大鼠钠尿肽B受体cDNA探针克隆载体的构建

目的 :构建大鼠钠尿肽 B受体 c DNA探针克隆载体 ,为研究该受体基因表达奠定基础。方法 :设计钠尿肽 B受体特异的 PCR引物 ,提取大鼠肺组织总 RNA,进行反转录 PCR,扩增目的片段 ,回收目的片段 ,将其克隆入p GEM- T载体 ,酶切鉴定并用双脱氧法测序。结果 :PCR反应产物经琼脂糖凝胶电泳鉴定与所设计引物大小一致 ,克隆入 p GEM- T载体后酶切可切出所需片段 ,测序证实为所设计序列。结论 :构建的 p GEM- T重组质粒为进一步研究该受体基因表达、分布等创造了条件     

糖尿病大鼠瘦素受体OB-Ra表达的研究

目的 探讨瘦素受体0B-RamRNA在糖尿病(DM)大鼠多个组织中的表达及胰岛素(Ins)分泌减少对其表达的影响。方法 从大鼠脾组织中提取总RNA，采用逆转录PCR技术扩增了约1．44kb的0B-Ra基因片段；以四氧嘧啶诱导DM模型大鼠；检测和比较正常和DM大鼠多个组织中0B-Ra mRNA的表达。结果 0B-RamRNA在大鼠体内广泛表达，脾、睾丸、腹部白色脂肪(简称脂肪)及大脑等组织中表达水平较高，而心、肺中几乎未见表达；DM大鼠的胰腺、脂肪和睾丸等组织0B-RamRNA的表达量与正常大鼠相比，分别减少了62％、15％和11％。结论 DM大鼠体内胰腺、脂肪和睾丸等组织中0B-RamRNA表达量的降低与Ins分泌减少及由此引起的代谢改变有关。     

姜黄素对肝纤维化大鼠瘦素及瘦素受体表达的影响

目的：探讨姜黄素对实验性肝纤维化大鼠肝脏组织中瘦素（leptin）以及瘦素受体（leptin receptor）表达的影响。方法：将22只雄性SD大鼠随机分为3组：正常对照组、模型组、姜黄素治疗组。采用四氯化碳（CCl4）皮下注射制作大鼠肝纤维化模型。制备肝组织切片，行常规HE染色、Masson染色，观察各组大鼠肝脏病理学变化，采用肝纤维化病理分期进行病理学疗效评估；应用免疫组织化学法检测肝组织中瘦素以及瘦素受体的表达。结果：姜黄素对肝纤维化大鼠的肝脏病理分期具有明显的改善作用。并且免疫组织化学检测显示姜黄素能显著减少瘦素与瘦素受体的阳性表达。结论：姜黄素能明显改善实验性大鼠肝纤维化，同时下调肝纤维化大鼠肝脏中瘦素以及瘦素受体的水平，这可能是姜黄素抗肝纤维化作用的机制之一。     

人胸苷激酶基因cDNA克隆

目的　克隆人胸苷激酶基因cDNA。方法采用逆转录PCR方法扩增胸苷激酶cDNA ,用十二烷基氟波酯 (TPA)刺激人外周血细胞的mRNA为模板 ;扩增的PCR产物经TA克隆重组和内切酶鉴定 ,以及DNA序列分析确定克隆基因的序列。结果经逆转录PCR扩增出一个 70 0bp左右的DNA片段 ,TA克隆出现 1 6个阳性克隆 ,选择其中的一个克隆 (TKTA8)进行DNA序列分析 ,序列分析结果表明TKTA8克隆重组的序列是人胸苷激酶的cDNA序列。结论从外周血细胞中得到了hTK完整的开放读码框架。     

瘦素受体基因多态性与心血管及相关疾病的研究

疫素参与肥胖和相关疾病如冠心病、高血压、代谢综合征,随着研究的深入,人们发现了疫素受体基因多态性及其变异,由于瘦素通过瘦素受体发挥作用,因而,瘦素受体基因多态性成为了近年的研究趋势与热点.研究较多的为Lys109Arg,Gln223Arg,Lys656Asn这3种.瘦素受体基因多态性在人类可引起病态肥胖,其与高血压、糖...     

瘦素及其受体在大鼠酒精性肝病形成过程中的动态变化及意义

目的:观察酒精性肝病形成中大鼠血清瘦素水平、肝组织瘦素及其受体表达的动态变化,探讨瘦素及其受体在酒精性肝病形成中的发病机制.方法:SD雄性大鼠随机分为正常组和模型组,以白酒-玉米油-吡唑混合液灌胃建立酒精性肝病动物模型,分别在4、8、14周末光镜观察大鼠肝脏组织学变化情况,免疫组化法检测肝组织瘦素及其受体的表达,ELISA法检测大鼠血清瘦素水平.结果:模型组大鼠肝组织在4周时出现轻度脂肪变及腺泡内个别点状坏死灶,8周时脂肪变程度加重、腺泡内点状坏死灶增多、门管区炎细胞浸润,14周脂肪变和炎症程度进一步加重;模型组大鼠4周时,肝组织瘦素及其受体表达、血清瘦素水平与同期正常组比差异无显著性意义(P＞0.05),随着肝脏损伤程度的加重,三者均明显增加(P＜0.05,P＜0.01);肝组织中瘦素及其受体表达水平与肝脏脂肪变及炎症程度均呈明显正相关.结论:在白酒-玉米油-吡唑混合液灌服复制的大鼠酒精性肝病形成过程中,瘦素及其受体表达增加,且随肝脏损伤程度的加重而逐渐增加,提示其参与了酒精性肝病的发生发展.     

瘦素及瘦素功能性受体表达与大鼠肝硬化关系的研究

目的观察实验性大鼠肝硬化形成过程中,肝组织瘦素及瘦素功能性受体表达的动态变化以及与肝内胶原含量的关系。方法经皮下注射四氯化碳(0.3ml/100g体重,每周2次)复制肝硬化动物模型,于第0、3、6、9、12周各处死一批大鼠。用RT-PCR检测各期动物肝组织中瘦素及瘦素功能性受体(Ob-Rb)mRNA表达水平。用免疫组织化学方法检测各期动物肝组织中瘦素、瘦素受体的表碉和定位,用放射免疫法检测各期动物血浆中透明质酸(HA)、瘦素水平。运用MPIAS-500多媒体真彩色图像分析系统作肝内胶原定量分析。结果正常肝组织中有少量瘦素、瘦素功能性受体的mRNA表达,随着肝硬化的形成,各期表达量逐渐递增(与0周比较,P<0.01)。正常肝组织可见瘦素及瘦素受体在血管周围、汇管区及肝索间质细胞的细胞质和细胞膜有少量表达,随着肝硬化的形成,瘦素、瘦素受体表达增多、增强(P<0.01)。随着造模时间的增加,肝内胶原面积、血浆中HA和瘦素均逐渐增加,各期比较差异有统计学意义(P<0.05)。肝硬化形成过程中,肝脏中瘦素、瘦素功能性受体的mRNA表达水平与血浆中HA水平及肝内胶原含量呈正相关(瘦素与HA:r=0.726,P=0.00;瘦素与胶原:r=0.732,P=0.00;Ob-Rb与HA:r=0.705,P=0.00;Ob-Rb与胶原:r=0.764,P=0.00)。结论在四氯化碳诱导的大鼠肝硬化形成过程中,瘦素及功能性受体的基因和蛋白表达增加,并随肝硬化程度的加重而逐步升高,从而促进肝硬化的发生。     

瘦素和瘦素受体mRNA表达与哮喘和肥胖的关系

目的探讨瘦素(LEP)和瘦素受体(LEPR)mRNA表达与哮喘和肥胖的关系。方法采用实时荧光定量PCR(RT-qPCR)方法测定各组PBMC LEP和LEPR mRNA表达水平,分析LEP mRNA与血浆瘦素浓度、EOS%、IgE和FEV1占预计值(%)的关系。结果哮喘、肥胖、哮喘合并肥胖组的PBMC LEP mRNA表达量与健康对照组相比明显升高(P均0.01),中、重度哮喘与轻度哮喘相比明显升高(P均0.05);肥胖组LEPR mRNA表达量与其余各组相比明显升高(P均0.01);哮喘组PBMC LEP mRNA表达量与血浆LEP浓度呈正相关(r=0.211,P=0.012),与FEV1占预计值(%)成负相关(r=-0.314,P=0.043),与EOS%、IgE无相关关系(r=0.002、0.001,P=0.58、0.96)。结论 LEP mRNA表达升高可能参与哮喘和肥胖的发生,并且导致较为严重的哮喘表型,其机制可能与血浆瘦素浓度增高有关,其相关的哮喘是非嗜酸细胞性、非过敏性的;LEPR mRNA高表达参与肥胖发生,但与哮喘无关。     

高脂喂养大鼠血清瘦素水平与肝脏瘦素受体mRNA的表达

与正常大鼠相比,肥胖大鼠血清瘦素升高(P<0.05),而肝内瘦素受体mRNA表达降低(P<0.05),两者呈负相关(r=-0.823, -0.827,均P<0.05),提示肥胖大鼠高血清瘦素对瘦素受体mRNA表达有抑制作用。     

重症急性胰腺炎大鼠瘦素、功能性瘦素受体表达的动态变化

重症急性胰腺炎（SAP）病情危重，发展快、病死率高。SAP发病机制不甚清楚，近年研究认为，细胞因子网络和免疫功能紊乱在SAP中发挥重要作用，过度活化的炎性细胞及炎症因子对SAP的预后有重要影响。瘦素作为一种重要的调节炎症和免疫反应的细胞因子，在急性胰腺炎（AP）时水平明显升高，炎症诱导瘦素产生，瘦素可能发挥炎性细胞因子的作用。本研究探讨瘦素及其受体（OB-R）在SAP中的动态变化。     

长胞内域瘦素受体全长mRNA表达增加对骨胳肌细胞葡萄糖氧化的影响

目的 观察增加骨胳肌细胞全长长胞内域瘦素受体(OBRb)mRNA表达以后,瘦素对原代培养的骨胳肌细胞葡萄糖氧化代谢的影响。方法 分离SD乳鼠的骨胳肌细胞,分4组进行细胞培养,其中转染重组质粒、转染空载质粒和非转染3组(分别加瘦素10ng/ml和胰岛素10mU/ml),另外1组为非转染未加瘦素和胰岛素的对照组。将载有全长OBRb cDNA的真核表达质粒或空载质粒在脂质体的介导下导入骨胳肌细胞。48h后加入瘦素和胰岛素,孵育1h,再加入D-[U-~(14)C]-葡萄糖,继续培养2h后终止反应,收集~(14)CO_2,液体闪烁计数。同时以RT-PCR检测转染细胞的OBRb mRNA表达水平。结果 重组质粒转染、空载质粒转染和非转染3组,特异性条带和β-actin条带积分光密度比值分别为1.22±0.10、0.41±0.08和0.49±0.09,前者较其它两组明显升高(均P<0.001)。葡萄糖氧化前者明显高于其他3组(均P<0.005)。结论 增加SD乳鼠骨胳肌细胞OBRb mRNA的表达,明显改善细胞葡萄糖氧化,提示OBRbmRNA的高度表达能改善瘦素的作用。     

Leptin, leptin gene and leptin receptor gene polymorphism in heart failure with preserved ejection fraction

Heart failure with a normal ejection fraction (HFNEF) is common in obesity and coronary artery disease (CAD). Both ischemia and reperfusion induce leptin (LEP) and leptin receptor (LEPR) gene expression. We aimed to investigate the possible associations of serum leptin, leptin gene and leptin receptor gene polymorphism with HFNEF in patients with CAD. 100 Egyptian CAD patients with HFNEF and 100 healthy subjects (the control group) were genotyped for LEP and LEPR polymorphism. Leptin levels were measured. Serum leptin levels were significantly increased in patients compared to the control group. There was a significant increase in the leptin gene (AA genotype) and the leptin receptor gene (RR genotype) in HFNEF patients compared to the control group. Leptin levels, leptin gene (AA genotype) and LEPR (RR genotype) were more associated with NYHA III than with NYHA I and II. We thus concluded that HFNEF is associated with increased serum leptin levels, and the LEP AA genotype or LEPR RR genotype carries at least a threefold increased risk of developing HFNEF.     

Leptin gene and leptin receptor gene polymorphisms are associated with sweet preference and obesity

Leptin is an adipocyte-secreted hormone that regulates food intake and body weight, and that was recently reported to suppress sweet sensitivity in an animal model. We investigated the associations among sweet preference, obesity, and polymorphisms of the leptin gene (LEP) or leptin receptor gene (LEPR). A total of 3,653 residents randomly selected from among the citizens of Suita City, Osaka, Japan were enlisted as subjects, in whom we investigated sweet preference, clinical characteristics, including obesity and serum leptin level, and the polymorphisms of LEP and LEPR (G-2548A and A19G for LEP; R109K, R223Q, and rs3790439 for LEPR). We determined the associations among the parameters using logistic regression analysis, in order to consider potential confounding factors for sweet preference and/or obesity. The LEP A19G and LEPR R109K polymorphisms were associated with sweet preference, whereas the serum leptin level was not. Further, the LEPR 109KK genotype was found to be associated with obesity along with sweet preference. In conclusion, our results are the first to show associations of LEP and LEPR polymorphisms with sweet preference, and may provide useful information for diagnosis and treatment of lifestyle-related diseases.     

瘦素受体基因Gln223Arg多态性与原发性高血压相关性研究

目的:研究高血压候选基因瘦素受体基因(LEPR)在中国北方汉族人群的分布,探讨LEPR基因Gln223Arg多态性与原发性高血压(EH)的关系。方法:采集健康体检人群临床资料,分析体质量指数、血脂、血糖等临床指标,同时调查受检者吸烟和饮酒等生活习惯;Taq Man-MGB法分析LEPR基因Gln223Arg多态性在中国北方汉族人群(样本总数1 273例,其中原发性高血压病患者774例,血压正常的健康体检者499例)的分布及其与临床指标的相关性。结果:LEPR基因Gln223Arg基因型在两组总体的差异具有统计学意义(P=0.036)。按照血尿酸水平进行亚组分析,高尿酸亚组基因型(P=0.039)和等位基因频率(P=0.037)的差异具有统计学意义;调整相关因素的影响后,多因素Logistic回归模型分析显示:LEPR基因Gln223Arg多态性与EH相关[(GG+AG)vs.AA模型,OR=3.23,95%CI:1.01~10.34,P=0.008;GG vs.AA模型,OR=3.43,95%CI:1.35~8.75,P=0.010];高尿酸亚组也显示该多态性与EH发病密切相关[G vs.A模型,OR=1.89,95%CI:1.09~3.28,P=0.023;(GG+AG)vs.AA模型,OR=7.76,95%CI:1.18~49.80,P=0.033;GG vs.AA模型,OR=8.50,95%CI:1.29~56.12,P=0.026;GG vs.AG vs.AA模型,OR=1.94,95%CI:1.10~3.43,P=0.023]。结论:在中国北方汉族人群中,LEPR基因Gln223Arg多态性与原发性高血压相关。     

Role of serum leptin levels and leptin receptor gene polymorphisms in systemic lupus erythematosus

Clinical Rheumatology - Serum leptin and leptin receptor gene polymorphisms may play a role in the etiopathogenesis of SLE. This study was undertaken to explore the relationship between serum...     

Hypertension in obesity and the leptin receptor gene locus

Recent animal studies indicate that leptin is involved in the regulation of blood pressure through the leptin receptor. Therefore, 51-yr-old men (N = 284) were selected; and anthropometric, endocrine, metabolic, and hemodynamic variables were examined in relation to polymorphisms of the leptin receptor gene (LEPR), by restriction fragment length polymorphism technique. Three polymorphisms were examined: Lys109Arg in exon 4, Gln223Arg in exon 6, and Lys656Asn in exon 14. In comparison with Lys109 homozygotes, Arg109 homozygotes (9%) showed lower body mass index (BMI) and abdominal sagittal diameter, as well as lower systolic (10.0 mm Hg) and diastolic (7.8 mm Hg) blood pressure. Additionally, Arg223 homozygotes (26.8%) showed lower blood pressure (7.6/5.7 mm Hg) than Gln223 homozygotes. These lower blood pressure levels were independent of other variables. No differences were found with the Lys656Asn polymorphism. Measurements of body fat mass correlated with leptin concentration in Lys109 homozygotes and in Lys109 heterozygotes but not in Arg109 homozygotes. Blood pressure correlated with leptin only in men carrying the wild-type allele Lys109. With both elevated BMI and leptin, Lys109 homozygotes had higher blood pressure than the Arg109 homozygous men (12.4/6.9 mm Hg). Men with blood pressure > or = 140/90 mm Hg had, in comparison with normotensive men, increased BMI and leptin levels, and Lys109 homozygotes were significantly more prevalent. These results suggest that leptin is associated with blood pressure regulation in men through the leptin receptor. When BMI and leptin are elevated, increased blood pressure is found only with the most prevalent LEPR genotype at codons 109 and 223, whereas variants of this receptor seem to protect from hypertension. This might explain why not all obese men are hypertensive.     

Interaction between leptin and leptin receptor in gastric carcinoma: gene ontology analysis.

Gastric carcinoma is a rare but important malignancy. The link between leptin, a cytokine that is elevated in obese individuals, and cancer development has been proposed. It is noted that leptin and its receptor may play a positive role in the progression in gastric cancer. However, the exact mechanism resulting form the interaction between leptin and leptin receptor has never been clarified. Here, the author used a new gene ontology technology to predict the molecular function and biological process due to the interaction between leptin and leptin receptor. Comparing to leptin and leptin receptor, the leptin-leptin receptor poses the same function and biological process as leptin receptor. This can confirm that leptin receptor has a significant suppressive effect on the expression of leptin. Loss of hormone activity and disturbance of normal cell signaling pathway of leptin can be seen. Blocking of receptor might be rational therapeutic strategy.     

Expression of leptin receptor gene in developing and adult zebrafish

Interactions of leptin and leptin receptors play crucial roles during animal development and regulation of appetite and energy balance. In this study we analyzed expression pattern of a zebrafish leptin receptor gene in both developing and adult zebrafish using in situ hybridization and Q-PCR methods. Zebrafish leptin receptor message (lepr) was detected in all embryonic and larval stages examined, and in adult zebrafish. In embryonic zebrafish, lepr was mainly expressed in the notochord. As development proceeded, lepr expression in the notochord decreased, while its expression in several other tissues, including the trunk muscles and gut, became evident. In both larval and adult brains, large lepr expressing cells were detected in similar regions of the hindbrain. In adult zebrafish, lepr expression was also observed in several other brain regions including the hypothalamic lateral tuberal nucleus, the fish homolog of the arcuate nucleus. Q-PCR experiments confirmed lepr expression in the adult fish brain, and also showed lepr expression in several adult tissues including liver, muscle and gonads. Our results showed that lepr expression was both spatially and temporally regulated.