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

【摘要】： 目的:應(yīng)用特殊設(shè)計(jì)的寡核苷酸鏈糾正點(diǎn)突變這一技術(shù)是近年發(fā)展起來的一項(xiàng)分子生物學(xué)新技術(shù),應(yīng)用該技術(shù)能在基因組堿基序列中任意位點(diǎn)糾正或引入一個(gè)指定的堿基。較之傳統(tǒng)的基因重組技術(shù),該技術(shù)對(duì)基因組本身的結(jié)構(gòu)幾乎沒有任何影響,且不會(huì)產(chǎn)生因病毒載體和質(zhì)粒載體引起的免疫反應(yīng)以及隨機(jī)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(diǎn)點(diǎn)突變引起的。本研究旨在建立應(yīng)用特殊設(shè)計(jì)的寡核苷酸鏈糾正點(diǎn)突變這一新技術(shù)的體內(nèi)外研究方法,探討影響寡核苷酸鏈的細(xì)胞核內(nèi)轉(zhuǎn)染率以及突變糾正率的因素,并將該技術(shù)初步應(yīng)用于誘導(dǎo)大鼠肝細(xì)胞Ⅸ因子突變,為今后將該技術(shù)應(yīng)用于血友病等由基因點(diǎn)突變而引起的疾病的基因治療提供實(shí)驗(yàn)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(xué)載體合成。通過減少孵育時(shí)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(cè)定L-PEI中的半乳糖殘基含量,計(jì)算其乳糖化率。第二部分為體內(nèi)、外轉(zhuǎn)染實(shí)驗(yàn)。體外實(shí)驗(yàn)是將熒光標(biāo)記的L-PEI與寡核苷酸鏈復(fù)合物分別轉(zhuǎn)染大鼠正常肝細(xì)胞(BRL)、人正常肝細(xì)胞(HL-7702)以及人肝癌細(xì)胞(SMMC-7721),共聚焦顯微鏡觀察比較三株細(xì)胞之間寡核苷酸鏈核內(nèi)轉(zhuǎn)染率的差異。體內(nèi)實(shí)驗(yàn)則是將復(fù)合物直接注射入SD大鼠肝尾狀葉,注射后不同時(shí)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(shí)驗(yàn),分體內(nèi)、體外兩部分。體內(nèi)實(shí)驗(yàn)是將適量的寡核苷酸鏈復(fù)合物經(jīng)尾靜脈注射入SD大鼠體內(nèi),注射后不同時(shí)間采血測(cè)定凝血功能指標(biāo)包括凝血酶原時(shí)間(PT)、活化部分凝血活酶時(shí)間(APTT);提取肝臟DNA,PCR產(chǎn)物測(cè)序來驗(yàn)證點(diǎn)突變。體外實(shí)驗(yàn)是將寡核苷酸鏈復(fù)合物和細(xì)胞孵育一定時(shí)間后提取DNA,PCR產(chǎn)物測(cè)序。 結(jié)論:通過減少孵育時(shí)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(biāo)記的L-PEI-ODN復(fù)合物對(duì)BRL、HL-7702、SMMC-7721三種細(xì)胞的轉(zhuǎn)染率分別為51%、73%、46%。結(jié)果表明,ODN對(duì)HL-7702細(xì)胞的轉(zhuǎn)染率較高,與另外兩種細(xì)胞比較有顯著性差異。轉(zhuǎn)染細(xì)胞熒光強(qiáng)度定量分析結(jié)果表明,三種細(xì)胞平均熒光強(qiáng)度的強(qiáng)弱與其細(xì)胞核轉(zhuǎn)染率高低呈相同趨勢(shì),提示寡核苷酸鏈轉(zhuǎn)運(yùn)入細(xì)胞核的能力在不同種類的細(xì)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(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)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(dǎo)的人凝血因子IX基因治療血友病B小鼠研究[J];中國(guó)科學(xué)(C輯:生命科學(xué));2003年05期

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

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本文編號(hào)：1751519