低密度脂蛋白受体基因点突变染色体原位修复及调控表达的研究

发布时间：2018-09-18 13:43

【摘要】： 目的家族性高胆固醇血症(familial cholesterolemia,FH)的致病基因是位于19号染色体上的低密度脂蛋白受体(LDLR)基因,大多为点突变,是最常见的单基因遗传病之一,属公认的遗传性疾病基因治疗的首选病种。已进行的研究表明现行的基因治疗方法在安全性和持久性方面尚有不少问题。新近报道的对点突变进行染色体(靶向)原位修复是一种全新的理念,有望获得重大突破。修饰的单链DNA寡核苷酸(modified single-stranded DNA oligonucleotide,MSO)被证实可以较低频率地修复报告基因中的点突变。本研究的目的是建立LDLR基因点突变的细胞模型,进行MSO靶向基因原位修复试验,为探索FH的基因治疗打基础,由此建立的策略方法也适用于其他以点突变为主的单基因遗传性疾病,具有较高理论价值和应用前景。同时本研究还建立了LDLR基因四环素调控稳定细胞系,为研究LDLR基因功能和FH的基因治疗提供了新的细胞模型。方法低密度脂蛋白受体基因点突变细胞模型构建为了构建质粒表达载体pIRES-Hyg-WT-LDLR-EGFP、pIRES-Hyg-556-LDLR-EGFP和pIRES-Hyg-660-LDLR-EGFP,人低密度脂蛋白受体基因的cDNA编码序列被插入到质粒表达载体pIRES-Hyg的多克隆位点。WT即野生型LDLR基因,556是LDLR基因在12外显子1730位有G→C点突变,660是LDLR基因在14外显子2043位有C→A点突变。三种质粒表达载体分别制备,纯化后的质粒DNA通过脂质体介导分别转染CHO、HepG2和Vero细胞,600μg/mlHygromycin筛选抗性细胞克隆,通过(1)荧光显微镜观察EGFP的表达;(2)提取阳性克隆细胞的基因组作为模板,PCR鉴定SNP位点;(3)western blot检测LDLR-EGFP融合蛋白的表达;(4)检测LDLR受体对Dil标记的人低密度脂蛋白(DiI-LDL)的摄取功能;对构建的稳定细胞模型进行鉴定。低密度脂蛋白受体基因点突变原位修复对构建的稳定细胞模型Vero-660的LDLR基因点突变进行原位修复,以突变点为中心,分别合成15 nt、25 nt、31 nt、35 nt和45 nt长度,末端6个碱基硫代修饰的MSO,每个长度的链合成4条,分别为两条修复链:与编码链一致为反义链;与模板链一致为正义链;两条对照链:C链与编码链一致,但未纠正突变位点;NC链为无关对照链。利用脂质体转染细胞系,流式细胞仪检测细胞EGFP的表达,初步确定修复效率。通过流式细胞仪分选EGFP~+细胞,利用Dil-LDL内移实验验证LDLR的功能,培养一定时间后,提取核酸和蛋白质分别进行焦磷酸测序分析和Western Blot检测,确定最终的修复效率。低密度脂蛋白受体基因调控表达首先构建质粒表达载体pIRES-TetR-Hyg,即以pcDNA6/TR质粒核酸为模板,设计引物,PCR扩增Tet R片段,插入到质粒表达载体pIRES-Hyg的多克隆位点。同时构建质粒表达载体pCDNA4/TO-WT-LDLR-EGFP,即人低密度脂蛋白受体基因的cDNA编码序列插入质粒表达载体pCDNA4/TO的多克隆位点,WT即野生型LDLR基因。将构建好的两种质粒表达载体pIRES-TetR-Hyg和pCDNA4/TO-WT-LDLR-EGFP通过脂质体介导分别共转染CHO和HepG2细胞,用600μg/ml Hygromycin和400μg/ml Zeocin筛选抗性细胞克隆。以2μg/ml四环素诱导LDLR基因表达,通过(1)荧光显微镜观察EGFP的表达;(2)western blot检测LDLR-EGFP蛋白的表达;(3)检测LDLR受体对DiI-LDL的摄取功能;对构建的低密度脂蛋白受体基因调控细胞模型进行鉴定。结果实验结果显示野生型和点突变LDLR基因的稳定转染细胞模型构建成功,稳定转染pIRES-Hyg-WT-LDLR-EGFP质粒的细胞系,分别命名为HepG2-WT、CHO-WT和Vero-WT;稳定转染pIRES-Hyg-556-LDLR-EGFP质粒的细胞系,分别命名为HepG2-556、CHO-556和Vero-556;稳定转染pIRES-Hyg-660-LDLR-EGFP质粒的细胞系,分别命名为HepG2-660、CHO-660和Vero-660。其中HepG2-WT、CHO-WT和Vero-WT细胞系中有正常野生型LDLR基因,而LDLR受体是膜受体,因此荧光显微镜下可观察到荧光主要分布在细胞膜上,将Dil标记的人低密度脂蛋白与细胞相互作用后,激光共聚焦显微镜观察结果显示,外源性的低密度脂蛋白受体具有正常的与配基相结合的功能。同时Western blot结果进一步提示,细胞中有大量的外源性低密度脂蛋白受体蛋白的表达。HepG2-556、CHO-556和Vero-556细胞系中的LDLR基因在12外显子1730位有G→C点突变,由于该突变,导致LDLR受体虽然能表达但不能定位于细胞膜,因此荧光显微镜下可观察到荧光主要分布在细胞质中。HepG2-660、CHO-660和Vero-660细胞系中的LDLR基因在14外显子2043位有C→A点突变,该突变导致提前出现终止密码子,使LDLR受体及下游的EGFP蛋白均无法表达,因此荧光显微镜观察无荧光可见。对构建的稳定细胞模型Vero-660的LDLR基因点突变进行原位修复,由于Vero-660的LDLR基因点突变属于无义突变,能较简便判别修复效率,不能修复者,突变位点下游部分均不能表达。凡能纠正该点突变者即可获得包括EGFP的完整表达产物,修复效率越高,EGFP的表达越多。本研究以此为模型,通过流式细胞仪检测EGFP的表达,分析比较不同MSO的修复效果,结果发现35 A-MSO具有较好的效果,EGFP阳性比例为4.99±0.74%。但通过Pyrosequencing测序,结果显示荧光转阳细胞克隆的突变修复率为10.72±0.93%,因此确切的突变修复率应为0.53%左右。实验结果还显示我们成功构建了LDLR基因四环素调控表达细胞模型,HepG2和CHO四环素调控细胞分别命名为HepG2-TetR-WT,CHO-TetR-WT。调控细胞加入四环素前,荧光显微镜观察无绿色荧光可见,加入四环素后,荧光显微镜可见细胞内有大量绿色荧光,且荧光主要分布在细胞膜上,将Dil-LDL与细胞相互作用后,激光共聚焦显微镜观察结果显示,外源性的低密度脂蛋白受体具有正常的与配基相结合的功能。同时Western blot结果进一步提示,细胞中有大量的外源性低密度脂蛋白受体蛋白的表达,表明构建的细胞模型LDLR基因受四环素调控且不影响受体功能。结论成功制备LDLR基因点突变的多种细胞模型,使用简易有效的MSO对LDLR的点突变进行定点原位修复方面开展研究,为探索FH安全有效的基因治疗新途径打下基础。四环素调控的LDLR基因稳定细胞系的建立,为研究LDLR基因功能和FH的基因治疗提供了新的细胞模型。
[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.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R346

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