【摘要】：基因治療是將基因導入人體細胞中,以期產(chǎn)生有益的蛋白質來治療疾病的一種方法。1995年,美國國立衛(wèi)生組織(NIH)提出基因治療目前存在的三大關鍵問題之重點問題便是:具有良好選擇性、安全性和高效性的基因導入系統(tǒng)(即基因載體)。非病毒載體由于具有低毒,低免疫反應,靶向性和易于組裝等優(yōu)點,但轉染效率一直不高。如何提高DNA在基因轉染和基因治療中的轉染效率成為-基因轉導技術的關鍵問題。因此尋找新的非病毒載體成為目前的研究熱點。 本研究自行組裝構建了一種新型非病毒基因載體脂質-HAP-DNA復合體(Lipid—Hydroxyapatite—DNA,LHD)并驗證其用作基因轉導的可行性。本論文分為三個部分: 第一研究了HAP納米粒子的制備與改性。通過該部分研究我們得到如下結論: 1.運用化學沉淀法合成了HAP納米顆粒并進行了表面改性。得到了Ca/P比接近1.67的HAP納米顆粒,在緩沖液中穩(wěn)定性及分散性良好、粒徑均勻、符合下一步研究需要。 2.利用有機大分子A作為穩(wěn)定劑,改性后HAP表面Zeta電位為正,在緩沖液中可長時間穩(wěn)定存在。 3.化學沉淀法合成納米HAP時,反應溫度、PH值對產(chǎn)物HAP的影響顯著,溶液濃度對產(chǎn)物HAP影響不大。 4.超聲處理、滴加速度及攪拌強度作為輔助手段會對制備所得納米HAP晶體尺寸產(chǎn)生影響,而穩(wěn)定劑種類和劑量會顯著影響生成納米HAP的表面特性。 第二進行了LHD復合體的制備研究。得到如下結論: 1.用正電荷的納米HAP分別與不同比例的DNA結合,最終確定b DNA與HAP的較為合適的結合比例。 2.用反相蒸發(fā)法將DNA—HAP復合物包裹進入脂質膜中,制備得到LHD復合體。觀察到合成的LHD復合體成球形、具有一定包封率,并且對被包裹的DNA起到有效的保護作用。 3.在LHD復合體的制備中,進行了溫度變化和光線照射對卵磷脂氧化率和LHD的包封率的影響的研究。確定了實驗的防氧化措施及貯存方法。
[Abstract]:Gene therapy is a method of introducing genes into human cells in order to produce beneficial proteins to treat diseases. The National Health Organization (NIH) has put forward three key problems in gene therapy: a gene transfer system (gene vector) with good selectivity, safety and efficiency. Non-viral vectors have the advantages of low toxicity, low immune response, targeting and easy assembly, but the transfection efficiency is not high. How to improve the transfection efficiency of DNA in gene transfection and gene therapy has become a key issue in gene transduction technology. Therefore, the search for new non-viral vectors has become a hot research topic. A novel non-viral gene vector lipid-HAP-DNA complex (Lipid-Hydroxyapatite-DNA LHD) was constructed and its feasibility for gene transduction was verified. This thesis is divided into three parts: firstly, the preparation and modification of HAP nanoparticles are studied. Through this part of the study, we get the following conclusions: 1. HAP nanoparticles were synthesized and modified by chemical precipitation method. The HAP nanoparticles with Ca/P ratio close to 1.67 have good stability and dispersion in buffer solution, and the particle size is uniform, which meets the need of further study. Using organic macromolecule A as stabilizer, the surface Zeta potential of modified HAP is positive, which can be stabilized in buffer solution for a long time. In the synthesis of nanometer HAP by chemical precipitation method, the pH value of the reaction temperature has a significant effect on the product HAP, while the concentration of solution has little effect on the product HAP. Ultrasonic treatment, drop acceleration and stirring intensity as auxiliary means will affect the size of the prepared nanocrystalline HAP crystal, and the type and dosage of stabilizer will significantly affect the surface characteristics of the nanocrystalline HAP. Secondly, the preparation of LHD complex was studied. The conclusions are as follows: 1. The proper binding ratio of b DNA and HAP was determined by combining positive charge nanometer HAP with different proportion of DNA respectively. The DNA-HAP complex was encapsulated into the lipid membrane by reversed-phase evaporation to prepare the LHD complex. It is observed that the synthesized LHD complex is spherical, has a certain encapsulation efficiency and has an effective protective effect on the encapsulated DNA. In the preparation of LHD complex, the effects of temperature and light irradiation on the oxidation rate of lecithin and the encapsulation efficiency of LHD were studied. The anti-oxidation measures and storage methods are determined.
【學位授予單位】：武漢理工大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R346

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