本文選題：寡核苷酸鏈 + 基因定點突變　； 參考：《蘇州大學》2006年碩士論文

【摘要】： 目的:應用特殊設計的寡核苷酸鏈糾正點突變這一技術是近年發(fā)展起來的一項分子生物學新技術,應用該技術能在基因組堿基序列中任意位點糾正或引入一個指定的堿基。較之傳統(tǒng)的基因重組技術,該技術對基因組本身的結構幾乎沒有任何影響,且不會產(chǎn)生因病毒載體和質(zhì)粒載體引起的免疫反應以及隨機整合。一些遺傳性疾病,如乙型血友病,有部分是由于Ⅸ因子核苷酸序列中特定位點點突變引起的。本研究旨在建立應用特殊設計的寡核苷酸鏈糾正點突變這一新技術的體內(nèi)外研究方法,探討影響寡核苷酸鏈的細胞核內(nèi)轉(zhuǎn)染率以及突變糾正率的因素,并將該技術初步應用于誘導大鼠肝細胞Ⅸ因子突變,為今后將該技術應用于血友病等由基因點突變而引起的疾病的基因治療提供實驗依據(jù)。 方法:本研究分為三部分,第一部分為寡核苷酸鏈的化學載體合成。通過減少孵育時間,以還原胺化法制備低乳糖化水平的聚乙烯亞胺(L-PEI),采用酚-硫酸法測定L-PEI中的半乳糖殘基含量,計算其乳糖化率。第二部分為體內(nèi)、外轉(zhuǎn)染實驗。體外實驗是將熒光標記的L-PEI與寡核苷酸鏈復合物分別轉(zhuǎn)染大鼠正常肝細胞(BRL)、人正常肝細胞(HL-7702)以及人肝癌細胞(SMMC-7721),共聚焦顯微鏡觀察比較三株細胞之間寡核苷酸鏈核內(nèi)轉(zhuǎn)染率的差異。體內(nèi)實驗則是將復合物直接注射入SD大鼠肝尾狀葉,注射后不同時間觀察寡核苷酸鏈核內(nèi)轉(zhuǎn)染率情況。第三部分為誘變實驗,分體內(nèi)、體外兩部分。體內(nèi)實驗是將適量的寡核苷酸鏈復合物經(jīng)尾靜脈注射入SD大鼠體內(nèi),注射后不同時間采血測定凝血功能指標包括凝血酶原時間(PT)、活化部分凝血活酶時間(APTT);提取肝臟DNA,PCR產(chǎn)物測序來驗證點突變。體外實驗是將寡核苷酸鏈復合物和細胞孵育一定時間后提取DNA,PCR產(chǎn)物測序。 結論:通過減少孵育時間,最終成功制備了乳糖化率為6.27%的低乳糖化水平的L-PEI。熒光標記的L-PEI-ODN復合物對BRL、HL-7702、SMMC-7721三種細胞的轉(zhuǎn)染率分別為51%、73%、46%。結果表明,ODN對HL-7702細胞的轉(zhuǎn)染率較高,與另外兩種細胞比較有顯著性差異。轉(zhuǎn)染細胞熒光強度定量分析結果表明,三種細胞平均熒光強度的強弱與其細胞核轉(zhuǎn)染率高低呈相同趨勢,提示寡核苷酸鏈轉(zhuǎn)運入細胞核的能力在不同種類的細胞之間是有差異的。L-PEI-ODN與
[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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R346

【參考文獻】

相關期刊論文 前2條

1 葉晨波,高嘯波,侍鼎,陳立,邱信芳,薛京倫;微小腺病毒介導的人凝血因子IX基因治療血友病B小鼠研究[J];中國科學(C輯:生命科學);2003年05期

2 何睿,韓忠朝;基因治療血友病B的研究進展[J];中華血液學雜志;2003年03期

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本文編號：1751519