枯草溶菌素转换酶9基因新突变的发现及其功能初步研究

发布时间：2018-06-15 02:01

本文选题：家族性高胆固醇血症 + 枯草溶菌素转换酶9　；参考：《南华大学》2011年博士论文

【摘要】：研究背景家族性高胆固醇血症(familia hypercholesterlolemia, FH)是显性遗传代谢性疾病,可由多种脂代谢相关基因突变所导致。家系遗传分析研究证实低密度脂蛋白受体(low-density lipoprotein receptor,LDL-R)、枯草溶菌素转换酶9(proprotein convertase subtilisin kexin 9,PCSK9)等基因突变引发LDL-R功能障碍均可导致相同的临床表型:纯合子发病率1/百万,血浆LDL胆固醇大幅度增高导致早发动脉粥样硬化,儿童期即可导致严重的冠心病而死亡。杂合子发病率1/500,出生时即有LDL-R的功能障碍,机体长期暴露于高胆固醇水平,动脉硬化进展加速。通常50%男性患者在50岁之前、30%女性在60岁之前发生心梗,在小于60岁的心梗患者FH杂合子患者中约占5%。在已知的致病基因中LDL-R基因突变最为常见,约占FH病例的50%-70%,其它5种突变约占20%。然而,在FH患者中仍有约10-15%检测不到上述6种致病基因突变,推测尚有一些新的致病基因有待发现随着近年对PCSK9基因研究的不断深入,人们对LDL-胆固醇的代谢机制又有了新的认识。最新研究结果发现PCSK9基因对LDL-R代谢有重要调节作用;同为PCSK9基因的不同位点突变可导致完全相反的两种现象:PCSK9功能获得性突变与高胆固醇血症有关;PCSK9功能丧失突变与低胆固醇血症有关。本课题从一个FH家系入手,筛查致病突变,并于体外研究突变体功能,探讨该突变体作用机制,为高胆固醇血症的临床诊断及脂质代谢的机制提供新的线索。第一部分家族性高胆固醇血症患者临床资料分析及基因突变筛查 [目的] 筛查中国汉族家族性高胆固醇血症家系中的基因突变新类型,分析基因型与表型间的关系。 [方法] 以一FH家系为研究对象,详细调查患者饮食、生活习惯及家族史并进行心血管系统全面检查;提取外周血白细胞DNA,核苷酸序列测定法对一个汉族FH家系LDL-R、apoB和PCSK9基因进行突变检测;采用蛋白分析系统ExPASy预测PCSK9基因野生型和R306S突变体编码蛋白的二、三级结构。 [结果] 1、该FH家系先证者体检显示心血管系统严重动脉粥样硬化性改变,心脏缺血性损伤;2、LDL-R和apo B100基因未见突变;核苷酸序列测定法发现先证者及其父亲PCSK9基因第918位核苷酸G T改变,导致第6外显子第306位精基酸被丝氨酸取代;3、蛋白质二、三级结构的模拟发现,与野生型相比,PCSK9基因发生R306S突变后编码蛋白梭基末端结构域和催化亚基结构域两个主要的部分构像发生改变,且这2个结构域之间的距离拉大。 [结论] 1.在一汉族FH家系中发现PCSK9基因新错义突变R306S; 2.计算机结构模拟分析发现新突变体R306S编码蛋白二、三级结构发生改变。第二部分枯草溶菌素转换酶9基因新突变体表达及功能初步研究 [目的] 探讨枯草溶菌素转换酶9基因新突变对LDL代谢的作用及突变致FH的分子机制,并为突变患者采取早期治疗,防治心血管疾病发生和扩展LDL的代谢机制等提供理论和实验依据。 [方法] 从人肝肿瘤细胞系BEL-7402细胞获得野生型PCSK9基因全长cDNA(WT-PCSK9);构建真核表达载体并经核苷酸序列测定法鉴定;定点突变方法构建携带PCSK9基因致病型的重组真核表达质粒并测序及酶切法鉴定插入片段的大小及序列;以空白载体为对照,脂质体转染法将重组质粒转染BEL-7402细胞;RT-PCR检测LDL-R mRNA表达,Western blot检测PCSK9和LDL-R蛋白表达;荷脂实验观察对LDL代谢的影响;免疫荧光观察PCSK9基因与LDL-R细胞共定位;流式细胞法检测转染细胞LDL-R对荧光标记LDL的结合能力的变化;稳定转染肝细胞系,建立Tet on/off系统,定量观察PCSK9基因对LDL-R表达的影响。 [结果] 1、核苷酸序列测定法鉴定证实构建的表达载体插入片段大小和序列正确。2、各突变体转染BEL-7402细胞LDL-R mRNA水平与野生型比较无明显差异(p0.05)。3、Western blot检测各突变体转染BEL-7402细胞PCSK9前体蛋白和成熟蛋白无明显差异(p0.05);与对照组比较,转染野生型PCSK9质粒后LDL-R成熟蛋白表达降低(p0.05);转染阳性对照质粒后成熟LDL-R条带消失表达明显降低(p0.01);转染R306S突变体后成熟LDL-R降低(p0.01)。4、荷脂实验结果显示,与空白对照相比,PCSK9野生型肝细胞内脂质减少(p0.05);R306S组肝细胞脂质减少明显(p0.01),且低于野生型PCSK9 BEL肝细胞(p0.05)。5、免疫荧光检测肝细胞PCSK9与LDL-R共定位,转染前后分别主要位于细胞膜和胞内。6、流式细胞法检测转染细胞LDL-R对荧光标记LDL的结合活性的变化,与空白对照组细胞相比野生型PCSK9组荧光强度降低(p0.05),阳性对照F216R组平均荧光降低更为明显(p0.01),新突变体组平均荧光强度降低程度较野生型明显(p0.01)。7、突变体Tet on系统在BEL-7402细胞中高效、稳定表达。在PCSK9野生型及突变体Tet on系统中随着Doxycycline药物浓度的增加,PCSK9野生型和突变体开始表达,且表达量逐渐升高,LDL-R成熟蛋白部分逐渐减少,未糖基化的LDL-R未见显著变化;与野生型相比,R306S降低成熟LDL-R的能力显著提高(p0.05)。 [结论] 1.成功构建PCSK9基因野生和突变类型的真核表达载体; 2.体外实验中发现PCSK9基因新突变体R306S对LDL-R的转录无显著影响; 3.PCSK9基因新突变体R306S可明显降低细胞LDL-R成熟蛋白水平; 4.PCSK9基因新突变体R306S可减少其对LDL的摄取从而引发高胆固醇血症,可能是该FH家系致病基因。
[Abstract]:Research background
Familial hypercholesterolemia (Familia hypercholesterlolemia, FH) is a dominant genetic metabolic disease that can be caused by mutations in a variety of lipid metabolism related genes. Family genetic analysis has confirmed that low density lipoprotein receptor (low-density lipoprotein receptor, LDL-R), and withered lysozyme converting enzyme 9 (proprotein convertase subtilisin Kex) In 9, PCSK9) and other gene mutations cause LDL-R dysfunction to lead to the same clinical phenotype: the incidence of homozygote is 1/ million, the increase in plasma LDL cholesterol leads to early atherosclerosis, and childhood can lead to severe coronary heart disease. The incidence of heterozygotes is 1/ 500, LDL-R dysfunction at birth, and long-term body violence. When exposed to high cholesterol levels, arteriosclerosis progresses rapidly. Usually 50% male patients before 50 years of age, 30% women have myocardial infarction before 60 years of age, and the FH heterozygotes in patients with FH heterozygotes less than 60 years old are the most common 5%. mutations in the known pathogenetic genes, accounting for 50%-70% of the FH cases, and the other 5 mutations accounting for 20%., however, in F About 6 of the H gene mutations were not detected in 10-15% patients. It is speculated that there are still some new genes to be discovered.
With the development of PCSK9 gene research in recent years, people have new understanding of the metabolic mechanism of LDL- cholesterol. The latest research results show that the PCSK9 gene plays an important role in the regulation of LDL-R metabolism; the mutation of the loci of the same PCSK9 gene can lead to the completely opposite two images: PCSK9 functional acquired mutation and high cholesterol blood. It is related to disease; the loss of PCSK9 function is associated with hypocholesterolemia. This topic begins with a FH family, screening the pathogenic mutation, and studies the mutant function in vitro, and explores the mechanism of the mutant function, providing a new clue for the clinical diagnosis of hypercholesterolemia and the mechanism of lipid metabolism.
Part one clinical data analysis and gene mutation screening in patients with familial hypercholesterolemia
[Objective]
To screen new types of gene mutations in Chinese Han families with hypercholesterolemia, and to analyze the relationship between genotype and phenotype.
[method]
A FH family was used as the study object to investigate the patients' diet, life habits and family history and to carry out a comprehensive examination of cardiovascular system. The DNA of peripheral blood leucocyte was extracted and nucleotide sequencing method was used to detect the mutation of LDL-R, apoB and PCSK9 genes in a FH family of Han nationality, and the egg white analysis system ExPASy was used to predict the wild type and R306S of PCSK9 gene. The two, three grade structure of the mutant protein.
[results]
1, the FH family precursor showed severe atherosclerotic changes in the cardiovascular system, ischemic injury of the heart, and no mutation in 2, LDL-R and apo B100 genes; the nucleotide sequencing method found that the precursor and the 918th nucleotide G T of the father PCSK9 gene were changed, resulting in the replacement of 306th arginine acids in sixth exons; 3, protein. Two, the simulation of the three stage structure found that, compared with the wild type, the R306S mutation of the PCSK9 gene changed after the mutation of the terminal domain of the encoding protein and the two major parts of the subunit structure, and the distance between the 2 domains was enlarged.
[Conclusion]
1. a new missense mutation R306S of PCSK9 gene was found in a Han FH family.
2. computer simulation analysis showed that the new mutant R306S encoded protein two and the three grade structure changed.
The second part is a preliminary study on the expression and function of a new mutant of the lysozyme converting enzyme 9 gene.
[Objective]
The effect of the new mutation of the lysozyme 9 gene on the metabolism of LDL and the molecular mechanism of FH induced by mutation were investigated, and the theoretical and experimental basis for the early treatment of the mutant patients, the prevention and treatment of cardiovascular disease and the expansion of the metabolic mechanism of LDL were provided.
[method]
The full length cDNA (WT-PCSK9) of the wild type PCSK9 gene was obtained from the human liver tumor cell line BEL-7402 cells; the eukaryotic expression vector was constructed and identified by the nucleotide sequencing method. The recombinant eukaryotic expression plasmid carrying the PCSK9 gene pathogenicity was constructed and the size and sequence of the inserted fragments were sequenced and the enzyme digestion method was used to determine the size and sequence of the inserted fragments. The recombinant plasmid was transfected into BEL-7402 cells by liposome transfection, the expression of LDL-R mRNA was detected by RT-PCR, the expression of PCSK9 and LDL-R protein was detected by Western blot, the effect of lipid test on LDL metabolism, the co localization of PCSK9 gene and LDL-R cell was observed by immunofluorescence, and the binding ability of the transfected cell LDL-R to fluorescent labeling was detected by flow cytometry. The Tet on/off system was established and the effect of PCSK9 gene on LDL-R expression was quantitatively observed.
[results]
1, the nucleotide sequencing method confirmed that the constructed expression vector inserted the fragment size and sequence correctly.2. The LDL-R mRNA level of each mutant transfected BEL-7402 cells was not significantly different from that of the wild type (P0.05).3, and there was no significant difference between the BEL-7402 cell PCSK9 precursor protein and the mature protein (P0.05) by Western blot detection. After transfection of wild type PCSK9 plasmid, the expression of LDL-R mature protein decreased (P0.05), and the vanished expression of mature LDL-R bands decreased significantly (P0.01) after transfection of positive control plasmid, and the mature LDL-R decreased (P0.01).4 after transfection of R306S mutant, and lipid reduction (P0.05) in PCSK9 wild type hepatocytes compared with blank control (P0.05); R (P0.05); The liver cell lipid decreased significantly in 306S group (P0.01), and was lower than that of wild type PCSK9 BEL hepatocyte (P0.05).5. Immunofluorescence detection of hepatocyte PCSK9 and LDL-R was Co located. The transfection was mainly located in the cell membrane and intracellular.6. Flow cytometry was used to detect the changes in the binding activity of LDL-R to fluorescent LDL, and the cell phase in the blank control group. The fluorescence intensity of the PCSK9 group was lower than that of the wild type group (P0.05), and the average fluorescence reduction of the positive control F216R group was more obvious (P0.01). The decrease of the average fluorescence intensity in the new mutant group was significantly higher than that of the wild type (P0.01).7, and the mutant Tet on system was highly efficient and stable in BEL-7402 cells. The increase of drug concentration, the PCSK9 wild type and the mutant began to express, and the expression level increased gradually, the LDL-R mature protein part gradually decreased, and the LDL-R of the non glycosylation was not significantly changed. Compared with the wild type, the ability of R306S to reduce the maturity of LDL-R was significantly improved (P0.05).
[Conclusion]
1. the wild and mutant eukaryotic expression vectors of PCSK9 gene were successfully constructed.
2. in vitro, it was found that R306S, a new mutant of PCSK9 gene, had no significant effect on LDL-R transcription.
R306S, a new mutant of 3.PCSK9 gene, significantly reduced the level of LDL-R mature protein.
R306S, a new mutant of 4.PCSK9 gene, can reduce its intake of LDL and cause hypercholesterolemia, which may be the pathogenic gene of FH family.
【学位授予单位】：南华大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R346

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