【摘要】： 目的 家族性高膽固醇血癥(familial cholesterolemia,FH)的致病基因是位于19號(hào)染色體上的低密度脂蛋白受體(LDLR)基因,大多為點(diǎn)突變,是最常見(jiàn)的單基因遺傳病之一,屬公認(rèn)的遺傳性疾病基因治療的首選病種。已進(jìn)行的研究表明現(xiàn)行的基因治療方法在安全性和持久性方面尚有不少問(wèn)題。新近報(bào)道的對(duì)點(diǎn)突變進(jìn)行染色體(靶向)原位修復(fù)是一種全新的理念,有望獲得重大突破。修飾的單鏈DNA寡核苷酸(modified single-stranded DNA oligonucleotide,MSO)被證實(shí)可以較低頻率地修復(fù)報(bào)告基因中的點(diǎn)突變。本研究的目的是建立LDLR基因點(diǎn)突變的細(xì)胞模型,進(jìn)行MSO靶向基因原位修復(fù)試驗(yàn),為探索FH的基因治療打基礎(chǔ),由此建立的策略方法也適用于其他以點(diǎn)突變?yōu)橹鞯膯位蜻z傳性疾病,具有較高理論價(jià)值和應(yīng)用前景。同時(shí)本研究還建立了LDLR基因四環(huán)素調(diào)控穩(wěn)定細(xì)胞系,為研究LDLR基因功能和FH的基因治療提供了新的細(xì)胞模型。 方法 低密度脂蛋白受體基因點(diǎn)突變細(xì)胞模型構(gòu)建 為了構(gòu)建質(zhì)粒表達(dá)載體pIRES-Hyg-WT-LDLR-EGFP、pIRES-Hyg-556-LDLR-EGFP和pIRES-Hyg-660-LDLR-EGFP,人低密度脂蛋白受體基因的cDNA編碼序列被插入到質(zhì)粒表達(dá)載體pIRES-Hyg的多克隆位點(diǎn)。WT即野生型LDLR基因,556是LDLR基因在12外顯子1730位有G→C點(diǎn)突變,660是LDLR基因在14外顯子2043位有C→A點(diǎn)突變。三種質(zhì)粒表達(dá)載體分別制備,純化后的質(zhì)粒DNA通過(guò)脂質(zhì)體介導(dǎo)分別轉(zhuǎn)染CHO、HepG2和Vero細(xì)胞,600μg/mlHygromycin篩選抗性細(xì)胞克隆,通過(guò)(1)熒光顯微鏡觀察EGFP的表達(dá);(2)提取陽(yáng)性克隆細(xì)胞的基因組作為模板,PCR鑒定SNP位點(diǎn);(3)western blot檢測(cè)LDLR-EGFP融合蛋白的表達(dá);(4)檢測(cè)LDLR受體對(duì)Dil標(biāo)記的人低密度脂蛋白(DiI-LDL)的攝取功能;對(duì)構(gòu)建的穩(wěn)定細(xì)胞模型進(jìn)行鑒定。 低密度脂蛋白受體基因點(diǎn)突變?cè)恍迯?fù) 對(duì)構(gòu)建的穩(wěn)定細(xì)胞模型Vero-660的LDLR基因點(diǎn)突變進(jìn)行原位修復(fù),以突變點(diǎn)為中心,分別合成15 nt、25 nt、31 nt、35 nt和45 nt長(zhǎng)度,末端6個(gè)堿基硫代修飾的MSO,每個(gè)長(zhǎng)度的鏈合成4條,分別為兩條修復(fù)鏈:與編碼鏈一致為反義鏈;與模板鏈一致為正義鏈;兩條對(duì)照鏈:C鏈與編碼鏈一致,但未糾正突變位點(diǎn);NC鏈為無(wú)關(guān)對(duì)照鏈。利用脂質(zhì)體轉(zhuǎn)染細(xì)胞系,流式細(xì)胞儀檢測(cè)細(xì)胞EGFP的表達(dá),初步確定修復(fù)效率。通過(guò)流式細(xì)胞儀分選EGFP~+細(xì)胞,利用Dil-LDL內(nèi)移實(shí)驗(yàn)驗(yàn)證LDLR的功能,培養(yǎng)一定時(shí)間后,提取核酸和蛋白質(zhì)分別進(jìn)行焦磷酸測(cè)序分析和Western Blot檢測(cè),確定最終的修復(fù)效率。 低密度脂蛋白受體基因調(diào)控表達(dá) 首先構(gòu)建質(zhì)粒表達(dá)載體pIRES-TetR-Hyg,即以pcDNA6/TR質(zhì)粒核酸為模板,設(shè)計(jì)引物,PCR擴(kuò)增Tet R片段,插入到質(zhì)粒表達(dá)載體pIRES-Hyg的多克隆位點(diǎn)。同時(shí)構(gòu)建質(zhì)粒表達(dá)載體pCDNA4/TO-WT-LDLR-EGFP,即人低密度脂蛋白受體基因的cDNA編碼序列插入質(zhì)粒表達(dá)載體pCDNA4/TO的多克隆位點(diǎn),WT即野生型LDLR基因。將構(gòu)建好的兩種質(zhì)粒表達(dá)載體pIRES-TetR-Hyg和pCDNA4/TO-WT-LDLR-EGFP通過(guò)脂質(zhì)體介導(dǎo)分別共轉(zhuǎn)染CHO和HepG2細(xì)胞,用600μg/ml Hygromycin和400μg/ml Zeocin篩選抗性細(xì)胞克隆。以2μg/ml四環(huán)素誘導(dǎo)LDLR基因表達(dá),通過(guò)(1)熒光顯微鏡觀察EGFP的表達(dá);(2)western blot檢測(cè)LDLR-EGFP蛋白的表達(dá);(3)檢測(cè)LDLR受體對(duì)DiI-LDL的攝取功能;對(duì)構(gòu)建的低密度脂蛋白受體基因調(diào)控細(xì)胞模型進(jìn)行鑒定。 結(jié)果 實(shí)驗(yàn)結(jié)果顯示野生型和點(diǎn)突變LDLR基因的穩(wěn)定轉(zhuǎn)染細(xì)胞模型構(gòu)建成功,穩(wěn)定轉(zhuǎn)染pIRES-Hyg-WT-LDLR-EGFP質(zhì)粒的細(xì)胞系,分別命名為HepG2-WT、CHO-WT和Vero-WT;穩(wěn)定轉(zhuǎn)染pIRES-Hyg-556-LDLR-EGFP質(zhì)粒的細(xì)胞系,分別命名為HepG2-556、CHO-556和Vero-556;穩(wěn)定轉(zhuǎn)染pIRES-Hyg-660-LDLR-EGFP質(zhì)粒的細(xì)胞系,分別命名為HepG2-660、CHO-660和Vero-660。其中HepG2-WT、CHO-WT和Vero-WT細(xì)胞系中有正常野生型LDLR基因,而LDLR受體是膜受體,因此熒光顯微鏡下可觀察到熒光主要分布在細(xì)胞膜上,將Dil標(biāo)記的人低密度脂蛋白與細(xì)胞相互作用后,激光共聚焦顯微鏡觀察結(jié)果顯示,外源性的低密度脂蛋白受體具有正常的與配基相結(jié)合的功能。同時(shí)Western blot結(jié)果進(jìn)一步提示,細(xì)胞中有大量的外源性低密度脂蛋白受體蛋白的表達(dá)。HepG2-556、CHO-556和Vero-556細(xì)胞系中的LDLR基因在12外顯子1730位有G→C點(diǎn)突變,由于該突變,導(dǎo)致LDLR受體雖然能表達(dá)但不能定位于細(xì)胞膜,因此熒光顯微鏡下可觀察到熒光主要分布在細(xì)胞質(zhì)中。HepG2-660、CHO-660和Vero-660細(xì)胞系中的LDLR基因在14外顯子2043位有C→A點(diǎn)突變,該突變導(dǎo)致提前出現(xiàn)終止密碼子,使LDLR受體及下游的EGFP蛋白均無(wú)法表達(dá),因此熒光顯微鏡觀察無(wú)熒光可見(jiàn)。 對(duì)構(gòu)建的穩(wěn)定細(xì)胞模型Vero-660的LDLR基因點(diǎn)突變進(jìn)行原位修復(fù),由于Vero-660的LDLR基因點(diǎn)突變屬于無(wú)義突變,能較簡(jiǎn)便判別修復(fù)效率,不能修復(fù)者,突變位點(diǎn)下游部分均不能表達(dá)。凡能糾正該點(diǎn)突變者即可獲得包括EGFP的完整表達(dá)產(chǎn)物,修復(fù)效率越高,EGFP的表達(dá)越多。本研究以此為模型,通過(guò)流式細(xì)胞儀檢測(cè)EGFP的表達(dá),分析比較不同MSO的修復(fù)效果,結(jié)果發(fā)現(xiàn)35 A-MSO具有較好的效果,EGFP陽(yáng)性比例為4.99±0.74%。但通過(guò)Pyrosequencing測(cè)序,結(jié)果顯示熒光轉(zhuǎn)陽(yáng)細(xì)胞克隆的突變修復(fù)率為10.72±0.93%,因此確切的突變修復(fù)率應(yīng)為0.53%左右。 實(shí)驗(yàn)結(jié)果還顯示我們成功構(gòu)建了LDLR基因四環(huán)素調(diào)控表達(dá)細(xì)胞模型,HepG2和CHO四環(huán)素調(diào)控細(xì)胞分別命名為HepG2-TetR-WT,CHO-TetR-WT。調(diào)控細(xì)胞加入四環(huán)素前,熒光顯微鏡觀察無(wú)綠色熒光可見(jiàn),加入四環(huán)素后,熒光顯微鏡可見(jiàn)細(xì)胞內(nèi)有大量綠色熒光,且熒光主要分布在細(xì)胞膜上,將Dil-LDL與細(xì)胞相互作用后,激光共聚焦顯微鏡觀察結(jié)果顯示,外源性的低密度脂蛋白受體具有正常的與配基相結(jié)合的功能。同時(shí)Western blot結(jié)果進(jìn)一步提示,細(xì)胞中有大量的外源性低密度脂蛋白受體蛋白的表達(dá),表明構(gòu)建的細(xì)胞模型LDLR基因受四環(huán)素調(diào)控且不影響受體功能。 結(jié)論 成功制備LDLR基因點(diǎn)突變的多種細(xì)胞模型,使用簡(jiǎn)易有效的MSO對(duì)LDLR的點(diǎn)突變進(jìn)行定點(diǎn)原位修復(fù)方面開(kāi)展研究,為探索FH安全有效的基因治療新途徑打下基礎(chǔ)。四環(huán)素調(diào)控的LDLR基因穩(wěn)定細(xì)胞系的建立,為研究LDLR基因功能和FH的基因治療提供了新的細(xì)胞模型。
[Abstract]:objective
Familial hypercholesterolemia (FH) is a low density lipoprotein receptor (LDLR) gene located on chromosome 19. Most of the LDLR genes are point mutations. It is one of the most common monogenic inherited diseases. It is recognized that FH is the preferred gene therapy for inherited diseases. Modified single-stranded DNA oligonucleotides (MSO) have been shown to repair reporter genes at lower frequencies. The aim of this study is to establish a cell model of point mutation of LDLR gene and carry out MSO-targeted gene in situ repair test to lay a foundation for exploring gene therapy of FH. The strategy and method established by this study can also be applied to other single gene inherited diseases with point mutation as the dominant factor, and has high theoretical value and application prospect. A stable cell line regulated by tetracycline of LDLR gene was established, which provided a new cell model for studying the function of LDLR gene and gene therapy of FH.
Method
Construction of point mutation cell model of low density lipoprotein receptor gene
In order to construct plasmid expression vectors pIRES-Hyg-WT-LDLR-EGFP, pIRES-Hyg-556-LDLR-EGFP and pIRES-Hyg-660-LDLR-EGFP, the cDNA coding sequence of the human LDLR gene was inserted into the polyclonal site of the plasmid expression vector pIRES-Hyg. WT is the wild type LDLR gene, 556 is the point mutation of the LDLR gene in exon 12 1730, 660 is the G-C point mutation. LDLR gene has C_A point mutation at exon 2043. Three plasmid expression vectors were prepared, and the purified plasmid DNA was transfected into CHO, HepG2 and Vero cells by liposome mediation respectively. Resistant cell clones were screened by 600 ug/ml Hygromycin, and the expression of EGFP was observed by fluorescence microscope; (2) Genome of positive cloned cells was extracted as a model. SNP sites were identified by PCR; (3) LDLR-EGFP fusion protein expression was detected by Western blot; (4) Dil-labeled human low density lipoprotein (DiI-LDL) uptake by LDLR receptors was detected; and the stable cell model was identified.
In situ repair of point mutation of low density lipoprotein receptor gene
The LDLR gene point mutation of Vero-660 was repaired in situ. The length of 15 nt, 25 nt, 31 nt, 35 nt and 45 nt, and the length of 6 base thio-modified MSO were synthesized with the center of the mutation point. Four repair chains were synthesized with each length, which were antisense chains consistent with the coding chains and justice consistent with the template chains. The expression of EGFP was detected by flow cytometry. EGFP~+ cells were sorted by flow cytometry. The function of LDLR was verified by Dil-LDL translocation assay. After culture for a certain time, the cells were extracted. Pyrophosphate sequencing and Western Blot analysis were performed to determine the final repair efficiency.
Low density lipoprotein receptor gene regulation expression
Firstly, the plasmid expression vector pIRES-TetR-Hyg was constructed. The primers were designed using pcDNA6/TR plasmid nucleic acid as template. The TetR fragment was amplified by PCR and inserted into the polyclonal site of the plasmid expression vector pIRES-Hyg. At the same time, the plasmid expression vector pCDNA4/TO-WT-LDLR-EGFP was constructed. Two plasmid expression vectors, pIRES-TetR-Hyg and pCDNA4/TO-WT-LDLR-EGFP, were co-transfected into CHO and HepG2 cells via liposome mediation. Resistant cell clones were screened by 600 ug/ml Hygromycin and 400 ug/ml Zeocin. LDLR gene expression was induced by 2 ug/ml tetracycline. The expression of EGFP was observed by fluorescence microscope; (2) the expression of LDLR-EGFP protein was detected by Western blot; (3) the uptake of DiI-LDL by LDLR receptor was detected; and the low density lipoprotein receptor gene regulatory cell model was identified.
Result
The results showed that the stable transfection cell model of wild type and point mutation LDLR gene was successfully constructed, and the stable transfection cell lines of pIRES-Hyg-WT-LDLR-EGFP plasmid were named HepG2-WT, CHO-WT and Vero-WT, respectively; the stable transfection cell lines of pIRES-Hyg-556-LDLR-EGFP plasmid were named HepG2-556, CHO-556 and Vero-556, respectively. Hyg-660-LDLR-EGFP plasmid cell lines were named HepG2-660, CHO-660 and Vero-660, respectively. HepG2-WT, CHO-WT and Vero-WT cell lines had normal wild-type LDLR genes, and LDLR receptors were membrane receptors. Therefore, fluorescence mainly distributed on the cell membrane, and Dil-labeled human low-density lipoprotein interacted with cells. Laser confocal microscopy showed that the exogenous LDL receptor had normal binding to the ligand. Western blot analysis further indicated that there were a large number of LDL receptor proteins in HepG2-556, CHO-556 and Vero-556 cell lines. There is a G_C point mutation at exon 1730, which results in the expression of LDLR receptor but not localization on the cell membrane. Fluorescence can be observed mainly in the cytoplasm under fluorescence microscope. In HepG2-660, CHO-660 and Vero-660 cell lines, the LDLR gene has a C_A point mutation at exon 1443, which leads to the early appearance of LDLR receptor. The termination of codon made LDLR receptor and downstream EGFP protein unable to express, so there was no fluorescence visible under fluorescence microscope.
Point mutation of LDLR gene in Vero-660 was repaired in situ. Because point mutation of LDLR gene in Vero-660 belonged to nonsense mutation, it was easy to judge the repairing efficiency, and the downstream part of the mutation site could not be expressed if it could not be repaired. The higher the efficiency was, the more EGFP was expressed. In this study, the expression of EGFP was detected by flow cytometry, and the repairing effect of different MSOs was compared. The results showed that 35 A-MSO had a better effect, and the positive rate of EGFP was 4.99 (+ 0.74%). However, the mutant repairing rate of fluorescent Transpositive cell clones was 10. 72 + 0.93%, so the exact rate of mutation repair should be around 0.53%.
The results also showed that we successfully constructed the cell model of LDLR gene tetracycline-regulated expression. HepG2 and CHO tetracycline-regulated cells were named HepG2-TetR-WT, respectively, and CHO-TetR-WT. The results of confocal laser scanning microscopy showed that the exogenous LDL receptor had normal binding function with ligand. The Western blot results further indicated that there were a large number of exogenous LDL receptor eggs in the cells. The expression of white indicates that the LDLR gene is regulated by tetracycline and does not affect receptor function.
conclusion
Successful preparation of a variety of LDLR gene point mutation cell models, the use of simple and effective MSO site-directed repair of LDLR point mutation research, to explore a safe and effective gene therapy for FH lay the foundation. Tetracycline-regulated LDLR gene stable cell lines to study the function of LDLR gene and FH gene therapy A new cell model is provided.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R346

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7 劉剛;雌激素與膽囊結(jié)石及癌變相關(guān)[N];中國(guó)醫(yī)藥報(bào);2004年

8 萬(wàn)同己;黃芪當(dāng)歸合劑治療原發(fā)性腎病綜合征[N];中國(guó)醫(yī)藥報(bào);2002年

9 付東紅 孫揚(yáng);跟腱等部位結(jié)節(jié)預(yù)示心腦血管疾病[N];中國(guó)醫(yī)藥報(bào);2003年

10 喻維民;遺傳代謝病的治療原則及進(jìn)展[N];大眾衛(wèi)生報(bào);2000年

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