OATP1B1 521T>C基因多态性与血脂异常的相关性研究
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摘要
血脂是血浆中所含的脂肪类物质,主要成分包括总胆固醇(TC)、甘油三酯(TG)、磷脂及游离脂肪酸等。血脂异常主要是指血中TC、TG和低密度脂蛋白胆固醇(LDL-C)的升高以及高密度脂蛋白胆固醇(HDL-C)的降低。已有的证据表明,血清TC、TG及LDL-C水平过高、HDL-C水平过低等血脂异常在动脉粥样硬化的发生及发展中起重要作用。大量的流行病学研究显示血中TC、TG以及LDL-C和HDL-C水平与冠心病的发病关系密切。美国Framingham研究证实LDL-C升高与冠心病发病呈正相关,HDL-C升高则与冠心病发病呈负相关。越来越多的资料显示,血中TG、LDL-C水平升高和HDL-C水平的降低,对冠心病发病危险有协同作用,同一水平的HDL-C和LDL-C所表现的冠心病危险,取决于血清TG水平的高低。病理研究显示,脂质在血管壁的积聚是动脉粥样硬化的重要病理基础,而且脂质的含量多少与动脉粥样硬化斑块的稳定性关系密切。脂质含量多、炎性细胞浸润明显、纤维成分少的斑块稳定性较差,容易发生破裂,引起急性心肌缺血、梗塞和猝死。临床上可以见到随着血脂异常的改善,不稳定性心绞痛、急性心肌梗死等冠状动脉事件的发生率,以及对经皮冠状动脉腔内成形术(PTCA)及冠状动脉旁路移植术(CABG)的需求都有明显的减少。通过药物或非药物的方法对血脂水平进行干预,可显著地降低冠心病发病和死亡的危险。
血脂异常与遗传、饮食、生活习惯等因素有关,其中遗传因素的影响越来越引起人们的重视。研究血脂异常的遗传易感基因对于开展针对性的人群预防,延缓或防止血脂异常的发生具有重要的意义。随着分子生物学的迅速发展及人类基因组计划的进行,寻找血脂异常易感基因并从基因水平阐明血脂异常发病机制成为医学研究的热点。早在1985年,诺贝尔奖获得者Goldstein和Brown发现了LDL受体基因突变而导致的家族性高胆固醇血症。后来多种载脂蛋白和血管紧张素转化酶、胆固醇7α-羟化酶等的基因多态性与血脂异常的相关性陆续被报道,而有机阴离子转运多肽1B1 (organic anion transporting polypeptide 1B1,OATP1B1)基因多态性与血脂异常是否具有相关性尚少报道。
OATP是摄入型转运体中的一大类,隶属于溶质转运体超家族(superfamily of solute carriers,SLC),对于内、外源性物质,尤其是药物的吸收、分布、消除具有重要影响,其编码基因统称为SLCO基因。OATP家族成员转运的底物广泛而具有重叠性,即某种底物通常可通过一种以上的转运体进行转运。除胆汁酸等内源性物质外,OATP转运体还参与多种外源性物质和药物的转运。尤其是OATP1B1作用最为重要,它们特异性表达于人肝细胞基底外侧膜上,可从门脉系统吸收和转运多种内源性物质和药物进入肝细胞进而代谢和清除,包括胆红素、胆汁酸、甲状腺激素、白三烯、前列腺素、硫酸盐和葡萄糖醛酸结合产物、肽类、甲氨蝶呤、瑞格列奈和他汀类药物。
人类OATP1B1基因定位于12号染色体短臂21区2带,全长10.86kb,包括15个外显子和14个内含子,其编码基因SLCO1B1的cDNA包含2073个碱基,编码691个氨基酸,存在20个以上的功能遗传多态性。近年来,SLCO1B1基因上已陆续发现多个单核苷酸多态性(SNP),其中有些SNP已经在体外或体内实验中证实与转运功能异常有关,研究多集中在388A>G和521T>C两个位点的突变。
在OATP1B1转运的众多底物中,胆红素、胆汁酸、甲状腺素等均与血脂异常的发病相关。胆红素能干扰胆固醇转运,降低血脂,卵磷脂胆固醇酯酰转移酶(LCAT)在HDL的胆固醇的逆转运过程中起重要作用,而有文献报道低胆红素水平可致LCAT活性减弱,从而推断出胆红素能影响体内血脂代谢；胆汁酸是胆固醇在肝脏中代谢的产物,肝内胆固醇的主要去路是通过肝细胞合成胆汁酸,同时胆汁酸进入胆汁排泄后,又促进胆固醇的有效乳化,形成混合微团,帮助消化吸收,因此,胆汁酸的合成与分泌的质和量都对胆固醇在体内的含量有明显影响,且与脂类代谢密切相关；甲状腺素能刺激脂蛋白脂肪酶受体活性,甲减时其清除率下降,血清LDL颗粒的清除被延迟,LDL水平即升高,血清TC水平亦随之升高,所以甲减常合并血脂异常；综上所述,OATP1B1转运的部分内源性底物均参与了血脂的调节,从功能意义上可考虑OATP1B1与血脂异常的发病机制相关,推测OATP1B1521T>C基因和血脂异常的易感性可能存在联系。而且人们在对健康志愿者的研究显示OATP1B1521T>C增加胆固醇的合成率,推测OATP1B1基因多态性与高胆固醇血症具有相关性。有研究表明,OATP1B1中分布较低的c.521CC基因型(在白种人中占2-5%)比c.521TT基因型更能增加人体内普伐他汀、瑞舒伐他汀、辛伐他汀、阿托伐他汀的血药浓度,已有大量的研究证明了OATP1B1对他汀类药物的药代动力学的影响,但很少研究了解OATP1B1基因多态性是否对血脂的发生产生影响,本研究采用实时荧光定量Taqman MGB探针法对321名血脂异常患者和259名正常对照者进行了OATP1B1 521T>C基因的基因分型研究,试图寻找OATP1B1 521T>C基因突变型在两者间可能的不同分布,从而初步寻找OATP1B1 521T>C基因的遗传多态性与血脂异常间可能存在的关联性,从基因角度来了解血脂异常的发病机制,为寻找血脂异常的易感基因建立一定的临床证据。
目的
探讨OATP1B1 521T>C基因多态性与血脂异常的发病是否存在相关性。
对象及方法
对象
所有研究对象均来自广州军区广州总医院2008年10月至2009年6月心血管内科的住院病人及体检中心的体检人群。入选标准：1、血脂异常组：a符合血脂异常诊断标准,即禁食12小时后测血清TC≥5.72mmol/L或LDL-C≥3.36mmol/L或者TG≥1.70mmol/L,或者HDL-C≤0.9 mmol/L,血脂中TG、TC、LDL-C、HDL-C至少2个或2个以上指标异常才被入选；b肝肾功能正常；c无烟酒嗜好；d无糖尿病等内分泌代谢疾病以及其它严重的全身性疾病。2、对照组：各项血脂指标正常、血尿常规、凝血功能、胸部X光及血生化、心电图等检查正常,无肝肾疾病,无烟酒嗜好,无糖尿病等内分泌代谢疾病及其它严重的全身性疾病。两组研究对象近期均无服用任何降脂药物,女性近期无使用钙剂及围绝经期激素替代治疗。本实验共抽取1500份血标本,经筛选符合纳入标准的共有580份,其中血脂异常组321例,男162例,女159例,年龄53.54±12.06岁；对照组259例,男111例,女148例,年龄52.63±11.68岁。对所有对象均询问病史、吸烟史、酗酒史、体检、实验室检查及辅助检查。两组性别构成、年龄具有可比性,血脂异常组及对照组所有研究对象均为汉族,研究个体间无血缘关系。
方法
实验对象空腹12小时以上,取静脉血采用标准酶比色法测定TC、TG、LDL-C和HDL-C,用免疫浊度法测定载脂蛋白A1(apoA1)和载脂蛋白B(apoB)及脂蛋白(a)。另取5ml静脉血置于EDTA-K2抗凝管按人血液DNA提取试剂盒操作步骤提取白细胞基因组DNA,所提DNA采用Rotor-Gene Q 6000实时荧光定量PCR仪检测,运用核酸扩增结合双色荧光标记的TaqMan MGB探针来检测OATP1B1 521T>C位点的基因型。采用卡方检验对血脂异常组和对照组OATP1B1 521T>C基因型及等位基因分布频率进行比较,再用方差分析分别分析两组中OATP1B1 521T>C基因多态性同TC、TG、LDL-C、HDL-C各个血脂水平间的关系,最后用Logistic回归分析对影响血脂异常的相关因素进行回归分析,P<0.05为有统计学意义。
结果
1.血脂异常组和对照组均可检测出OATP1B1 521T>C野生型纯合子(TT)、突变型杂合子(TC)、突变型纯合子(CC)三种基因型。
2.随机选取8个DNA样本,送往上海英骏生物技术有限公司进行DNA测序,测序结果与Taqman MGB探针法检测基本吻合。
3.血脂异常组OATP1B1 521T>C的T/T、T/C、C/C基因型检出率分别为74.8%、23.7%、1.5%,对照组的分别为76.8%、21.2%、2.0%,经χ2检验差异无统计学意义(χ2=0.575,P=0.750)；血脂异常组OATP1B1 521T>C的等位基因即T和C等位基因分布频率分别为86.6%和13.4%,对照组OATP1B1 521T>C的等位基因即T和C等位基因分布频率分别为87.5%和12.5%,经χ2检验差异无统计学意义(χ2=0.182,P=0.670)；
4.采用方差分析分别分析两组间OATP1B1 521T>C基因多态性同TC、TG、LDL-C、HDL-C各个血脂水平间的关系,但均P>0.05；
5.用Logistic回归分析对影响血脂异常的相关因素进行回归分析,见除了年龄和性别是血脂异常发病的相关因素外,OATP1B1 521T>C各基因型与血脂异常无显著相关性(P>0.05)。
结论
1.汉族人群中可检测到OATP1B1 521T>C基因型：TT野生型、TC杂合突变型和CC纯合突变型共三种基因型。
2.OATP1B1 521T>C基因多态性与血脂异常之间未发现相关性。
3.OATP1B1 521T>C基因多态性对各个单一的血脂指标无明显影响。
Plasma lipids are fatty substances, the main components include total cholesterol (TC)、triglyceride (TG)、phospholipids、free fatty acids and so on. Dyslipidemia mainly refers to the blood lipids get more than the normal range, including the TC、TG and low-density lipoprotein cholesterol (LDL-C) levels and high density lipoprotein cholesterol (HDL-C) reduction. Existing evidence suggests that the dyslipidemia such as higher lever of the TC、TG、LDL-C and lower level of HDL-C plays an important role in the development of atherosclerosis. A large number of epidemiological studies have shown that the level of TC、TG、LDL-C and HDL-C is closely linked to coronary heart disease. U.S. Framingham study has confirmed the elevated LDL-C and coronary heart disease was positively correlated,and increased HDL-C was negatively correlated. More and more information have shown that the risk of increased TG、LDL-C levels and the decreased HDL-C levels has a strong synergy to the coronary heart disease.At the same level of HDL-C and LDL-C, the risk of coronary heart disease depends on the level of serum TG level. Pathological studies have shown that the accumulation of lipids in the blood vessel wall is an important pathologic basis of atherosclerosis, and the number of lipid levels is closely related with the stability of atherosclerotic plaque.The plaque stability is less poor which is rich in lipid content, inflammatory cell infiltration, and poor in fibrous components, which prones to rupture, acute myocardial ischemia, infarction and sudden death. When the dyslipidemia has been clinical improved, the incidence of coronary events such as unstable angina, acute myocardial infarction, as well as the demand of percutaneous transluminal coronary angioplasty (PTCA) and coronary artery bypass grafting (CABG) have significantly reduced. To intervent the plasma lipids by drugs or not drugs can significantly reduce the risk of coronary heart disease and death.
Dyslipidemia has become a common and frequently-occurring disease, but the pathogenesis of dyslipidemia has not been reported. We have only to understand the pathogenesis of dyslipidemia in order to better prevent the occurrence of disease and reinforce the treatment of disease. Dyslipidemia is caused by heredity, diet, lifestyle and other factors, and genetic factors has drawn increasing attention. Studying the genetic susceptibility genes of dyslipidemia is important for people to carry out targeted prevention, delay or prevent the occurrence of the disease. With the rapid development of molecular biology and human genome project, to find the susceptibility genes of abnormal blood lipid levels and to clarify the pathogenesis of dyslipidemia from genes have become hotspot for medical research. As early as 1985, Nobel Laureate Brown and Goldstein had found that the gene mutations of LDL receptor identified result of familial hypercholesterolemia. Later, it has been reported one after another that the gene polymorphism of a variety of apolipoproteins angiotensin-converting enzyme, and cholesterol 7α-hydroxylase are associated with dyslipidemia, but whether Organic anion transporting polypeptide1B1 (OATP1B1) gene polymorphism and lipid abnormalities is relevanted have not been reported.
OATP is the intake-type transporter in a large class, belonging to the superfamily of solute carriers(SLC), it plays an important influence in the absorption、distribution、elimination of the endogenous and exogenous substances, the drug in particular,and its coding gene referred to as SLCO gene.The substrate of OATP transporter family members is broad and overlap, which is a substrate usually by more than one transporter for transport. In addition to bile acids and other endogenous substances, OATP transporter is also involved in the transit of various exogenous substances and drugs. OATP1B1 especially plays the most important role, which was specifically expressed in human liver cells in the basolateral membrane, and a variety of endogenous substances and drugs can be absorbed and transported from the portal system into the liver cells to be metabolized and cleared, including bilirubin, bile acids, thyroid hormones, leukotrienes, prostaglandins, sulfate and glucuronide products, peptides, methotrexate, repaglinide, and statins.
Human OATP1B1 gene was located on the short arm of 12 chromosome with 21 zone 2, full 10.86kb, including 15 exons and 14 introns, the cDNA of its coding gene SLCO1B1 contains 2073 bp,encoding 691 amino acids, there is more than 20 functional genetic polymorphism. In recent years, SLCO1B1 gene has been discovered more than a single nucleotide polymorphism (SNP), some of which have been confirmed that they are associated with the transfer function in vitro or in vivo experiments, now studies were focused on the mutations of 388A> G and 521T> C.
Many transporter substrates in the OATP1B1 such as the bilirubin, bile acids, and thyroid hormone are all connected with the pathogenesis of dyslipidemia. Bilirubin can interfere with cholesterol transport and lower blood lipids, lecithin cholesterol ester acyl transferase (LCAT) plays an important role in the reversal transport of HDL cholesterol, while the reported about low level of bilirubin can lead to a lack of LCAT activity, which conclude that bilirubin can affect the lipid metabolism in the body; Bile acid is a product of cholesterol metabolism in the liver, cholesterol in the liver mainly synthesizes bile acid by liver cells, while after bile acid excretes into the bile, it can promote emulsified efficient of cholesterol to form mixed micelles, which helps digestion and absorption, so, both quantity and quality of bile acid synthesis and secretion have significant effect to content of cholesterol in the body, and closely related with lipid metabolism also; Thyroid hormone stimulates lipoprotein lipase receptor activity, the clearance rate of which decreases when hypothyroidism, the clearance of serum LDL particles is delayed, then LDL level is higher also, with an increasing level of serum TC, so hypothyroidism is often associated with dyslipidemia; In conclusion, most of the endogenous substrates transported by OATP1B1 are involved in the regulation of blood lipids, it can be considered that OATP1B1 and pathogenesis of dyslipidemia is correlated from the functional significance, suggesting that OATP1B1 521T> C gene may link to susceptibility of lipid abnormalities.Studies in healthy volunteers showed that OATP1B1 521T> C could increase cholesterol synthesis rate, which suggesting that OATP1B1 gene polymorphisms is relevanted with hypercholesterolemia. Extensive researches have been proved that the OATP1B1 on statin pharmacokinetics, but few studies about the incidence of OATP 1B1 on blood lipids. In this study,321 dyslipidemia patients and 259 healthy controls were detected by the TaqMan-MGB probe real-time fluorescence quantitative PCR, trying to find the difference of OATP1B1 521T> C gene mutations between groups so as to search initially the association between OATP1B1 521T> C gene polymorphism and lipid abnormalities and to explore the pathogenesis of dyslipidemia.
Objective
Exploring the relationship between the OATP1B1 521T>C gene polymorphism and dyslipidemia.
Subjects and methods
1. Subjects
1500 blood samples were taken from the General Hospital of Guangzhou Military Command from October 2008 to June 2009, and 580 blood samples were screened. The inclusion criteria of dyslipidemia:a consistent diagnostic criteria for dyslipidemia, or 12 hours after the two measured fasting serum TC≥5.72mmol/L or LDL-C≥3.36mmol/L or TG≥1.70mmol/L, or HDL-C≥0.9 mmol/L; Those subjects who meet the diagnostic criteria of dyslipidemia were classified into dyslipidemia group, but those subjects who the lipid levels were normal into a control group. All objects are individuals of the Han nationality, and there is not kinship between all individuals. And those who sufferred from liver, kidney, tumor, thyroid and diabetes diseases were excluded. There were 321 cases in the dyslipidemia group: 162 cases of male and 159 cases of female, age 53.54±12.06 years;and there were 259 cases in the control group:111 cases of male and 148 cases of female, age 52.63±11.68 years. By statistical analysis, the age, sex rate and other conditions in two groups were no significant difference.
2. methods
Subjects after fasting more than 12 hours were taken venous blood. TC、TG、LDL-C and HDL-C were detected by standard blood enzyme colorimetric, and apolipoprotein A1(apoA1)、apolipoprotein B(apoB) and lipoprotein (a) were measured by immune turbidimetry. Another 5ml venous blood was taken to extract leukocyte genomic DNA by the blood DNA extraction kit. The extracted DNA was detected by the TaqMan-MGB probe real-time fluorescence quantitative PCR,and the results were compared with those of DNA sequencing.Two Independent-Samples T-Test was used to analyze the distinction of age between dyslipidemia group and control group, and test was employed to compare the baseline information such as the percentage of men、the incidence of CHD、hypertension in two groups. Chi-Square test was employed to compare OATP1B1 521T>C genotype frequencies and allele frequencies in two groups, and analysis of variance was used to compare the genotypes of TG、TC、LDL-C、HDL-C between the two groups,in the end, Logistic regression analysis was used to analyze the factors related to abnormal blood lipid.A probability value less than 0.05 was considered statistically significant.
Results
1.Dyslipidemia and control groups can be detected three OATP1B1 521T> C genotypes:homozygous wild-type(TT), heterozygous mutant(TC), homozygous mutant (CC).
2.8 randomly selected DNA samples were sent to the Biotechnology Co., Ltd. Shanghai Ying Chun for DNA sequencing, the conclusions of sequencing and Taqman MGB probe assay are basically consistent.
3. In the dyslipidemia group OATP1B1 521T> C of the T/T, T/C, C/C genotype detection rates were 74.8%,23.7%,1.5%,and in the control group were 76.8%, 21.2%,2.0%, it showed no significant difference byχ2 test (χ2=0.575, P=0.750); In the dyslipidemia group the frequencies of OATP1B1 521T> C allele T and C allele were 86.6% and 13.4%,and in the control group were 87.5% and 12.5%, it showed no significant difference byχ2 test(χ2=0.182, P=0.670);
4. Using analysis of variance to analyze the relationship of OATP1B1 521T> C gene polymorphism and TC, TG, LDL-C, HDL-C in all serum lipid levels between the two groups, but all P> 0.05;
5. Useing logistic regression analysis to analyze related factors with dyslipidemia, it show that in addition to age and gender are related to the pathogenesis of dyslipidemia factors, OATP1B1 521T> C genotypes and lipid abnormalities was no significant correlation (P>0.05).
Conclusion
1. In Han Chinese, it can detecte three kinds of OATP1B1 521T> C genotype:TT wild type, TC heterozygous and CC homozygous mutant.
2. It found no correlation between OATP1B1 521T> C polymorphism and dyslipidemia.
3. OATP1B1 521T>C gene polymorphism had no effect on all single serum lipid.
血脂异常与遗传、饮食、生活习惯等因素有关,其中遗传因素的影响越来越引起人们的重视。研究血脂异常的遗传易感基因对于开展针对性的人群预防,延缓或防止血脂异常的发生具有重要的意义。随着分子生物学的迅速发展及人类基因组计划的进行,寻找血脂异常易感基因并从基因水平阐明血脂异常发病机制成为医学研究的热点。早在1985年,诺贝尔奖获得者Goldstein和Brown发现了LDL受体基因突变而导致的家族性高胆固醇血症。后来多种载脂蛋白和血管紧张素转化酶、胆固醇7α-羟化酶等的基因多态性与血脂异常的相关性陆续被报道,而有机阴离子转运多肽1B1 (organic anion transporting polypeptide 1B1,OATP1B1)基因多态性与血脂异常是否具有相关性尚少报道。
OATP是摄入型转运体中的一大类,隶属于溶质转运体超家族(superfamily of solute carriers,SLC),对于内、外源性物质,尤其是药物的吸收、分布、消除具有重要影响,其编码基因统称为SLCO基因。OATP家族成员转运的底物广泛而具有重叠性,即某种底物通常可通过一种以上的转运体进行转运。除胆汁酸等内源性物质外,OATP转运体还参与多种外源性物质和药物的转运。尤其是OATP1B1作用最为重要,它们特异性表达于人肝细胞基底外侧膜上,可从门脉系统吸收和转运多种内源性物质和药物进入肝细胞进而代谢和清除,包括胆红素、胆汁酸、甲状腺激素、白三烯、前列腺素、硫酸盐和葡萄糖醛酸结合产物、肽类、甲氨蝶呤、瑞格列奈和他汀类药物。
人类OATP1B1基因定位于12号染色体短臂21区2带,全长10.86kb,包括15个外显子和14个内含子,其编码基因SLCO1B1的cDNA包含2073个碱基,编码691个氨基酸,存在20个以上的功能遗传多态性。近年来,SLCO1B1基因上已陆续发现多个单核苷酸多态性(SNP),其中有些SNP已经在体外或体内实验中证实与转运功能异常有关,研究多集中在388A>G和521T>C两个位点的突变。
在OATP1B1转运的众多底物中,胆红素、胆汁酸、甲状腺素等均与血脂异常的发病相关。胆红素能干扰胆固醇转运,降低血脂,卵磷脂胆固醇酯酰转移酶(LCAT)在HDL的胆固醇的逆转运过程中起重要作用,而有文献报道低胆红素水平可致LCAT活性减弱,从而推断出胆红素能影响体内血脂代谢；胆汁酸是胆固醇在肝脏中代谢的产物,肝内胆固醇的主要去路是通过肝细胞合成胆汁酸,同时胆汁酸进入胆汁排泄后,又促进胆固醇的有效乳化,形成混合微团,帮助消化吸收,因此,胆汁酸的合成与分泌的质和量都对胆固醇在体内的含量有明显影响,且与脂类代谢密切相关；甲状腺素能刺激脂蛋白脂肪酶受体活性,甲减时其清除率下降,血清LDL颗粒的清除被延迟,LDL水平即升高,血清TC水平亦随之升高,所以甲减常合并血脂异常；综上所述,OATP1B1转运的部分内源性底物均参与了血脂的调节,从功能意义上可考虑OATP1B1与血脂异常的发病机制相关,推测OATP1B1521T>C基因和血脂异常的易感性可能存在联系。而且人们在对健康志愿者的研究显示OATP1B1521T>C增加胆固醇的合成率,推测OATP1B1基因多态性与高胆固醇血症具有相关性。有研究表明,OATP1B1中分布较低的c.521CC基因型(在白种人中占2-5%)比c.521TT基因型更能增加人体内普伐他汀、瑞舒伐他汀、辛伐他汀、阿托伐他汀的血药浓度,已有大量的研究证明了OATP1B1对他汀类药物的药代动力学的影响,但很少研究了解OATP1B1基因多态性是否对血脂的发生产生影响,本研究采用实时荧光定量Taqman MGB探针法对321名血脂异常患者和259名正常对照者进行了OATP1B1 521T>C基因的基因分型研究,试图寻找OATP1B1 521T>C基因突变型在两者间可能的不同分布,从而初步寻找OATP1B1 521T>C基因的遗传多态性与血脂异常间可能存在的关联性,从基因角度来了解血脂异常的发病机制,为寻找血脂异常的易感基因建立一定的临床证据。
目的
探讨OATP1B1 521T>C基因多态性与血脂异常的发病是否存在相关性。
对象及方法
对象
所有研究对象均来自广州军区广州总医院2008年10月至2009年6月心血管内科的住院病人及体检中心的体检人群。入选标准：1、血脂异常组：a符合血脂异常诊断标准,即禁食12小时后测血清TC≥5.72mmol/L或LDL-C≥3.36mmol/L或者TG≥1.70mmol/L,或者HDL-C≤0.9 mmol/L,血脂中TG、TC、LDL-C、HDL-C至少2个或2个以上指标异常才被入选；b肝肾功能正常；c无烟酒嗜好；d无糖尿病等内分泌代谢疾病以及其它严重的全身性疾病。2、对照组：各项血脂指标正常、血尿常规、凝血功能、胸部X光及血生化、心电图等检查正常,无肝肾疾病,无烟酒嗜好,无糖尿病等内分泌代谢疾病及其它严重的全身性疾病。两组研究对象近期均无服用任何降脂药物,女性近期无使用钙剂及围绝经期激素替代治疗。本实验共抽取1500份血标本,经筛选符合纳入标准的共有580份,其中血脂异常组321例,男162例,女159例,年龄53.54±12.06岁；对照组259例,男111例,女148例,年龄52.63±11.68岁。对所有对象均询问病史、吸烟史、酗酒史、体检、实验室检查及辅助检查。两组性别构成、年龄具有可比性,血脂异常组及对照组所有研究对象均为汉族,研究个体间无血缘关系。
方法
实验对象空腹12小时以上,取静脉血采用标准酶比色法测定TC、TG、LDL-C和HDL-C,用免疫浊度法测定载脂蛋白A1(apoA1)和载脂蛋白B(apoB)及脂蛋白(a)。另取5ml静脉血置于EDTA-K2抗凝管按人血液DNA提取试剂盒操作步骤提取白细胞基因组DNA,所提DNA采用Rotor-Gene Q 6000实时荧光定量PCR仪检测,运用核酸扩增结合双色荧光标记的TaqMan MGB探针来检测OATP1B1 521T>C位点的基因型。采用卡方检验对血脂异常组和对照组OATP1B1 521T>C基因型及等位基因分布频率进行比较,再用方差分析分别分析两组中OATP1B1 521T>C基因多态性同TC、TG、LDL-C、HDL-C各个血脂水平间的关系,最后用Logistic回归分析对影响血脂异常的相关因素进行回归分析,P<0.05为有统计学意义。
结果
1.血脂异常组和对照组均可检测出OATP1B1 521T>C野生型纯合子(TT)、突变型杂合子(TC)、突变型纯合子(CC)三种基因型。
2.随机选取8个DNA样本,送往上海英骏生物技术有限公司进行DNA测序,测序结果与Taqman MGB探针法检测基本吻合。
3.血脂异常组OATP1B1 521T>C的T/T、T/C、C/C基因型检出率分别为74.8%、23.7%、1.5%,对照组的分别为76.8%、21.2%、2.0%,经χ2检验差异无统计学意义(χ2=0.575,P=0.750)；血脂异常组OATP1B1 521T>C的等位基因即T和C等位基因分布频率分别为86.6%和13.4%,对照组OATP1B1 521T>C的等位基因即T和C等位基因分布频率分别为87.5%和12.5%,经χ2检验差异无统计学意义(χ2=0.182,P=0.670)；
4.采用方差分析分别分析两组间OATP1B1 521T>C基因多态性同TC、TG、LDL-C、HDL-C各个血脂水平间的关系,但均P>0.05；
5.用Logistic回归分析对影响血脂异常的相关因素进行回归分析,见除了年龄和性别是血脂异常发病的相关因素外,OATP1B1 521T>C各基因型与血脂异常无显著相关性(P>0.05)。
结论
1.汉族人群中可检测到OATP1B1 521T>C基因型：TT野生型、TC杂合突变型和CC纯合突变型共三种基因型。
2.OATP1B1 521T>C基因多态性与血脂异常之间未发现相关性。
3.OATP1B1 521T>C基因多态性对各个单一的血脂指标无明显影响。
Plasma lipids are fatty substances, the main components include total cholesterol (TC)、triglyceride (TG)、phospholipids、free fatty acids and so on. Dyslipidemia mainly refers to the blood lipids get more than the normal range, including the TC、TG and low-density lipoprotein cholesterol (LDL-C) levels and high density lipoprotein cholesterol (HDL-C) reduction. Existing evidence suggests that the dyslipidemia such as higher lever of the TC、TG、LDL-C and lower level of HDL-C plays an important role in the development of atherosclerosis. A large number of epidemiological studies have shown that the level of TC、TG、LDL-C and HDL-C is closely linked to coronary heart disease. U.S. Framingham study has confirmed the elevated LDL-C and coronary heart disease was positively correlated,and increased HDL-C was negatively correlated. More and more information have shown that the risk of increased TG、LDL-C levels and the decreased HDL-C levels has a strong synergy to the coronary heart disease.At the same level of HDL-C and LDL-C, the risk of coronary heart disease depends on the level of serum TG level. Pathological studies have shown that the accumulation of lipids in the blood vessel wall is an important pathologic basis of atherosclerosis, and the number of lipid levels is closely related with the stability of atherosclerotic plaque.The plaque stability is less poor which is rich in lipid content, inflammatory cell infiltration, and poor in fibrous components, which prones to rupture, acute myocardial ischemia, infarction and sudden death. When the dyslipidemia has been clinical improved, the incidence of coronary events such as unstable angina, acute myocardial infarction, as well as the demand of percutaneous transluminal coronary angioplasty (PTCA) and coronary artery bypass grafting (CABG) have significantly reduced. To intervent the plasma lipids by drugs or not drugs can significantly reduce the risk of coronary heart disease and death.
Dyslipidemia has become a common and frequently-occurring disease, but the pathogenesis of dyslipidemia has not been reported. We have only to understand the pathogenesis of dyslipidemia in order to better prevent the occurrence of disease and reinforce the treatment of disease. Dyslipidemia is caused by heredity, diet, lifestyle and other factors, and genetic factors has drawn increasing attention. Studying the genetic susceptibility genes of dyslipidemia is important for people to carry out targeted prevention, delay or prevent the occurrence of the disease. With the rapid development of molecular biology and human genome project, to find the susceptibility genes of abnormal blood lipid levels and to clarify the pathogenesis of dyslipidemia from genes have become hotspot for medical research. As early as 1985, Nobel Laureate Brown and Goldstein had found that the gene mutations of LDL receptor identified result of familial hypercholesterolemia. Later, it has been reported one after another that the gene polymorphism of a variety of apolipoproteins angiotensin-converting enzyme, and cholesterol 7α-hydroxylase are associated with dyslipidemia, but whether Organic anion transporting polypeptide1B1 (OATP1B1) gene polymorphism and lipid abnormalities is relevanted have not been reported.
OATP is the intake-type transporter in a large class, belonging to the superfamily of solute carriers(SLC), it plays an important influence in the absorption、distribution、elimination of the endogenous and exogenous substances, the drug in particular,and its coding gene referred to as SLCO gene.The substrate of OATP transporter family members is broad and overlap, which is a substrate usually by more than one transporter for transport. In addition to bile acids and other endogenous substances, OATP transporter is also involved in the transit of various exogenous substances and drugs. OATP1B1 especially plays the most important role, which was specifically expressed in human liver cells in the basolateral membrane, and a variety of endogenous substances and drugs can be absorbed and transported from the portal system into the liver cells to be metabolized and cleared, including bilirubin, bile acids, thyroid hormones, leukotrienes, prostaglandins, sulfate and glucuronide products, peptides, methotrexate, repaglinide, and statins.
Human OATP1B1 gene was located on the short arm of 12 chromosome with 21 zone 2, full 10.86kb, including 15 exons and 14 introns, the cDNA of its coding gene SLCO1B1 contains 2073 bp,encoding 691 amino acids, there is more than 20 functional genetic polymorphism. In recent years, SLCO1B1 gene has been discovered more than a single nucleotide polymorphism (SNP), some of which have been confirmed that they are associated with the transfer function in vitro or in vivo experiments, now studies were focused on the mutations of 388A> G and 521T> C.
Many transporter substrates in the OATP1B1 such as the bilirubin, bile acids, and thyroid hormone are all connected with the pathogenesis of dyslipidemia. Bilirubin can interfere with cholesterol transport and lower blood lipids, lecithin cholesterol ester acyl transferase (LCAT) plays an important role in the reversal transport of HDL cholesterol, while the reported about low level of bilirubin can lead to a lack of LCAT activity, which conclude that bilirubin can affect the lipid metabolism in the body; Bile acid is a product of cholesterol metabolism in the liver, cholesterol in the liver mainly synthesizes bile acid by liver cells, while after bile acid excretes into the bile, it can promote emulsified efficient of cholesterol to form mixed micelles, which helps digestion and absorption, so, both quantity and quality of bile acid synthesis and secretion have significant effect to content of cholesterol in the body, and closely related with lipid metabolism also; Thyroid hormone stimulates lipoprotein lipase receptor activity, the clearance rate of which decreases when hypothyroidism, the clearance of serum LDL particles is delayed, then LDL level is higher also, with an increasing level of serum TC, so hypothyroidism is often associated with dyslipidemia; In conclusion, most of the endogenous substrates transported by OATP1B1 are involved in the regulation of blood lipids, it can be considered that OATP1B1 and pathogenesis of dyslipidemia is correlated from the functional significance, suggesting that OATP1B1 521T> C gene may link to susceptibility of lipid abnormalities.Studies in healthy volunteers showed that OATP1B1 521T> C could increase cholesterol synthesis rate, which suggesting that OATP1B1 gene polymorphisms is relevanted with hypercholesterolemia. Extensive researches have been proved that the OATP1B1 on statin pharmacokinetics, but few studies about the incidence of OATP 1B1 on blood lipids. In this study,321 dyslipidemia patients and 259 healthy controls were detected by the TaqMan-MGB probe real-time fluorescence quantitative PCR, trying to find the difference of OATP1B1 521T> C gene mutations between groups so as to search initially the association between OATP1B1 521T> C gene polymorphism and lipid abnormalities and to explore the pathogenesis of dyslipidemia.
Objective
Exploring the relationship between the OATP1B1 521T>C gene polymorphism and dyslipidemia.
Subjects and methods
1. Subjects
1500 blood samples were taken from the General Hospital of Guangzhou Military Command from October 2008 to June 2009, and 580 blood samples were screened. The inclusion criteria of dyslipidemia:a consistent diagnostic criteria for dyslipidemia, or 12 hours after the two measured fasting serum TC≥5.72mmol/L or LDL-C≥3.36mmol/L or TG≥1.70mmol/L, or HDL-C≥0.9 mmol/L; Those subjects who meet the diagnostic criteria of dyslipidemia were classified into dyslipidemia group, but those subjects who the lipid levels were normal into a control group. All objects are individuals of the Han nationality, and there is not kinship between all individuals. And those who sufferred from liver, kidney, tumor, thyroid and diabetes diseases were excluded. There were 321 cases in the dyslipidemia group: 162 cases of male and 159 cases of female, age 53.54±12.06 years;and there were 259 cases in the control group:111 cases of male and 148 cases of female, age 52.63±11.68 years. By statistical analysis, the age, sex rate and other conditions in two groups were no significant difference.
2. methods
Subjects after fasting more than 12 hours were taken venous blood. TC、TG、LDL-C and HDL-C were detected by standard blood enzyme colorimetric, and apolipoprotein A1(apoA1)、apolipoprotein B(apoB) and lipoprotein (a) were measured by immune turbidimetry. Another 5ml venous blood was taken to extract leukocyte genomic DNA by the blood DNA extraction kit. The extracted DNA was detected by the TaqMan-MGB probe real-time fluorescence quantitative PCR,and the results were compared with those of DNA sequencing.Two Independent-Samples T-Test was used to analyze the distinction of age between dyslipidemia group and control group, and test was employed to compare the baseline information such as the percentage of men、the incidence of CHD、hypertension in two groups. Chi-Square test was employed to compare OATP1B1 521T>C genotype frequencies and allele frequencies in two groups, and analysis of variance was used to compare the genotypes of TG、TC、LDL-C、HDL-C between the two groups,in the end, Logistic regression analysis was used to analyze the factors related to abnormal blood lipid.A probability value less than 0.05 was considered statistically significant.
Results
1.Dyslipidemia and control groups can be detected three OATP1B1 521T> C genotypes:homozygous wild-type(TT), heterozygous mutant(TC), homozygous mutant (CC).
2.8 randomly selected DNA samples were sent to the Biotechnology Co., Ltd. Shanghai Ying Chun for DNA sequencing, the conclusions of sequencing and Taqman MGB probe assay are basically consistent.
3. In the dyslipidemia group OATP1B1 521T> C of the T/T, T/C, C/C genotype detection rates were 74.8%,23.7%,1.5%,and in the control group were 76.8%, 21.2%,2.0%, it showed no significant difference byχ2 test (χ2=0.575, P=0.750); In the dyslipidemia group the frequencies of OATP1B1 521T> C allele T and C allele were 86.6% and 13.4%,and in the control group were 87.5% and 12.5%, it showed no significant difference byχ2 test(χ2=0.182, P=0.670);
4. Using analysis of variance to analyze the relationship of OATP1B1 521T> C gene polymorphism and TC, TG, LDL-C, HDL-C in all serum lipid levels between the two groups, but all P> 0.05;
5. Useing logistic regression analysis to analyze related factors with dyslipidemia, it show that in addition to age and gender are related to the pathogenesis of dyslipidemia factors, OATP1B1 521T> C genotypes and lipid abnormalities was no significant correlation (P>0.05).
Conclusion
1. In Han Chinese, it can detecte three kinds of OATP1B1 521T> C genotype:TT wild type, TC heterozygous and CC homozygous mutant.
2. It found no correlation between OATP1B1 521T> C polymorphism and dyslipidemia.
3. OATP1B1 521T>C gene polymorphism had no effect on all single serum lipid.
引文
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