)勢。通過進一步對SM在MMR+細胞中作用機制的研究,證明SM是通過MMR受體介導,并通過網(wǎng)格蛋白及內(nèi)陷小窩兩條入胞途徑進入細胞中,其MMR介導需要鈣離子的存在。而對SM/DNA復合物的胞內(nèi)轉(zhuǎn)運及內(nèi)涵體逃逸的研究表明,SM/DNA可以成功從細胞內(nèi)涵體中逃逸,并將DNA釋放進入細胞核,而SM本身并不進入細胞核中。還進一步將SM基因傳遞體系應用在了原代骨髓源樹突狀細胞中,證明BMDC對SM的攝取率要遠高于其他非病毒載體,并成功實現(xiàn)對BMDC的轉(zhuǎn)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.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R943
本文編號：2218603
[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.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R943
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