应用特殊设计的寡核苷酸链纠正点突变的初步研究

发布时间：2018-04-14 23:28

本文选题：寡核苷酸链 + 基因定点突变　；参考：《苏州大学》2006年硕士论文

【摘要】： 目的:应用特殊设计的寡核苷酸链纠正点突变这一技术是近年发展起来的一项分子生物学新技术,应用该技术能在基因组碱基序列中任意位点纠正或引入一个指定的碱基。较之传统的基因重组技术,该技术对基因组本身的结构几乎没有任何影响,且不会产生因病毒载体和质粒载体引起的免疫反应以及随机整合。一些遗传性疾病,如乙型血友病,有部分是由于Ⅸ因子核苷酸序列中特定位点点突变引起的。本研究旨在建立应用特殊设计的寡核苷酸链纠正点突变这一新技术的体内外研究方法,探讨影响寡核苷酸链的细胞核内转染率以及突变纠正率的因素,并将该技术初步应用于诱导大鼠肝细胞Ⅸ因子突变,为今后将该技术应用于血友病等由基因点突变而引起的疾病的基因治疗提供实验依据。方法:本研究分为三部分,第一部分为寡核苷酸链的化学载体合成。通过减少孵育时间,以还原胺化法制备低乳糖化水平的聚乙烯亚胺(L-PEI),采用酚-硫酸法测定L-PEI中的半乳糖残基含量,计算其乳糖化率。第二部分为体内、外转染实验。体外实验是将荧光标记的L-PEI与寡核苷酸链复合物分别转染大鼠正常肝细胞(BRL)、人正常肝细胞(HL-7702)以及人肝癌细胞(SMMC-7721),共聚焦显微镜观察比较三株细胞之间寡核苷酸链核内转染率的差异。体内实验则是将复合物直接注射入SD大鼠肝尾状叶,注射后不同时间观察寡核苷酸链核内转染率情况。第三部分为诱变实验,分体内、体外两部分。体内实验是将适量的寡核苷酸链复合物经尾静脉注射入SD大鼠体内,注射后不同时间采血测定凝血功能指标包括凝血酶原时间(PT)、活化部分凝血活酶时间(APTT);提取肝脏DNA,PCR产物测序来验证点突变。体外实验是将寡核苷酸链复合物和细胞孵育一定时间后提取DNA,PCR产物测序。结论:通过减少孵育时间,最终成功制备了乳糖化率为6.27%的低乳糖化水平的L-PEI。荧光标记的L-PEI-ODN复合物对BRL、HL-7702、SMMC-7721三种细胞的转染率分别为51%、73%、46%。结果表明,ODN对HL-7702细胞的转染率较高,与另外两种细胞比较有显著性差异。转染细胞荧光强度定量分析结果表明,三种细胞平均荧光强度的强弱与其细胞核转染率高低呈相同趋势,提示寡核苷酸链转运入细胞核的能力在不同种类的细胞之间是有差异的。L-PEI-ODN与
[Abstract]:Objective: using specially designed oligonucleotide chain correction point mutation is a new molecular biological technique developed in recent years. It can correct or introduce a specified base at any site in the genome base sequence.Compared with traditional gene recombination technique, this technique has little effect on the structure of genome itself, and does not produce immune response and random integration caused by virus vector and plasmid vector.Some hereditary diseases, such as type B hemophilia, are due in part to specific point mutations in the nucleotide sequence of factor IX.The purpose of this study was to establish a new technique of correcting point mutation of oligonucleotide chain in vitro and in vivo, and to explore the factors affecting the transfection rate and mutation correction rate of oligonucleotide chain.The technique was applied to induce the mutation of factor IX in rat hepatocytes, which provided experimental basis for gene therapy of hemophilia and other diseases caused by point mutation of gene.Methods: this study was divided into three parts. The first part was the synthesis of oligonucleotide chains.By reducing the incubation time, the low lactose level polyethylene imide L-PEI was prepared by reducing amination method. The content of galactose residues in L-PEI was determined by phenol-sulfuric acid method, and the lactating rate was calculated.The second part is in vivo and in vitro transfection experiment.In vitro, fluorescent labeled L-PEI and oligonucleotide chain complexes were transfected into rat normal hepatocytes, human normal hepatocytes (HL-7702) and human hepatoma cells (SMMC-7721) respectively. Confocal microscopy was used to observe and compare the oligonucleotide chain nuclei among the three cells.The difference of internal transfection rate.In vivo, the complex was injected directly into the caudate lobe of the liver of SD rats, and the transfection efficiency of oligonucleotide chain nucleus was observed at different time after injection.The third part is mutagenesis experiment, which is divided into two parts: in vivo and in vitro.In vivo, the oligonucleotide chain complex was injected into SD rats via tail vein.At different time after injection, the parameters of blood coagulation function including prothrombin time (PTT), activated partial thromboplastin time (APTTT), and PCR products from liver DNA were sequenced to verify point mutation.In vitro, the oligonucleotide chain complex and the cells were incubated for a certain time, and the PCR products were sequenced.Conclusion: L-PEI with low lactating rate of 6.27% was successfully prepared by reducing incubation time.The transfection efficiency of fluorescent labeled L-PEI-ODN complex to BRL HL-7702 and SMMC-7721 cells was 51and 7346g, respectively.The results showed that the transfection efficiency of HL-7702 cells was higher than that of the other two cells.The results of quantitative analysis of fluorescence intensity of transfected cells showed that the average fluorescence intensity of the three kinds of cells showed the same trend as the nuclear transfection efficiency.These results suggest that the ability of oligonucleotide chains to transport into the nucleus is different among different types of cells.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

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