本文選題：家族性高膽固醇血癥 + 枯草溶菌素轉(zhuǎn)換酶9　； 參考：《南華大學》2011年博士論文

【摘要】：研究背景 家族性高膽固醇血癥(familia hypercholesterlolemia, FH)是顯性遺傳代謝性疾病,可由多種脂代謝相關(guān)基因突變所導致。家系遺傳分析研究證實低密度脂蛋白受體(low-density lipoprotein receptor,LDL-R)、枯草溶菌素轉(zhuǎn)換酶9(proprotein convertase subtilisin kexin 9,PCSK9)等基因突變引發(fā)LDL-R功能障礙均可導致相同的臨床表型:純合子發(fā)病率1/百萬,血漿LDL膽固醇大幅度增高導致早發(fā)動脈粥樣硬化,兒童期即可導致嚴重的冠心病而死亡。雜合子發(fā)病率1/500,出生時即有LDL-R的功能障礙,機體長期暴露于高膽固醇水平,動脈硬化進展加速。通常50%男性患者在50歲之前、30%女性在60歲之前發(fā)生心梗,在小于60歲的心梗患者FH雜合子患者中約占5%。在已知的致病基因中LDL-R基因突變最為常見,約占FH病例的50%-70%,其它5種突變約占20%。然而,在FH患者中仍有約10-15%檢測不到上述6種致病基因突變,推測尚有一些新的致病基因有待發(fā)現(xiàn) 隨著近年對PCSK9基因研究的不斷深入,人們對LDL-膽固醇的代謝機制又有了新的認識。最新研究結(jié)果發(fā)現(xiàn)PCSK9基因?qū)DL-R代謝有重要調(diào)節(jié)作用;同為PCSK9基因的不同位點突變可導致完全相反的兩種現(xiàn)象:PCSK9功能獲得性突變與高膽固醇血癥有關(guān);PCSK9功能喪失突變與低膽固醇血癥有關(guān)。本課題從一個FH家系入手,篩查致病突變,并于體外研究突變體功能,探討該突變體作用機制,為高膽固醇血癥的臨床診斷及脂質(zhì)代謝的機制提供新的線索。 第一部分家族性高膽固醇血癥患者臨床資料分析及基因突變篩查 [目的] 篩查中國漢族家族性高膽固醇血癥家系中的基因突變新類型,分析基因型與表型間的關(guān)系。 [方法] 以一FH家系為研究對象,詳細調(diào)查患者飲食、生活習慣及家族史并進行心血管系統(tǒng)全面檢查;提取外周血白細胞DNA,核苷酸序列測定法對一個漢族FH家系LDL-R、apoB和PCSK9基因進行突變檢測;采用蛋白分析系統(tǒng)ExPASy預測PCSK9基因野生型和R306S突變體編碼蛋白的二、三級結(jié)構(gòu)。 [結(jié)果] 1、該FH家系先證者體檢顯示心血管系統(tǒng)嚴重動脈粥樣硬化性改變,心臟缺血性損傷;2、LDL-R和apo B100基因未見突變;核苷酸序列測定法發(fā)現(xiàn)先證者及其父親PCSK9基因第918位核苷酸G T改變,導致第6外顯子第306位精基酸被絲氨酸取代;3、蛋白質(zhì)二、三級結(jié)構(gòu)的模擬發(fā)現(xiàn),與野生型相比,PCSK9基因發(fā)生R306S突變后編碼蛋白梭基末端結(jié)構(gòu)域和催化亞基結(jié)構(gòu)域兩個主要的部分構(gòu)像發(fā)生改變,且這2個結(jié)構(gòu)域之間的距離拉大。 [結(jié)論] 1.在一漢族FH家系中發(fā)現(xiàn)PCSK9基因新錯義突變R306S; 2.計算機結(jié)構(gòu)模擬分析發(fā)現(xiàn)新突變體R306S編碼蛋白二、三級結(jié)構(gòu)發(fā)生改變。 第二部分枯草溶菌素轉(zhuǎn)換酶9基因新突變體表達及功能初步研究 [目的] 探討枯草溶菌素轉(zhuǎn)換酶9基因新突變對LDL代謝的作用及突變致FH的分子機制,并為突變患者采取早期治療,防治心血管疾病發(fā)生和擴展LDL的代謝機制等提供理論和實驗依據(jù)。 [方法] 從人肝腫瘤細胞系BEL-7402細胞獲得野生型PCSK9基因全長cDNA(WT-PCSK9);構(gòu)建真核表達載體并經(jīng)核苷酸序列測定法鑒定;定點突變方法構(gòu)建攜帶PCSK9基因致病型的重組真核表達質(zhì)粒并測序及酶切法鑒定插入片段的大小及序列;以空白載體為對照,脂質(zhì)體轉(zhuǎn)染法將重組質(zhì)粒轉(zhuǎn)染BEL-7402細胞;RT-PCR檢測LDL-R mRNA表達,Western blot檢測PCSK9和LDL-R蛋白表達;荷脂實驗觀察對LDL代謝的影響;免疫熒光觀察PCSK9基因與LDL-R細胞共定位;流式細胞法檢測轉(zhuǎn)染細胞LDL-R對熒光標記LDL的結(jié)合能力的變化;穩(wěn)定轉(zhuǎn)染肝細胞系,建立Tet on/off系統(tǒng),定量觀察PCSK9基因?qū)DL-R表達的影響。 [結(jié)果] 1、核苷酸序列測定法鑒定證實構(gòu)建的表達載體插入片段大小和序列正確。2、各突變體轉(zhuǎn)染BEL-7402細胞LDL-R mRNA水平與野生型比較無明顯差異(p0.05)。3、Western blot檢測各突變體轉(zhuǎn)染BEL-7402細胞PCSK9前體蛋白和成熟蛋白無明顯差異(p0.05);與對照組比較,轉(zhuǎn)染野生型PCSK9質(zhì)粒后LDL-R成熟蛋白表達降低(p0.05);轉(zhuǎn)染陽性對照質(zhì)粒后成熟LDL-R條帶消失表達明顯降低(p0.01);轉(zhuǎn)染R306S突變體后成熟LDL-R降低(p0.01)。4、荷脂實驗結(jié)果顯示,與空白對照相比,PCSK9野生型肝細胞內(nèi)脂質(zhì)減少(p0.05);R306S組肝細胞脂質(zhì)減少明顯(p0.01),且低于野生型PCSK9 BEL肝細胞(p0.05)。5、免疫熒光檢測肝細胞PCSK9與LDL-R共定位,轉(zhuǎn)染前后分別主要位于細胞膜和胞內(nèi)。6、流式細胞法檢測轉(zhuǎn)染細胞LDL-R對熒光標記LDL的結(jié)合活性的變化,與空白對照組細胞相比野生型PCSK9組熒光強度降低(p0.05),陽性對照F216R組平均熒光降低更為明顯(p0.01),新突變體組平均熒光強度降低程度較野生型明顯(p0.01)。7、突變體Tet on系統(tǒng)在BEL-7402細胞中高效、穩(wěn)定表達。在PCSK9野生型及突變體Tet on系統(tǒng)中隨著Doxycycline藥物濃度的增加,PCSK9野生型和突變體開始表達,且表達量逐漸升高,LDL-R成熟蛋白部分逐漸減少,未糖基化的LDL-R未見顯著變化;與野生型相比,R306S降低成熟LDL-R的能力顯著提高(p0.05)。 [結(jié)論] 1.成功構(gòu)建PCSK9基因野生和突變類型的真核表達載體; 2.體外實驗中發(fā)現(xiàn)PCSK9基因新突變體R306S對LDL-R的轉(zhuǎn)錄無顯著影響; 3.PCSK9基因新突變體R306S可明顯降低細胞LDL-R成熟蛋白水平; 4.PCSK9基因新突變體R306S可減少其對LDL的攝取從而引發(fā)高膽固醇血癥,可能是該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.
【學位授予單位】：南華大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R346

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6 申淳;我國發(fā)現(xiàn)水稻黃綠葉突變基因[N];中國知識產(chǎn)權(quán)報;2007年

7 近物所;甘肅重離子輻照玉米誘變育種有新突破[N];農(nóng)資導報;2008年

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10 科綜;水稻“長生不老”可被制約[N];大眾科技報;2008年

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