【作者】 杨天赐；

【导师】 彭宣宪； 李祺福；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 根据自主知识产权的专利工艺路线,合成具有分支结构、分子量30kD的阳离子聚合物,以此为基础构建sofast基因载体,用于大分子核酸的体外和体内的基因传递。通过sofast/DNA复合物形成、粒径、zeta电位、抗核酸酶能力、酸碱缓冲能力、结合核酸能力,以及基因融合肽dilNF-7和工作介质盐浓度的影响等分析,对sofast基因载体的转染机制做了初步的探讨。在体外试验中,采用sofast基因载体将质粒pEGFPN1、pCMV-lac Z和pGL3分别转染到HEK297细胞,通过所表达的绿色荧光蛋白、β-半乳糖苷酶和荧光素酶的检测,证实其具有高转染效率:通过HUV-EC等众多细胞株的转染,结果表明其广泛适用于众多原代培养细胞和转化细胞株的转染:采用MTT法进行其在最高效转染时的细胞毒性研究,结果表明细胞存活率高于90%,证实其细胞毒性极低;通过sofast基因载体抗血清特性研究,表明其具有抗血清特性,可以直接加到细胞培养基中,转染前后无需更换培养基,简化转染程序;通过sofast基因载体稳定性试验,结果表明sofast基因载体性质极其稳定,适合常温运送,4℃较长时间(3年以上)储存。在体内试验研究中,急性毒性试验、亚急性毒性试验、慢性毒性试验、半致死量测定、过敏试验以及热源检测等均表明sofast基因载体体内应用毒性极低,生物兼容性好;通过豚鼠尾静脉注射sofast/pEGFPN1复合物在体内表达试验,结果表明sofast基因载体体内应用仍然具有较高的转染效率,略优于目前市场占有率最高的阳离子脂质体Lipofectamine 2000。因此,所构建的sofast基因载体是一种高转染效率、低细胞毒性、稳定性好,且适用细胞类型较广泛的基因载体,适合体内外基因转染应用,是新一代阳离子聚合物基因载体的代表。而其简单,快捷的试验步骤,同样具有很好的优越性。在成功构建sofast阳离子聚合物用于大分子核酸基因传递基础上,自主合成一带巴西烯酸侧链、分子量50kD的脂质-阳离子聚合物(lipid-cationic polymer),构建lipid-cationic polymer siRNA基因传递载体,专用于体内外小分子siRNA基因传递。通过lipid-cationic polymer/siRNA复合物形成、粒径、zeta电位、抗核酸酶能力、酸碱缓冲能力、结合核酸能力,以及基因融合肽diINF-7和质子泵抑制剂的影响等分析,对lipid-cationic polymer基因载体的转染机制做了初步的探讨。结果表明lipid-cationic polymer以阳离子聚合物为基本骨架,在阳离子聚合物侧链交联上巴西烯酸,同时具备阳离子聚合物和阳离子脂质体的某些特性,发挥运载siRNA功能。采用lipid-cationic polymer基因载体将针对GFP的siRNA转染到HT1080-EGFP和HeLa-EGFP细胞,结果绿色荧光蛋白抑制率分别为73%和72%,高于lipofectamine2000的抑制率:进一步的特异性、细胞毒性、生物毒性、生物兼容性、可降解性、理化性质稳定性等诸多特性的研究,表明所构建的脂质-阳离子聚合物介导的RNAi基因沉默技术是安全、特异和稳定的。采用靶向环氧化酶-2(COX-2)基因的siRNA特异诱导COX-2基因转录后沉默,mRNA水平的变化提示基因载体的有效性。进一步应用自己制备的COX-2多克隆抗体和单克隆抗体进行West-blot Test和免疫组化分析,从蛋白表达水平证实了基因载体介导的基因沉默是有效的。因此,lipid-cationicpolymer基因载体可作为胚胎发育、病毒感染(如SARS、肝炎)、肿瘤发生、药物筛选、基因功能及基因治疗研究的技术平台,并可望拓宽基因治疗药物中基因传递载体这一长期困扰基因治疗技术成熟的瓶颈。此外,我们还对肿瘤患者的调节型免疫复合物的免疫调节作用进行了研究。更多还原

【Abstract】 According to the independent intellectual property patent process route,a branched and 30KD cationic polymer was synthesized,which the sofast gene carrier was constructed based on.The sofast gene carrier can be applied as big molecular nucleic acids carrier both in vitro and in vivo.In this paper we investigated its transfection mechanism as a gene carrier through formation of sofast/DNA complex,particle size,zeta potential analysis,anti-nuclease assays,buffering capacity assays,DNA combining assayes and effect of diINF-7 and salt concentration of working solutions.The in vivtro asssays results showed that the sofast gene carrier had high transfection efficiency,can be applied to various cell lines transfection.It also had the property of low cytotoxicity,anti-serum capacity,long-time stability(4℃for 3 years) and simple operation.In in vivo assayes, acute toxicity test,sbuacute and chronic toxicity test,LD50 assays,allergic assays and pyrogen assays were taken.The results indicated the sofast gene carrier had low in vivo cytotoxicity.The in vivo transfection assays results showed that its tansfection efficiency was higher than cationic-liposome Lipofectamine 2000 which prevailed in the market. So the sofast gene carrier is an new kinds of easy handling,widely-fitting with the property of high efficiency,low toxicity,long stability,lipid-cationic polymer gene carrier.Based on the construction of sofast gene carrier,we synthesized 50KD brassidic acid branched lipid-cationic polymer,then constructed the lipid-cationic polymer siRNA gene carrier to mediated siRNA transfection both in vitro and in vivo.In this paper we investigated its transfection mechanism as a gene carrier through formation of sofast/DNA complex,particle size,zeta potential analysis,anti-nuclease assays,buffering capacity assays,DNA combining assayes and effect of diINF-7 and proton pump inhibitor. It showed that the lipid cationic polymer gene carrier is composed of the cationic polymer backbone and the branched brassidic acid.So it bore the characteristics of cationic polymer and cationic liposome.It’s transfection efficiency is higher than the Lipofectamine 2000.Further specificity test,cytotoxicity test,bio-compatibility test, degradation test,stability test and suitable cell line test indicated that the lipid-cationic polymer gene carrier mediated siRNA transfection is safe,efficient,specific and stable. The siRNA induced silence after the transcriptiom of cox-2 showed the effectiveness of gene carrier in mRNA level.The western blotting analysis and immunohistochemistry analysis using self-made COX-2 polyclonal and monoclonal antibodies showed the effectiveness of the gene carrier mediated gene silence in protein expression level.So the lipid-cationic polymer gene carrier can be applied in areas of embryo development,virus infection,carcinogenesis,drug screening,gene function and genetic therapy etc.And it is hoped that the lipid-cationic polymer gene carrier can break the bottle neck in genetic therapy the gene carrier in gene therapeutic drugs.更多还原