脂蛋白脂肪酶基因突变的可能影响

脂蛋白脂肪酶（LPL）是水解富含甘油三酯脂蛋白中甘油三酯的关键酶,其基因突变常可造成严重的高甘油三酯血症和胰腺炎等疾病。近年的研究发现,LPL基因突变的影响是广泛的,不仅可能与冠心病,高血压,阿尔茨海默病,围产期疾病等相关,而且某些突变可能延缓冠状动脉疾病和阿尔茨海默病的发生和发展。     

脂蛋白脂肪酶的分子生物学

脂蛋白脂肪酶（ＬＰＬ）是一种催化乳糜微粒和极低密度脂蛋白中甘油三酯分解的糖蛋白,表达基因定位于８ｐ２２,由１０个外显子和９个内含子组成,非编码区域存在多种遗传多态位点。其表达受到多种位点的调控。错义和无义突变是ＬＰＬ缺乏症的常见突变类型。     

脂蛋白脂肪酶基因敲除对急性高脂血症性胰腺炎所致肺损伤的影响及其机制研究

摘要 目的：研究脂蛋白脂肪酶（lipoprotein lipase,LPL）基因敲除对雨蛙素诱导的高脂血症急性胰腺炎小鼠肺损伤的影响。方法：将C57BL/6小鼠分为三组,Control组和AP-Model组为野生型C57 BL/6小鼠,LPL ko组为LPL基因敲除C57 BL/6小鼠;Control组小鼠正常饲养,AP-Model和LPL ko组小鼠建立高脂血症性急性胰腺炎模型,比较三组小鼠死亡率、胰腺和肺病理损伤以及血清淀粉酶（amylase, AMY）、丙二醛（malondialdehyde, MDA）、肿瘤坏死因子-α（Tumor necrosis factor-α, TNF-α）和白介素-6（Interleukin-6, IL-6）含量。结果：急性胰腺炎建立48 h后,Control组、AP-Model组和LPL ko组小鼠死亡率分别为0 %、20 %和40 %。与Control组相比,AP-Model组和LPL ko组小鼠急性胰腺炎诱导24和48 h后的胰腺和肺组织湿/干重比值,胰腺和肺组织病理评分,血清AMY、MDA、TNF-α和IL-6含量均显著升高（P<0.05）;与AP-Model组相比,LPL ko组小鼠急性胰腺炎诱导24和48 h后的胰腺和肺组织湿/干重比值,胰腺和肺组织病理评分,血清AMY、MDA、TNF-α和IL-6含量均显著升高（P<0.05）。结论：LPL基因敲除小鼠急性高脂血症性胰腺炎肺损伤更严重,其机制可能与LPL基因敲除引起更强的氧化应激和炎症有关。     

哺乳动物脂蛋白脂肪酶基因的进化研究

脂蛋白脂肪酶(LPL)是脂肪细胞、心肌细胞、骨骼肌细胞、乳腺细胞以及巨噬细胞等实质细胞合成和分泌的一种糖蛋白,其生理功能为促进乳糜微粒和极低密度脂蛋白中甘油三脂(TG)的分解,此外,lpl基因的异常可导致血TG水平的升高和高密度脂蛋白胆固醇(HDL-C)水平的降低,导致冠状动脉粥样硬化等疾病的形成和发展。采用生物信息学手段来分析在不同哺乳动物中lpl基因的长度多态性、终止密码子多态性、ORF多态性以及氨基酸序列的同源性,建立不同哺乳动物lpl基因上的进化关系图谱,为了解哺乳动物脂蛋白脂肪酶基因的演化关系,研究该基因的作用机理提供资料。     

脂蛋白脂肪酶在巨噬细胞代谢正常极低密度脂蛋白中的作用

家兔正常极低密度脂蛋白(N—VLDL)可使体外培养的小鼠腹腔巨噬细胞内甘油三酯(TG)增加6～16倍。加入脂蛋白脂肪酶(LPL)抑制剂苯硼酸(BBA)可使培养液内游离脂酸(FFA)水平降低,N—VLDL—TG含量升高、细胞内TG堆积被明显甚至完全抑制。证明了LPL在巨噬细胞摄取N—VLDL—TG中起主要作用。     

有氧运动对小鼠高脂血症及脂蛋白代谢的影响

本研究以高胆固醇饮食小鼠为实验对象,观察有氧运动对脂质水平异常的动物个体血脂及脂蛋白代谢的影响。结果显示,经１１周有氧耐力训练后,高脂膳食＋运动组小鼠血浆ＴＧ、ＴＣ及ＬＤＬ－Ｃ水平均显著低于高脂膳食组（Ｐ＜０．０５）,而ＨＤＬ－Ｃ／ＴＣ和ＨＤＬ－Ｃ／ＬＤＬ－Ｃ比值均显著高于高脂膳食组（Ｐ＜０．０５）。表明长期有氧耐力训练能显著改善高胆固醇饮食小鼠血脂及脂蛋白代谢状况。     

海水鱼真鲷脂蛋白脂肪酶基因cDNA序列与组织表达

为研究脊椎动物真鲷脂蛋白脂肪酶 (LPL)结构与功能关系以及探讨动脉粥样硬化形成机理 ,通过构建cDNA文库 ,克隆对动脉粥样硬化表现抗性的海水鱼真鲷LPL基因cDNA全序列 .再通过PCR方法扩增基因组DNA ,获取内含子 9及其两侧序列以确定外显子 10的大小 ,最后通过RT PCR ,以 β肌动蛋白为外参照 ,比较真鲷在食用两种脂肪含量不同饲料和摄食状态不同的处理条件下 ,肝脏和腹腔肠系膜脂肪组织LPLmRNA的相对水平 .从腹腔肠系膜脂肪组织cDNA文库中克隆出LPLcDNA序列 ,其完整的开放阅读框架由 15 36bp组成 ,编码 5 11个氨基酸残基 .与哺乳类不同 ,真鲷LPL基因外显子 10的开始部分是翻译的 .LPL的催化位点、二硫键位点、N 糖基化位点、肝素结合区、脂质结合位点、介导脂蛋白与低密度脂蛋白受体结合位点、二聚体形成位点等主要功能域在真骨鱼类真鲷与其它脊椎动物间基本保守 ,但肝素结合区的碱性氨基酸残基含量较人类减少 ,并在结合脂质底物的疏水环套中出现插入片段 .与哺乳类不同 ,真鲷LPL基因在成体肝脏存在诱导性表达 ,而在其腹腔肠系膜脂肪组织则存在与哺乳类相似的组成性表达 .当真鲷喂食高脂饲料时 ,其饱食状态下肝脏LPLmRNA水平升高 ,但对其腹腔肠系膜脂肪组织LPL表达没有影响 .当真鲷喂食标准商业饲料时 ,     

中国人群家族性高胆固醇血症LDLR基因突变研究进展

家族性高胆固醇血症(Familial hypercholesterolemia,FH)主要是由于低密度脂蛋白受体(Low-density lipoprotein receptor,LDLR)基因突变导致的单基因显性遗传性疾病。FH患者LDLR基因突变导致细胞膜表面LDLR减少或缺如,机体代谢胆固醇能力降低,血浆胆固醇增高并沉积在不同的组织和器官,常伴有全身黄色瘤和早发冠心病,因此FH也是最常见的严重代谢性疾病。世界范围内对LDLR基因突变的报道总共有1741种,经整理我国目前报道的140例FH指示病例,包括108种LDLR基因突变类型。文章对已报道的中国FH患者LDLR基因突变特点进行系统分析和综述,旨在为FH诊断治疗提供参考依据。     

脂蛋白酯酶基因PvuⅡ多态与中国人高脂血症和冠心病的Meta分析

国内多个研究报道了脂蛋白酯酶基因(LPL)PvuⅡ多态(rs285)与中国人高脂血症和冠心病的关系,但单个研究的样本量都较小(119～647),结果不尽一致.为了全面客观评价LPL基因PvuⅡ多态在中国人高脂血症和冠心病发病中的作用,文章对所有中国人群的研究进行了Meta分析.共11篇文献纳入研究,其中关于高脂血症的研究6项,包括患者943例,正常对照1 093例,关于冠心病的研究5项,包括患者821例,正常对照727例.入选研究无明显的发表偏倚,但经同质性检验存在明显的异质性.Meta分析结果显示LPL PvuⅡ多态的P+等位基因增加高脂血症的患病风险(OR=1.36,95%CI1.07～1.73,P=0.011),但与冠心病相关不显著(P=0.755).因此,文章结果表明LPL￡基因pvuⅡ多态与中国人高脂血症易感性相关联,与冠心病关联不显著.     

高脂血症患者脂蛋白脂酶基因外显子4区域变异的研究

为了探讨广东地区高脂血症患者脂蛋白脂酸(lipoprotein lipase,LPL)基因的分子变异,从258例高脂血症患者外周血白细胞中提取基因组DNA,用PCR-SSCP方法分析外显子4及其附近区域,对SSCP带型异常样品进行克隆和序列测定。在2名高脂血症患者LPL基因内含子3的3′端－6bp处发现C→T转换突变,252例正常对照中未发现该突变。IVS-3 的C→T突变可能与高脂血症有关。 Variants of Exon 4 and Its Flanking Region of LPL Genein Patients with Hyperlipidemia ZHAO Ying-she1,YANG Zhong-han2,FENG Jian-sheng1,JIANG Jian-wei1,WU Mei-yu1,ZHOU Tian-hong3 1.Department of Biochemistry,Medical College,Jinan University,Guangzhou 510632,China; 2.Department of Biochemistry,Sun Yat-sen University of Medical Sciences,Guangzhou 510089,China; 3.College of Life Sciences and Technology,Jinan University,Guangzhou 510632,China Abstract:To elucidate abnormalities of LPL gene in hyperlipidemia in the Chinese population in Guangdong,genomic DNA was extracted from leukocyte of 258 patients with primary hyperlipidemia.A segment of LPL gene including exon 4 and its flanking sequences was analyzed by PCR-SSCP.The PCR products with abnormal SSCP pattern were cloned and sequenced. As a C→T transition mutation at-6 bp of intron 3 was found in two Chinese with hyperlipidemia and the mutation was not found in 252 normolipidemic controls,the C→T transition in intron 3 may be related to hyperlipedemia. Key words：lipoprotein lipase;hyperlipidemia;polymerase chain reaction;single strand conformation polymorphism     

氧化LDL的不同组分在抑制巨噬细胞释放NO中的作用

研究表明：氧化ＬＤＬ（Ｏｘ－ＬＤＬ）可抑制ＬＰＳ诱导的巨噬细胞ＮＯ释放,而正常ＬＤＬ和乙酰化ＬＤＬ则无抑制作用。用谷胱甘肽地的酶模拟物ｅｂｓｅｌｅｎ清除Ｏｘ－ＬＤＬ上的脂氢过氧化物对其抑制作用没有影响。Ｏｘ－ＬＤＬ的蛋白组分对ＮＯ释放也没有影响,而脂质组分则有抑制作用。ＬＤＬ脂质的主要成分亚油酸和卵磷脂无论单独还是共同氧化后对ＮＯ释放都没有影响,而亚油酸和胆固醇一起氧化后则对ＮＯ的释放有很强的抑制     

Lipoprotein Lipase Gene is Linked and Associated with Hypertension in Taiwan Young-onset Hypertension Genetic Study

Summary Hypertriglyceridemia has been extensively associated with hypertension. However, the mechanism behind it is poorly understood. A positive linkage signal between Lipoprotein lipase (LPL) and young-onset hypertension has been identified by us as the strongest among 18 candidate genes. Here we report our fine mapping works with seven microsatellite markers flanking LPL, sequencing results for its promoter and exons, and an extended association study with the identified single nucleotide polymorphisms(SNP). First, using data from 213 individuals in 59 nuclear families of young-onset hypertension, multipoint analysis revealed a NPL score of 3.02 for the LPL (GZ-14/GZ-15) marker in intron 6. LPL marker (p<10⁻¹²) and the haplotypes containing its allele 1 (p<0.0001) were also significantly associated with young hypertension by transmission disequilibrium test. In-depth sequencing revealed no mutation in promoter and exon regions, except two cSNP: 7754C→ A (C/A: 0.91/0.09), a silent mutation in exon 8 and S447X (C/G: 0.92/0.08), a stop codon mutation in exon 9. Other 11 cSNPs documented in NCBI GenBank are absent in our sample. Constructed from the above 2 cSNPs, haplotype AC showed a moderate TDT association with elevated triglyceride (p=0.02) and with hypertension and elevated triglyceride combined (p=0.06). Again, in an extended case-control study, a significant association was found between S447X and patients with persistent hypertension and elevated triglyceride (p=0.02). We conclude that LPL variants may play a causal role in the development of hypertension in Taiwan Han Chinese. The moderate association with SNP haplotype suggests that other regulatory LPL variant may exist.     

脂蛋白脂酶基因的克隆、序列测定及定点突变

 以人的脂肪组织总RNA为模板 ,参考已报道的脂蛋白脂酶 (lipoproteinlipase ,LPL)cDNA设计引物 ,利用RT PCR方法扩增得到了LPLcDNA ,并经序列测定证实其序列是正确的 .在冠心病患者LPL基因第 5外显子的 830位碱基处发现了G→A的转换 ,该变异导致LPL基因第 192位的密码子CGA被CAA取代 ,使LPL第 192位精氨酸改变为谷氨酰胺 .在变异碱基附近设计合成两条引物 ,其中一条包含所要改变的碱基 ,利用基于PCR的定点突变技术和体外重组的方法获得了G830A变异的LPLcDNA     

A Pressure-dependent Model for the Regulation of Lipoprotein Lipase by Apolipoprotein C-II

Apolipoprotein C-II (apoC-II) is the co-factor for lipoprotein lipase (LPL) at the surface of triacylglycerol-rich lipoproteins. LPL hydrolyzes triacylglycerol, which increases local surface pressure as surface area decreases and amphipathic products transiently accumulate at the lipoprotein surface. To understand how apoC-II adapts to these pressure changes, we characterized the behavior of apoC-II at multiple lipid/water interfaces. ApoC-II adsorption to a triacylglycerol/water interface resulted in large increases in surface pressure. ApoC-II was exchangeable at this interface and desorbed on interfacial compressions. These compressions increase surface pressure and mimic the action of LPL. Analysis of gradual compressions showed that apoC-II undergoes a two-step desorption, which indicates that lipid-bound apoC-II can exhibit at least two conformations. We characterized apoC-II at phospholipid/triacylglycerol/water interfaces, which more closely mimic lipoprotein surfaces. ApoC-II had a large exclusion pressure, similar to that of apoC-I and apoC-III. However, apoC-II desorbed at retention pressures higher than those seen with the other apoCs. This suggests that it is unlikely that apoC-I and apoC-III inhibit LPL via displacement of apoC-II from the lipoprotein surface. Upon rapid compressions and re-expansions, re-adsorption of apoC-II increased pressure by lower amounts than its initial adsorption. This indicates that apoC-II removed phospholipid from the interface upon desorption. These results suggest that apoC-II regulates the activity of LPL in a pressure-dependent manner. ApoC-II is provided as a component of triacylglycerol-rich lipoproteins and is the co-factor for LPL as pressure increases. Above its retention pressure, apoC-II desorbs and removes phospholipid. This triggers release of LPL from lipoproteins.     

Association of the HindIII Polymorphism of the Lipoprotein Lipase Gene with Myocardial Infarction and the Life Span in Elderly Patients with Coronary Heart Disease

Allele and genotype frequencies of the HindIII polymorphism of the lipoprotein lipase (LPL) gene were studied in patients with myocardial infarction (MI) and stable angina of effort (SAE), including long-lived people (over 90). The polymorphism proved to be associated with MI and with the life span, genotype H+/H+ being predisposing to MI and allele H– being protective. The allele and genotype frequencies of long-lived people differed significantly from the Hardy–Weinberg proportions and from those of SAE patients aged up to 90. An excess of heterozygotes in this group suggests a selective pressure which eliminates homozygotes. Possibly, heterozygotes H+/H– have an adaptive advantage, which provides for their longevity.     

A quantitative assay measuring the function of lipase maturation factor 1

Newly synthesized lipoprotein lipase (LPL) and related members of the lipase gene family require an endoplasmic reticulum maturation factor for attainment of enzyme activity. This factor has been identified as lipase maturation factor 1 (Lmf1), and mutations affecting its function and/or expression result in combined lipase deficiency (cld) and hypertriglyceridemia. To assess the functional impact of Lmf1 sequence variations, both naturally occurring and induced, we report the development of a cell-based assay using LPL activity as a quantitative reporter of Lmf1 function. The assay uses a cell line homozygous for the cld mutation, which renders endogenous Lmf1 nonfunctional. LPL transfected into the mutant cld cell line fails to attain activity; however, cotransfection of LPL with wild-type Lmf1 restores its ability to support normal lipase maturation. In this report, we describe optimized conditions that ensure the detection of a complete range of Lmf1 function (full, partial, or complete loss of function) using LPL activity as the quantitative reporter. To illustrate the dynamic range of the assay, we tested several novel mutations in mouse Lmf1. Our results demonstrate the ability of the assay to detect and analyze Lmf1 mutations having a wide range of effects on Lmf1 function and protein expression.     

Evidence for Two Distinct Binding Sites for Lipoprotein Lipase on Glycosylphosphatidylinositol-anchored High Density Lipoprotein-binding Protein 1 (GPIHBP1)

GPIHBP1 is an endothelial membrane protein that transports lipoprotein lipase (LPL) from the subendothelial space to the luminal side of the capillary endothelium. Here, we provide evidence that two regions of GPIHBP1, the acidic N-terminal domain and the central Ly6 domain, interact with LPL as two distinct binding sites. This conclusion is based on comparative binding studies performed with a peptide corresponding to the N-terminal domain of GPIHBP1, the Ly6 domain of GPIHBP1, wild type GPIHBP1, and the Ly6 domain mutant GPIHBP1 Q114P. Although LPL and the N-terminal domain formed a tight but short lived complex, characterized by fast on- and off-rates, the complex between LPL and the Ly6 domain formed more slowly and persisted for a longer time. Unlike the interaction of LPL with the Ly6 domain, the interaction of LPL with the N-terminal domain was significantly weakened by salt. The Q114P mutant bound LPL similarly to the N-terminal domain of GPIHBP1. Heparin dissociated LPL from the N-terminal domain, and partially from wild type GPIHBP1, but was unable to elute the enzyme from the Ly6 domain. When LPL was in complex with the acidic peptide corresponding to the N-terminal domain of GPIHBP1, the enzyme retained its affinity for the Ly6 domain. Furthermore, LPL that was bound to the N-terminal domain interacted with lipoproteins, whereas LPL bound to the Ly6 domain did not. In summary, our data suggest that the two domains of GPIHBP1 interact independently with LPL and that the functionality of LPL depends on its localization on GPIHBP1.     

猪激素敏感脂肪酶基因外显子1的遗传多态性分析

采用PCR-SSCP方法检测猪激素敏感脂肪酶（hormone sensitive lipase,HSL）基因外显子1的多态性,并分析其与初生重、断奶重、6月龄重和背膘厚的关联性。根据猪HSL基因的DNA序列（AJ000483）设计5对引物,结果在P5引物对扩增的片段上发现了多态性,并对纯合子进行测序,发现外显子1的874bp处存在G-A转换,且存在3种基因型（AA、AB、BB）。统计结果表明,3种基因型在各品种中的分布不一致,长白猪和大白猪与莱芜猪和沂蒙黑猪比较差异极显著（P〈0．01）,长白猪与大白猪比较,莱芜猪与沂蒙黑猪比较差异均不显著（P〉0．05）。固定效应模型分析结果表明,背膘厚基因型间差异显著（P〈0．05）,而初生重、断奶重和6月龄重基因型间差异均不显著（P〉0．05）。最小二乘分析结果表明,BB基因型个体同AB和AA基因型个体比较背膘厚的差异显著（P〈0．05）,3种基因型在背膘厚的大小排列顺序为BB〈AB〈AA。因此,推测HSL基因对个体胴体瘦肉率存在一定的影响将HSL某因应用于猪育种过程中的标记辅助选择可以加快猪的育种进程。