本文關鍵詞：新型基因槍用羥基磷灰石微粒的研制　出處：《武漢理工大學》2010年碩士論文　論文類型：學位論文

  更多相關文章： 羥基磷灰石 基因轉染 基因槍 綠色熒光蛋白

【摘要】： 人類很多疾病是由基因上的缺陷所造成的,如果能從基因的水平上修復缺陷基因,將對傳統(tǒng)疾病治療方式產生革命性的影響。當前基因治療中的基因遞送系統(tǒng)主要分為病毒性載體和非病毒性載體兩大類。病毒性載體在臨床應用中具有潛在的危險性,而非病毒性載體轉染效率較低�；驑尲夹g是一種純物理的基因轉移技術,相比之下有著明顯的優(yōu)勢：操作簡單、傳遞基因迅速、安全等,這一系列優(yōu)點使其展現(xiàn)出誘人的應用前景,但基因槍技術中負載基因的金或鎢不能被生命機體代謝,具有潛在的安全性。考慮到羥基磷灰石生物相容性好、可代謝、無細胞毒性等優(yōu)點,本課題擬選用其作為金或鎢的替代材料,并探討其用于基因槍系統(tǒng)的有效性。實驗研究主要包括以下三個部分： 第一部分：穩(wěn)定劑H保護法制備HAP微粒 目的：制備出粒徑均勻,分散性良好,具有特定表面性質及特定粒徑范圍的HAP微粒。 方法：采用穩(wěn)定劑H保護沉淀法制備HAP微粒,并進行粒度測試、電位分析和形貌測定,以研究不同反應條件對HAP表面特性的影響。 結果：經Ca／P比值測定、電鏡測試和粒度、電位測試,所得HAP微粒穩(wěn)定,分散性良好,粒徑小且均勻。 結論：1、穩(wěn)定劑H保護法制備的羥基磷灰石呈針狀顆粒,粒徑小且均勻。 2、穩(wěn)定劑H用量、反應溫度、Ca(OH)2滴加速度、超聲處理等對產物粒徑、電位均有一定影響。 第二部分：pEGFP-C3質粒DNA的制備及HAP對DNA的吸附研究 目的：制備出純度、濃度都符合基因轉染要求的pEGFP-C3質粒DNA,并將其較好的負載在HAP微粒上。 方法：通過堿裂解法制備pEGFP-C3質粒DNA,并采用瓊脂糖凝膠電泳和紫外吸收法對其進行濃度、純度測定；然后采用紫外分光光度法測定不同操作條件對HAP吸附DNA的影響。 結果：1、檢測結果顯示,制備得到的質粒純度高,符合基因轉染的要求。 2、不同的加樣順序及亞精胺的pH調節(jié)對DNA在HAP上的吸附容量影響甚微。 結論：1、HAP吸附DNA時,可考慮加入亞精胺以增大其吸附容量。2、只要保證一定的HAP、DNA和亞精胺反應體系,均可獲得較好的負載。 第三部分：HAP納米載體介導的RSC96細胞體外基因轉染實驗 目的：摸索、研究羥基磷灰石納米粒作為基因轉移載體的有效性。 方法：將制備得到的pEGFP-C3質粒作為目的基因,采用不同的微彈材料和不同的基因槍儀器參數進行轉染,分析、比較其轉染效果的差別。 結果：1、金為載體時,視野中出現(xiàn)大量綠色熒光。2、HAP為載體時,視野中熒光量稍少。 結論：金負載DNA制成基因微彈,用于基因槍系統(tǒng),其轉染效果明顯。在一定的基因槍系統(tǒng)儀器參數條件下,HAP是能作為載體介導質粒DNA進入靶細胞內,并表達產生綠色熒光蛋白,以上說明HAP用于基因槍系統(tǒng)是可行的,但轉染效率和金相比略低。
[Abstract]:Many human diseases are caused by genetic defects, if can repair the defective gene from the gene level, will have a revolutionary impact on the traditional disease treatment. The gene therapy of gene delivery system is mainly divided into viral vectors and non viral vectors. Two kinds of viral vectors is dangerous the potential in clinical application, and the transfection efficiency of non viral vector is low. Gene gun technology is a kind of pure physical gene transfer technology, by contrast has obvious advantages: simple operation, safety, rapid gene transfer, a series of advantages, showing an attractive prospect, but the gene gun technology supported gold or tungsten can not gene by life metabolism, with potential safety. Considering the hydroxyapatite has good biocompatibility, can metabolism, cell toxicity and other advantages, this paper intends to use it as An alternative material for gold or tungsten and the effectiveness of its use in the gene gun system. The experimental study mainly includes the following three parts:
The first part: the preparation of HAP particles by the stabilizer H protection method
Objective: to prepare HAP particles with uniform particle size, good dispersibility and specific surface properties and specific particle size range.
Methods: HAP particles were prepared by stabilizer H protection precipitation method, and particle size test, potential analysis and morphology measurement were carried out to study the effect of different reaction conditions on the surface characteristics of HAP.
Results: through the determination of Ca / P ratio, electron microscope test, particle size and potential test, the obtained HAP particles are stable, good dispersion, small particle size and uniform.
Conclusion: 1, the hydroxyapatite prepared by the stabilizer H protection method is acicular particles with small and uniform particle size.
2, the amount of stabilizer H, the reaction temperature, the 2 drop acceleration of Ca (OH), and the ultrasonic treatment have a certain effect on the particle size and potential of the products.
The second part: the preparation of pEGFP-C3 plasmid DNA and the study of the adsorption of DNA by HAP
Objective: to prepare the pEGFP-C3 plasmid DNA with the purity and concentration which are in line with the gene transfection requirement and load it on HAP particles.
Methods: pEGFP-C3 plasmid DNA was prepared by alkaline lysis, and its concentration and purity were determined by agarose gel electrophoresis and ultraviolet absorption. Then, the influence of different operation conditions on HAP adsorption on DNA was determined by ultraviolet spectrophotometry.
Results: 1, the results showed that the purity of the prepared plasmid was high, which was in line with the requirements of gene transfection.
2, the different addition order and pH regulation of spermidine have little influence on the adsorption capacity of DNA on HAP.
Conclusion: 1. When HAP adsorbed DNA, it could consider adding spermidine to increase its adsorption capacity.2. As long as a certain HAP was guaranteed, DNA and spermidine reaction system could get better load.
The third part: HAP nanoscale mediated gene transfection of RSC96 cells in vitro
Objective: To explore the effectiveness of hydroxyapatite nanoparticles as a gene transfer carrier.
Methods: the pEGFP-C3 plasmid was used as target gene, and different microprojectile materials and different gene gun parameters were used to transfect, and the difference of transfection efficiency was analyzed.
Results: 1, when gold is the carrier, a large number of green fluorescent.2 appears in the field of vision. When HAP is the carrier, the fluorescence in the field of vision is slightly less.
Conclusion: DNA gene into gold supported micro bomb for gene gun system, its effect is obvious. The transfection of gene gun system of certain parameters, HAP can be used as a carrier mediated plasmid DNA into target cells, and the expression of green fluorescent protein, HAP described above for gene gun system is feasible, but the transfection efficiency and metallographic slightly lower.

【學位授予單位】：武漢理工大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R346

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本文編號：1358651