Genetic Screening of the Lipoprotein Lipase Gene for Mutations in Chinese Subjects with or without Hypertriglyceridemia

Objective： To investigate the association between the mutations in lipoprotein lipase gene and hypertriglyceridemia （HTG）. Methods： The lipoprotein lipase （LPL） gene was screened for mutations in 386 Chinese subjects with （108 cases in the HTG group） or without HTG （278 cases in the control group）, by single-strand conformation polymorphism （SSCP） analysis and DNA sequencing. Results： One novel silent mutation L103L, one missense mutation P207L, three splicing mutations Int3/3＇ -ass/C（-6）→T, and the common S447X polymorphism has been identified in the whole coding region and exon-intron junctions of the LPL gene were examined. Heterozygous P207L found in the HTG group was the first case reported in Asia and subsequently another P207L heterozygote was found in the proband＇s family, all of which suggested that P207L was one of the causes of familial combined hyperlipidemia, but was not so prevalent as that in French Canadian. Int3/3＇-ass/C（-6）→T was found in both groups in the present study although it was regarded as a pathogenic variant to HTG earlier on. Moreover about the beneficial polymorphism S447X, there was also some supportive evidence that the levels of triglycerides （TG） in S447X carriers were significantly lower than noncarders in the subjects without HTG. Conclusions： The association between the LPL variants and HTG is quite complicated and versatile, genotyping of LPL in a larger-scale screening should be necessary and justifiable.

Impact of serum levels of lipoprotein lipase, hepatic lipase, and endothelial lipase on the progression of coronary artery disease

Purpose: The purpose of this study was to investigate the relationship between serum levels of lipoprotein lipase(LPL), hepatic lipase(HL), and endothelial lipase(EL) and the progression of coronary artery disease(CAD).Materials and methods: According to the inclusion criteria, exclusion criteria, diagnostic criteria, angiography results, and the random matching scheme, the enrolled patients were divided into the following two groups: the progression-free group(n ? 47) and the progression group(n ? 15). The baseline characteristics and various biochemical parameters were obtained from the medical records and medical history. Serum LPL, HL, and EL levels were detected by ELISA. The correlation between serum LPL, HL, and EL levels and coronary lesions was statistically analyzed with SPSS software.Results: Significant differences were observed in serum levels of HL and EL between the progression-free group and the progression group(HL, 75.5 ? 39.2 ng/mL vs. 125.1 ? 42.1 ng/mL, P < 0.05;EL, 139.2 ? 59.6 pg/mL vs.175.1 ? 40.1 pg/mL, P < 0.05), while the difference in the LPL level was not significant(P > 0.05). Receiver operating characteristic curve(ROC) analysis showed that the area under the curve(AUC) values of LPL, HL, and EL were 0.506(95% CI: 0.369–0.642, P ? 0.9470), 0.792(95% CI: 0.664–0.888, P < 0.0001), and 0.693(95% CI:0.553–0.811, P ? 0.0095), respectively. Additionally, logistic regression analysis showed that the serum level of HL was an independent risk factor for coronary artery lesion progression.Conclusion: Serum levels of EL and HL, but not the serum level of LPL, were positively correlated with the progression of CAD. The serum level of HL was an independent risk factor for the progression of CAD, while the serum level of EL or LPL was not an independent risk factor for the progression of CAD. For the diagnosis of CAD progression, the serum level of HL was better than the serum level of EL or LPL.

Establishment of Real-Time TaqMan-Fluorescence Quantitative RT-PCR Assay for Detection and Quantification of Porcine Lipoprotein Lipase mRNA

Porcine lipoprotein lipase （LPL） cDNA was cloned as the standard for real-time quantifying LPL mRNA and the TaqMan-fluorescence quantitative PCR assay for detection was established. The total RNA extracted from Longissimus dorsi of porcine was reverse-transcribed to cDNA. LPL cDNA was ligated with pGM-T vector and transformed into Escherichia coli TOP10. Plasmid DNA extracted from positive clones was verified by PCR amplification and sequenced. LPL was amplified by real-time fluorescence quantitative PCR from the plasmid DNA. The concentration of DNA template purified was detected by analyzing absorbance in 260 nm and then the combined plasmid was diluted to series as standard for fluorescence quantitative PCR （FQ-PCR）. The method of LPL mRNA real-time PCR was well established, which detected as low as 103 with the linear range 10^3 to 10^10 copies. The standard curves showed high correlations （R2 = 0.9871）. A series of standards for real-time PCR analysis have been constructed successfially, and real-time TaqMan-fluorescence quantitative RT-PCR is reliable to quantitatively evaluate FQ-PCR mRNA in L. dorsi of porcine.

POLYMORPHISMS OF THE HUMAN LIPOPROTEIN LIPASE GENE:POSSIBLE ASSOCIATION WITH LIPID LEVELS IN PATIENTS WITH CORONARY HEART DISEASE IN BEIJING AREA

The polymorphisms(Pvu Ⅱand Hind Ⅲ) on the lipoprotein lipase(LPL) gene locus was investigated in a sample of 100 patients surviving previous myocardial infarction and 100 age matched healthy individuals selected from Han Chinese of Beijing area.In patient group a strong association was found between H+allele of Hind Ⅲ polymorphism and raised TG levels(P<0.01).In control group P-P-genotype was observed to be associated with higher TG levels compared with P+P genotype of Pvu Ⅱ polymorphism(P<0.05).Combination of H+H+ genotype with P-P-genotype showed the highest TG levels among all nine kinds of genotype combinations in patient group(P<0.01).However,comparison of distribution of alleles and genotypes of these polymorphisms between patient group and control group demonstrated no significant difference. Our data suggest that the polymorphisms at the LPL gene,as the linkage markers with an aetiologic mutation at or around LPL gene,may constitute one of the genetic determinants for the population variation in plasma TG levels,as well as for the common dyslipidemia in Chinese population.

Effect of Homogenization Temperature and Pressure on Lipoprotein Lipase Activity and Free Fatty Acids Accumulation in Milk

This study demonstrated that homogenization did not increase the activity of lipoprotein lipase (LPL) in spite of a fast accumulation of free fatty acids (FFA). Two homogenization pressures (100 and 170 bar) and two temperatures (40℃and 50℃) were examined. The activity of LPL was analyzed and the formation of FFA was measured with two different methods, the B.D.I.-method and a nonesterified fatty acids (NEFA) method. A homogenization temperature of 50℃ resulted in a decreased LPL activity compared to 40℃. No effect of homogenization pressure was found. Analyzing FFA concentration with the B.D.I.-method resulted in significant effect of homogenization temperature and no effect of pressure. The largest formation of FFA was found in milk homogenized at 40℃. Using the NEFA method, another result was obtained, indicating no effect of homogenization temperature and a larger FFA accumulation at 100 bar than at 170 bar. Both analytic methods demonstrated significant production of FFA during 60 min incubation at homogenization temperature after treatment. The level of FFA in the milk samples immediately after homogenization was very high, demonstrating that LPL cleaves the triglycerides very rapidly when the native membrane was damaged. The regression between the B.D.I.-method and the NEFA was fair in the interval between 4 and 14 mmol/100 g fat, whereas at higher concentrations, the correlation was poor.

Impact of lipoprotein lipase gene polymorphisms on ulcerative colitis

AIM： To examine the influence of lipoprotein lipase （LPL） gene polymorphism in ulcerative colitis （UC） patients.METHODS： Peripheral blood was obtained from 131 patients with UC and 106 healthy controls for DNA extraction. We determined LPL gene polymorphisms affecting the enzyme at Ser447stop, as well as Hind Ⅲ and Pvu Ⅱ polymorphisms using PCR techniques. PCR products were characterized by PCR-RFLP and direct sequencing. Polymorphisms were examined for association with clinical features in UC patients. Genotype frequencies for LPL polymorphisms were also compared between UC patients and controls.RESULTS： In patients with onset at age 20 years or younger, C/G and G/G genotypes for Ser447stop polymorphism were more prevalent than C/C genotype （OR = 3.13, 95% CI = 0.95-10.33）. Patients with H^＋/- or H^-/- genotype for Hind Ⅲ polymorphism also were more nu merous than those with H^＋/＋ genotype （OR = 2.51, 95% CI = 0.85-7.45）. In the group with H^＋/＋ genotype for Hind Ⅲ polymorphism, more patients had serum triglyceride concentrations over 150 mg/dL than patients with H^＋/- or H^- genotype （P 〈 0.01, OR = 6.46, 95% CI = 1.39-30.12）. Hypertriglycemia was also more prevalent in patients with P^＋/＋ genotypes for Pvu Ⅱ polymorphism （P 〈 0.05, OR = 3.0, 95% CI = 1.06-8.50）. Genotype frequency for LPL polymorphism did not differ significantly between UC patients and controls.

Correlation between betatrophin/angiogenin-likeprotein3/lipoprotein lipase pathway and severity of coronary artery disease in Kazakh patients with coronary heart disease

BACKGROUND The results of previous animal experiments and clinical studies have shown that there is a correlation between expression of betatrophin and blood lipid levels.However,there are still differences studies on the correlation and interaction mechanism between betatrophin,angiogenin-likeprotein3(ANGPTL3)and lipoprotein lipase(LPL).In our previous studies,we found an increase in serum ANGPTL3 Levels in Chinese patients with coronary heart disease(CHD).Therefore,we retrospectively studied Kazakh CHD patients.AIM To explore the correlation between the betatrophin/ANGPTL3/LPL pathway and severity of coronary artery disease(CAD)in patients with CHD.METHODS Nondiabetic patients diagnosed with CHD were selected as the case group;79 were of Kazakh descent and 72 were of Han descent.The control groups comprised of 61 Kazakh and 65 Han individuals.The serum levels of betatrophin and LPL were detected by enzyme-linked immunosorbent assay(ELISA),and the double antibody sandwich ELISA was used to detect serum level of ANGPTL3.The levels of triglycerides,total cholesterol,and fasting blood glucose in each group were determined by an automatic biochemical analyzer.At the same time,the clinical baseline data of patients in each group were included.RESULTS Betatrophin,ANGPTL3 and LPL levels of Kazakh patients were significantly higher than those of Han patients(P=0.031,0.038,0.021 respectively).There was a positive correlation between the Gensini score and total cholesterol(TC),triglycerides(TG),low-density lipoprotein cholesterol(LDL-C),betatrophin,and LPL in Kazakh patients(r=0.204,0.453,0.352,0.471,and 0.382 respectively),(P=0.043,0.009,0.048,0.001,and P<0.001 respectively).A positive correlation was found between the Gensini score and body mass index(BMI),TC,TG,LDL-C,LPL,betatrophin in Han patients(r=0.438,0.195,0.296,0.357,0.328,and 0.446 respectively),(P=0.044,0.026,0.003,0.20,0.004,and P<0.001).TG and betatrophin were the risk factors of coronary artery disease in Kazakh patients,while BMI and betatrophin were the risk factors in Han patients.CONCLUSION There was a correlation between the betatrophin/ANGPTL3/LPL pathway and severity of CAD in patients with CHD.

Implication of lipoprotein lipase-deficient impaired learning and memory: focus on aging and testosterone deficiency

OBJECTIVE Normal male aging is associated with declines in levels of the sex steroid hormone testosterone. A large body of evidence supports a neurotrophic role for testosterone in central nervous system. Lipoprotein lipase(LPL) is also expressed in the brain with highest levels found in the pyramidal cells of the hippocampus, we previous reported LPL-deficient mice exhibited memory disfunction. Testosterone is known to be largely converted to estradiol following aromatization within the hippocampus. Although testosterone has been implicated in lipid metabolism, it remains elusive whether testosterone can regulate brain LPL through DNA methylation mechanism. In order to clarify DNA methylation control exerted by testosterone over LPL gene in central nervous system, and its effect on lipid metabolism, we examined the adult male rat hippocampus to determine whether castration induced testosterone deficiency can affect lipid profile and LPL gene expression through its altered methylation pattern. METHODS Model of aging with declines in levels of the sex steroid hormone testosterone was performed as our previous description. RESULTS(1) Serum testosterone and brain testosterone levels were significantly decreased, which were restored to the control level after testosterone replacement,respectively(P<0.01);(2) Androgen deficiency was not found in Morris water maze and motor performance, however, androgen deficiency increases neurological and cognitive impairment in aged rats.(3)Decreased expression of olfactory marker protein(OMP) in olfactory bulb of SD rats treated with androgen deficiency.(4) The expression of Fox O3 and OMP in the olfactory bulb of androgen deficient rats was down-regulated, accompanied by dysfunction of the olfactory limbic system.(5) Decreased LPL m RNA level and inversely increased LPL promoter methylation level were observed following androgen deficiency and reserved by testosterone replacement.(6) In contrast, androgen deficiency slightly increased estrogen receptor beta(ERβ) m RNA levels and significantly decreased its promoter methylation levels within the hippocampus, and reserved as well by testosterone replacement. CONCLUSION(1) LPL in synaptic plasticity and contributes to a better understanding of the LPL function in the brain, where altered LPL levels are related to learning and memory impairment.(2) Androgen and Fox O3 play an important role in the olfactory cognitive process of the nervous system.(3) LPL expression in hippocampus is actively maintained by sex steroid hormones and that DNA methylation modification may contribute to this homeostatic regulation.

Lipoprotein lipase and obesity

Obesity is one of the fast-growing major diseases in developed and developing countries. As has been persuasively argued, long-term imbalance between intake and expenditure of fat is a central factor in the etiology of obesity. Obesity aggravates insulin resistance and promotes cardiovascular diseases and atherosclerosis. We hypothesized that elevating lipoprOtein lipase (LPL) activity in skeletal muscle would cause an improvement of obesity. To test this hypothesis, we studied the effects of the LPL activator NO-1886 inobese animals. NO-1886 elevated LPL activity in skeletal muscle, and improved obesity as well as insulin resistance in obese rats. Furthermore, NO-1886 mitigated body weight gain induced by pioglitazone without suppressive effect on the adiponectin-increasing action of pioglitazone. LPL activators hold a lot of promise of curing several diseases shown above in clinical scene.

Effect of Lipoprotein Lipase Gene Polymorphism on Plasma Lipid Levels, BMI and Subcutaneous Fat Distribution in Simple Obesity Children

Objective To study the effects of Hind Ⅲ DNA polymorphism in the lipoprotein lipase (LPL)gene on plasma lipid levels, body mass index (BMI) and subcutaneous fat distribution in simple obesity children.Methods The LPL Hind Ⅲ genotypes were detected with the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques in 92 children with simple obesity. The levels of the plasma lipid, plasma lipoproteins, BMI and skinfold thickness in three regions (biceps, subscapular and abdominal wall) were also measured. Results It was shown that the levels of TG, TC, LDL C, ApoB, BMI, biceps and subscapular skinfold thickness, and the average value of the skinfold thickness in three regions were significantly higher in the obese children with H+H+ genotype than those with H+H-genotype. Conclusions It can be concluded that LPL Hind Ⅲ polymorphism may modify the levels of plasma lipid, plasma lipoprotein and BMI in children with simple obesity, and meanwhile alters the distribution of subcutaneous fat.

脂蛋白脂肪酶(LPL)基因缺陷相关疾病与治疗进展

脂蛋白脂肪酶(LPL)是人体脂蛋白中甘油三酯代谢的关键限速酶,由心肌、骨骼肌、脂肪细胞等实质细胞合成,分泌到细胞间隙与硫酸乙酰肝素蛋白多糖(HSPG)结合,在肝素的作用下解离,被毛细血管内皮细胞上的糖基化磷脂酰肌醇锚定高密度脂蛋白结合蛋白1(GPIHBP1)捕获转位入血,将血液中脂蛋白中核心甘油三酯水解,释放游离脂肪酸,供机体氧化利用或储存。其活性受多种互作蛋白调节,包括ApoCs、ANGPTLs、LMF1等。编码LPL的基因发生突变、蛋白合成加工过程出现错误或相关互作蛋白异常都会导致LPL缺失或功能异常,出现血脂升高或伴发胰腺炎等临床症状,严重威胁患者的生命健康。目前LPL缺陷相关疾病的临床常规治疗手段是降脂药与严格饮食控制相结合,治疗效果并不理想。近年来,以LPL及其互作蛋白基因为靶点的药物相继进入临床研究和上市阶段,取得了良好的临床效果,其中以基因替代、寡核苷酸和小RNA类药物最受关注。本综述结合LPL的特性、LPL缺乏症(LPLD)的病理机制,总结现有的治疗方法及药物研发进展,以期对LPLD的药物研发和医学干预提供思路。

兴义鸭LPL基因外显子8多态性与生长和肉质性状的关联性分析

脂蛋白脂酶(LPL)是动物脂质沉积和新陈代谢的关键酶,对生长和肉质发挥着重要作用。试验采用PCR-SSCP法和DNA直接测序技术相结合对兴义鸭LPL基因外显子8进行多态性检测,并分析其多态与生长和肉质性状的关联性。结果表明,在兴义鸭LPL基因外显子8首次检测到1个T1251C同义突变,产生三种基因型TT、TC和CC,2个等位基因T和C,基因型TT和等位基因T的频率分别为0.807 7和0.875 0,多态信息含量为0.194 8,表现为低度多态,卡方(χ2)检验表明T1251C位点的基因型分布在兴义鸭中未偏离HardyWeinberg平衡。最小二乘分析显示,TT基因型个体的宰前体重显著低于CC基因型(p<0.05),胸宽显著低于TC基因型(p<0.05),推测等位基因C可能是兴义鸭生长性状的有利等位基因,可作为生长性状的一个标记性辅助选择位点。

绵羊肌肉LPL基因表达的发育性变化及其对肌内脂肪含量的影响

【目的】研究绵羊肌肉中脂蛋白脂酶(lipoprotein lipase,LPL)基因mRNA的发育性变化规律及其对肌内脂肪沉积的影响。【方法】选取2、30、60、90和120日龄的雄性哈萨克羊和新疆细毛羊各6只(120日龄只有新疆细毛羊),屠宰后取背最长肌检测肌内脂肪含量,用荧光实时定量PCR法检测肌肉LPL基因mRNA表达水平,分析在不同时期的变化及其与肌内脂肪含量的关系。【结果】随着日龄的增加,雄性哈萨克羊的IMF(IntramuscularFat)含量持续上升,各生长时期差异显著(P<0.05),而新疆细毛羊的IMF含量在各生长时期无显著差异(P>0.05),雄性哈萨克羊的IMF含量30～90d期间极显著高于新疆细毛羊(P<0.01);雄性哈萨克羊肌肉LPL基因的表达量在2日龄时最高,60日龄时降到最低,然后逐渐上升,2日龄时LPL表达量极显著高于其它日龄(P<0.01)。新疆细毛羊的表达量在2日龄时较低,30日龄时升到最高,随后下降,到90日龄时降到最低,然后又逐渐上升;30日龄表达量与2、90、120日龄差异极显著(P<0.01),与60日龄差异显著(P<0.05);60日龄表达量与2、90、120日龄差异显著(P<0.05);哈萨克羊LPL基因2～60日龄的表达量与IMF含量呈负相关,相关系数为-0.625(P<0.05);新疆细毛羊IMF含量与LPL基因表达量无显著相关性。【结论】新疆细毛羊和哈萨克羊背最长肌肌内脂肪沉积的发育性变化不同,在生长发育早期,哈萨克羊随日龄的增加其IMF上升,新疆细毛羊在各时期保持一个稳定的水平;LPL基因在哈萨克羊生长早期对其肌内脂肪的沉积可能有负调控作用,而对新疆细毛羊无明显作用。

饮食诱导肥胖易感和肥胖抵抗大鼠HSL和LPL基因表达的研究

目的探讨高脂饮食诱导肥胖易感(OP)大鼠和肥胖抵抗(OR)大鼠,激素敏感性脂肪酶(HSL)和脂蛋白脂肪酶(LPL)基因表达的差别。方法健康雄性SD大鼠80只,高脂饲料喂饲5周后,筛选出OP大鼠和OR大鼠,再将所有大鼠转为基础饲料喂饲10周后,处死动物,收集血清和白色脂肪组织,应用RT-PCR方法比较白色脂肪组织HSL和LPL基因的表达。结果高脂饲料喂饲5周后,OP大鼠白色脂肪组织HSL基因表达显著低于OR大鼠,而LPL基因表达显著高于OR大鼠;而转成基础饲料喂饲10后,OP大鼠HSL基因表达与OR大鼠无显著差异,但LPL基因表达仍显著高于OR大鼠。结论HSL基因表达水平下降和LPL基因表达水平升高,通过抑制脂肪分解促进脂肪合成,导致高脂饮食诱导肥胖易感大鼠的形成。HSL基因高表达更多是由高脂饮食诱导产生的,而LPL基因表达升高才是肥胖大鼠的特征性基因表达改变。

FASN和LPL基因对淮南麻黄鸡肌内脂肪含量的影响

【目的】研究淮南麻黄鸡肌肉品质及脂肪酸合酶(FASN)、脂蛋白脂酶(LPL)基因表达量随日龄的变化规律及其与肌内脂肪(IMF)含量的相关性。【方法】选取90,120,150和180日龄淮南麻黄鸡为研究对象,测定各日龄公、母胸肌和腿肌肌肉品质(亮度(L*)、红度(a*)和黄度(b*)值及pH、蒸煮损失、剪切力)、IMF含量和血脂指标(低密度脂蛋白(LDL)、高密度脂蛋白(HDL)、总胆固醇(TC)、甘油三酯(TG)、游离脂肪酸(FFA)和极低密度脂蛋白(VLDL)浓度),荧光定量表达检测脂肪沉积相关基因FASN和LPL的表达量,Pearson法分析血脂指标和FASN、LPL基因表达量与IMF含量的相关性。【结果】公鸡和母鸡胸肌L*、pH、腿肌蒸煮损失在不同日龄间无显著差异,其余肉质指标在不同日龄间有显著差异。公鸡和母鸡胸肌IMF含量在180日龄显著高于90日龄(P<0.05),公鸡腿肌IMF含量在180日龄显著高于90和120日龄(P<0.05),母鸡腿肌IMF含量在150和180日龄显著高于90日龄(P<0.05)。除公鸡HDL浓度在不同日龄间无显著差异外,公母鸡各血脂指标在不同日龄间均有显著差异。公鸡胸肌IMF含量与血清HDL浓度显著正相关,与VLDL浓度显著负相关;腿肌IMF含量与HDL浓度极显著正相关,与TG和LDL浓度极显著负相关。母鸡腿肌IMF含量与血清TG浓度呈显著负相关,与其他血脂生化指标相关性不显著。公鸡肝脏、胸肌FASN和LPL基因相对表达量在不同日龄间有显著差异,腿肌FASN和LPL基因相对表达量在不同日龄间无显著差异;母鸡肝脏FASN和LPL基因相对表达量在不同日龄间无显著差异,胸肌和腿肌FASN和LPL基因表达量在不同日龄间有显著差异。公鸡和母鸡胸肌IMF含量均与胸肌FASN基因表达量显著正相关;公鸡胸肌IMF含量与肝脏FASN和LPL表达量均显著正相关;母鸡腿肌IMF含量与腿肌LPL表达量显著正相关。【结论】日龄对淮南麻黄鸡肉品质、血脂水平和脂肪沉积相关基因表达量有显著影响,可通过血脂TG浓度间接选择IMF含量。FASN参与调控公、母鸡胸肌IMF含量,LPL参与调控母鸡腿肌IMF含量。

猪LPL基因内含子3的克隆、测序及多态性研究

Lipoprotein lipase (LPL) gene is a multifunctional protein, playing a major role in the hydrolysis of triglycerides in chylomicrons and very low density lipoproteins (VLDL). According to cDNA of Landrace breed (GenBank accession: X62984), a pair of primers was designed for amplification of intron 3. Sequencing analysis indicated that a G1200A exits in Large White breed by cloning and sequencing. The mutation can be detected by PCR-EcoT22I-RFLP. Polymorphism analysis in a resource family showed that significant difference exits in carcass traits. Pigs with AA genotype had more 6th-7th rib fat thickness (13%,P< 0.05), thorax-waist-fat thickness (11.7%,P=0.065 3), buttock fat thickness (13.1%,P=0.073 0) and average fat thickness (10.4%,P=0.050 3) than pigs with BB genotype. LPL gene showed mainly in the pattern of additive effect and all the dominant effect were not significant. The value of additive effect of 6th-7th rib fat thickness, buttock fat thickness and average fat thickness was -0.17±0.07(P< 0.05),-0.12±0.06( P< 0.05),-0.12±0.06(P< 0.05),respectively. The allelic frequency is significantly different between indigenous Chinese breeds and European breeds.

配合饲料、冰鲜杂鱼对大口黑鲈生长和LPL基因mRNA表达的影响

以体质量为40 g的大口黑鲈Micropterus salmoides为试验对象,分别投喂冰鲜杂鱼(对照)、A配合饲料(脂肪含量8.2%)和B配合饲料(脂肪含量14.5%),饲养试验共进行3个月,试验结束时测量各组鱼的生长性状,并检测其肝脏中两种脂蛋白脂肪酶基因LPLtype1和LPLtype2 mRNA的表达量。结果表明:喂食冰鲜杂鱼的大口黑鲈平均体质量增加率显著高于喂食两种配合饲料的大口黑鲈(P<0.05);喂食B配合饲料的大口黑鲈肝脏中LPLtype1和LPLtype2 mRNA的相对表达量与对照组、喂食A配合饲料的组存在显著性差异(P<0.05),饲料脂肪水平越高,LPLtype1和LPLtype2 mRNA的表达量也越高。研究表明,脂蛋白脂肪酶LPLtype1和LPLtype2基因编码的蛋白都具有分解贮存在肝脏细胞中多余脂肪的功能,且LPLtype2与LPLtype1基因可能存在更加精细的分工,LPLtype2比LPLtype1更易受饲料脂肪含量的调节,推测LPLtype2在参与分解肝细胞的脂肪中可能起到更大的作用。

猪LPL基因多态性及其部分DNA片段的测序

脂蛋白脂肪酶（Ｌｉｐｏｐｒｏｔｅｉｎｌｉｐａｓｅ，ＬＰＬ） 是影响动物脂肪沉积的关键酶之一。本文利用ＰＣＲＳＳＣＰ法，对猪的ＬＰＬ基因进行了多态性分析，发现其５’端区域存在ＤＮＡ 多态性。脂肪型猪种梅山猪和通城猪表现出３种等位基因型（ＡＡ、ＡＢ、ＢＢ） ，其等位基因Ａ和Ｂ的频率分别为０．７６、０ ．２４ 和０．７７、０ ．２３ ；而瘦肉型猪种长白猪和大白猪表现出另外不同的３ 种等位基因型（ ＣＣ、ＣＤ、ＤＤ），其等位基因Ｃ和Ｄ 的频率分别为０．７６ 、０ ．２４ 和０．７２、０．２８ 。测序结果表明具四种等位基因纯合子的ＤＮＡ 片段之间存在５ 处碱基突变，分别表示为等位基因ＡＡＴＣＣＴ，ＢＡＴＴＣＴ，ＣＧＣＣＣＴ和ＤＧＣＣＧＣ，其中等位基因Ｄ 第＋ ３２ 处发生Ｔ／Ｃ 的突变导致亮氨酸到脯氨酸的改变（Ｌｅｕ→Ｐｒｏ） 。

草鱼LPL基因的表达及饥饿和再投喂对其影响

获得了草鱼脂蛋白脂酶(lipoprotein lipase,LPL)部分cDNA序列(GenBank注册号为FJ612596),并进行了序列同源性分析;采用半定量反转录聚合酶链式反应(SQ-RT-PCR)方法,检测了LPL基因在草鱼不同组织和不同发育阶段脂肪细胞中的表达状况;研究了饥饿和再投喂对草鱼肝胰脏LPL基因表达的影响。结果显示,所获得的草鱼LPL部分cDNA序列长度为360bp,与其它物种的同源性为70%～89%;LPL基因在肝胰脏、肌肉、心脏、鳃、腹腔脂肪组织中均表达,其中在腹腔脂肪组织中表达丰度最高,在鳃中表达丰度最低;在成熟脂肪细胞中的表达丰度显著高于基质脉管组分(stromal vascular fraction,SVF)细胞;草鱼LPL基因表达水平在饥饿48h后显著升高,再次投喂后12h,回复到0h的表达水平。研究表明,草鱼LPL在不同组织及脂肪细胞分化的起始和终末阶段均有表达,显示其在草鱼不同组织和脂肪细胞分化中具有重要作用,同时,其表达水平受营养状况的调控。论文首次克隆得到草鱼LPL基因部分cDNA序列,并对其进行了表达分析,研究结果可为LPL在鱼类脂质代谢中的作用研究提供参考和依据。

徐淮山羊LPL基因克隆及其表达产物亚细胞定位的研究

旨在克隆徐淮山羊脂蛋白酯酶(LPL)基因的cDNA,并通过EGFP融合蛋白实现该基因亚细胞水平的表达定位。本研究通过RT-PCR方法克隆徐淮山羊LPL基因cDNA,并初步进行生物信息学分析,构建含有增强型绿色荧光蛋白(EGFP)报告基因的融合表达载体pEGFP-C1-LPL。聚乙烯亚胺(PEI)介导pEGFP-C1-LPL转染NIH-3T3细胞,48h后在荧光倒置显微镜下观察,并用RT-PCR方法检测LPLmRNA在细胞内的表达。结果,成功克隆出山羊LPL基因1 530bp长的cDNA,完整阅读框大小为1 437bp,共编码478个氨基酸,GenBank登录号为GU082383。信号肽区域预测结果表明LPL蛋白含有一小段信号肽序列,其可能性为100%。信号肽酶切割位置在第23和24个氨基酸之间,其可能性达到65.9%。成功构建融合表达载体pEGFP-C1-LPL,并且RT-PCR显示转染后LPL在mRNA水平上表达明显。EGFP-LPL融合蛋白定位在NIT-3T3细胞外和细胞膜部分。结果表明,首次成功克隆出徐淮山羊LPL基因cDNA;重组质粒pEGFP-C1-LPL构建成功,并在NIH-3T3细胞中明显表达。