SCD-1基因过表达对鸭子宫上皮细胞钙离子和脂质含量的效应

硬脂酰辅酶A去饱和酶-1 (Stearoyl-CoA desaturase-1,SCD-1)是催化单不饱和脂肪酸合成的关键性蛋白酶,Ca²⁺是生物体内重要阳离子,在生物体内发挥着重要作用。为探讨SCD-1基因与脂质指标和钙离子含量之间的关联性,通过构建pcDNA3.1(+)+SCD-1+Flag真核过表达载体和培养鸭子宫上皮细胞并共转染,通过载体上Flag标签检测SCD-1基因的过表达量,用Fluo-3/AM钙离子荧光标记法检测Ca²⁺浓度,用脂质指标试剂盒检测细胞内的脂质含量。结果表明,鸭SCD-1基因过表达量与甘油三酯(TG)和高密度脂蛋白胆固醇(HDL-C)含量呈负相关,与Ca²⁺浓度、总胆固醇(TC)含量、极低密度脂蛋白胆固醇(VLDL-C)含量和低密度脂蛋白胆固醇(LDL-C)含量呈正相关;Ca²⁺与TG、LDL-C和HDL-C的含量呈正相关,与TC和VLDL-C含量呈负相关,研究结果揭示了SCD-1基因过表达能够调控鸭子宫上皮细胞中Ca²⁺浓度和脂质合成与转运。     

大白猪不同脂肪部位脂质代谢相关基因的表达差异分析

脂肪沉积决定于脂肪酸合成、转运和分解代谢的动态平衡。为比较猪(Sus scrofa domesticus)不同脂肪部位脂质代谢差异和不同性别间的差别,筛选与脂肪沉积能力显著关联的相关酶基因,本研究检测大白猪(Large White pigs)脂质代谢相关生化指标、肌内脂肪(intramuscular fat)含量,皮下脂肪、腹部脂肪和肾周脂肪组织间脂肪酸合成酶基因ACC,脂肪酸转运酶基因CPT1A和CD36,以及脂肪酸分解酶基因HSL和LPL的转录表达水平,分析目标基因与血清生化指标和肌内脂肪含量的相关性,比较在不同脂肪组织间的基因表达差异。结果表明,血清甘油三酯(triglycerides)、总胆固醇(total cholesterol)和肌内脂肪含量在不同性别大白猪中均无显著性差异(P>0.05)。相关性分析表明,大白猪公猪腹部脂肪中ACC基因表达量与甘油三酯含量呈显著正相关(P<0.05),皮下脂肪中HSL基因表达量与甘油三酯含量呈显著负相关(P<0.05);大白猪母猪腹部脂肪中ACC基因表达量与甘油三酯含量呈极显著正相关(P<0.01)。实时定量PCR结...     

介质中不同Ca~(2+)浓度对五种榕树幼苗钙含量的影响

选择高榕(Ficusaltissima)、木瓜榕(F.auriculata)、聚果榕(F.racemosa)、鸡嗉子榕(F.semicorda ta)以及对叶榕(F.hispida)5种榕树,对其幼苗进行不同Ca2+(CaCl2)浓度下的水培实验,结果表明,5种幼苗钙含量随介质Ca2+浓度的增加而增加,增加趋势大致相同。高Ca2+浓度下(10mmol/L)幼苗地上部的钙含量为低Ca2+浓度下(0.05mmol/L)的1.51～1.63倍。5种榕树幼苗钙含量随培养液Ca2+浓度变化的Yadj值的多重比较结果显示,对叶榕钙含量显著高于其他四种(P<0 01),高榕则显著低于其他四种(P<0 01),木瓜榕、聚果榕、鸡嗉子榕之间的差异不显著。不同榕树幼苗地上部分钙含量受介质钙浓度影响,但可能主要决定于基因型的差异,表明榕属植物富钙基因的存在,这为高钙榕树植物资源开发提供初步的理论依据。     

F1代苏香杂交猪GAS7、JHDM1A基因组织表达水平研究

[目的]研究GAS7、JHDM1A基因在组织中的表达分布情况。[方法]试验采用荧光定量PCR技术,测定了苏香杂交猪的心、肝、脾、胃、肾、脑、小肠和背最长肌在各组织的分布表达情况。[结果]GAS7基因在脑表达含量最高(5.23±2.14),在心脏中表达量最低(0.07±0.03),且与脑中的表达量呈极显著差异(P<0.01),JHDM1A基因在脑部表达含量最高(32.79±12.76),在心脏(0.18±0.08)和肝脏(0.18±0.09)中表达量最低,且与脑中的表达量呈极显著差异(P<0.01)。[结论]GAS7基因主要在脑、肝脏组织表达并发挥其生理作用,而JHDM1A基因主要在脑组织表达并发挥其生理作用,此研究结果为进一步分析GAS7基因、JHDM1A基因在肉质或生长性能方面具体的分子作用机理奠定基础。     

AHCYL1基因mRNA表达和遗传变异对三穗鸭蛋壳品质的影响

本研究旨在探讨S-腺苷基同型半胱氨酸水样蛋白1基因(AHCYL1)mRNA表达和遗传变异与鸭蛋壳品质的相关性。采用实时荧光定量PCR检测AHCYL1基因mRNA组织表达谱,结果表明：在三穗鸭[麻鸭属(Tadorna)蛋用品种之一]12个组织中均检测到AHCYL1基因mRNA的表达,其中子宫部和肝脏表现出高度特异性表达,表达量依次为子宫>肝脏>大脑>十二指肠>腿肌>肺>腺胃>胸肌>心脏>肾脏>脾脏>肌胃,子宫部的表达量与蛋壳强度和蛋壳重呈显著正相关(P<0.05);聚合酶链式反应(PCR)结合直接测序法鉴定AHCYL1基因SNP位点,发现3个SNPs,分别为位于内含子1的g.169244 A>T、 g.169265 T>C突变和外显子6的g.172066 G>A同义突变,g.169244 A>T突变位点TT基因型的蛋壳强度显著高于AT和AA基因型的(P<0.05), AA基因型个体的蛋壳重显著低于AT和TT基因型的(P<0.05), TT和AT基因型的mRNA表达水平显著高于AA基...     

鸭SCD1基因对PC3细胞脂质性状的效应分析

为了探究鸭SCD1基因对PC3细胞脂质性状的影响,本研究构建了鸭SCD1基因的真核表达载体,转染前列腺PC3癌细胞,测定SCD1基因的表达量及脂质指标,分析SCD1基因对各脂质指标的影响,结果表明,鸭SCD1基因表达与甘油三酯、总胆固醇及低密度脂蛋白(LDL)呈极显著正相关,与高密度脂蛋白(HDL)呈极显著负相关,这表明,鸭SCD1基因的表达能够促进PC3细胞的脂质代谢速率,能够为PC3细胞的快速癌变增值提供必要的脂质需求及能量供应,据此推测,鸭SCD1基因的表达有可能对鸭的癌症病变的发生与发展有一定影响,在今后畜禽疾病防控研究中,可以通过分子标记技术,加强SCD1基因的监测,为畜禽的高效生产提供保障。     

连续两个生长季大气CO2浓度升高对银杏希尔反应活力和叶绿体ATP酶活性的影响

近年来,随着温室气体浓度不断上升,有关CO2浓度升高对植物影响的研究已取得一定进展,但CO2浓度升高对植物光合作用的影响需要从生理生化水平上进一步深入的研究.以沈阳城市森林树种银杏(Ginkgo biloba L.)为研究对象,利用开顶式气室研究连续两个生长季大气CO2浓度升高对银杏光合特性的影响.结果表明,在大气CO2浓度为700μmol·mol-1条件下,与对照相比,第1个生长季CO2处理的银杏叶片净光合速率极显著增加(P<0.01),希尔反应活力极显著增大(P<0.01)、Ca2+/Mg2+-ATP酶活性显著(P<0.05)或极显著增强(P<0.01)、光合产物淀粉的含量极显著增多(P<0.01);第2生长季CO2处理的银杏叶片净光合速率显著增加(P<0.05),希尔反应活力在通气60d时极显著(P<0.01)增大,Ca2+/Mg2+-ATP酶活性在处理30d时显著降低(P<0.05),淀粉含量增多.与第1个生长季相比,第2个生长季CO2处理的银杏叶片净光合速率降低,希尔反应活力减小,Ca2+/Mg2+-ATP酶活性减弱,叶绿素含量增多,淀粉含量减少.试验中出现了光合适应现象.     

内质网蛋白44(ERp44)通过1, 4, 5-三磷酸肌醇受体介导基因转录

细胞核内钙离子浓度的增加可以引起包括钙离子激活的基因转录在内的很多生理功能.运用Western blot、免疫荧光、实时定量聚合酶链反应、钙成像以及外源三磷酸腺苷刺激细胞释放钙离子等试验方法,发现1,4,5-三磷酸肌醇受体和内质网蛋白44(ERp44)在内质网和核膜上都有很好的共定位.外源三磷酸腺苷可以通过1,4,5-三磷酸肌醇受体刺激核内钙瞬变并磷酸化环磷酸腺苷反应原件结合蛋白(CREB)、刺激原癌基因c-Myc的表达.但是,这些功能都能被1,4,5-三磷酸肌醇受体抑制剂2-氨乙氧基二苯酯硼酸(2-APB)和过表达内质网蛋白44(ERp44)所抑制.这些结果均提示在子宫颈癌HeLa细胞中内质网蛋白44(ERp44)通过1,4,5-三磷酸肌醇受体而介导基因转录.     

SREBP-1基因表达对血清生化指标的效应分析

为丰富SREBP-1基因的功能研究并更好地选育优质鸭种奠定理论基础。本研究以鸭为试验材料,运用q PCR技术研究SREBP-1基因组织表达规律,并分析其与所测血清生化指标间的关联性。结果表明,SREBP-1基因在鸭9个组织中存在差异性表达。其中,在腹脂中表达最高,皮脂和肾次之,在肝、脾、小肠、肌胃、心及肺中均有少量表达;SREBP-1基因与甘油三酯、低密度胆固醇、总胆固醇,总蛋白及低密度脂蛋白显著或极显著正相关,与高密度胆固醇显著负相关。推测SREBP-1基因对鸭脂质代谢具有调控作用。     

金属胁迫下灵芝寡肽转运蛋白基因家族的转录表达

【目的】寡肽转运蛋白(Oligopeptide transporters,OPTs)通过细胞质膜,将细胞外环境中的物质转入细胞内,从而参与各种生理活动。植物中OPTs蛋白参与金属运输和分配,从而促进植物对金属的利用和适应,但真菌中OPTs是否参与这一过程的相关报道较少。探究OPTs是否在灵芝对金属的适应过程中发挥作用。【方法】以灵芝荣保1号为材料,利用平板实验分析不同浓度的Cu2+、Fe2+和Se4+对菌丝体生长和生物量的影响,并采用实时荧光定量PCR分析不同金属离子胁迫下,两个培养阶段(15 d和30 d)灵芝寡肽转运蛋白基因(OPTs)的转录表达水平。【结果】Cu2+、Fe2+和Se4+三个金属离子在实验使用的浓度范围内抑制灵芝菌丝体的生长量和生物量,且在相同处理条件下的生长量与生物量的变化趋势相同。荧光定量PCR结果显示,除未检测到转录本的3个基因(OPT7、OPT8和OPT9),其余10个基因均表现出了差异性表达模式。在培养前期(15 d),有6个寡肽转运蛋白基因(OPT1、OPT3-6和OPT10)的相对表达量在Cu2+的胁迫下发生上调,而在Fe2+和Se4+的胁迫下,所有的OPTs基因的表达量均被下调。在培养后期(30 d),Se4+胁迫下的OPTs基因的表达量仍旧被下调,而Cu2+和Fe2+的胁迫下表达量均显著上调。【结论】金属能够在不同的程度上影响灵芝的生长量和生物量,同时寡肽转运蛋白基因对这些金属胁迫在转录水平上做出了响应,表明寡肽转运蛋白基因可能在灵芝对金属的适应过程中发挥作用。     

Anx7 is required for nutritional control of gene expression in mouse pancreatic islets of Langerhans

BACKGROUND: Gene expression in islets of Langerhans is profoundly sensitive to glucose and other nutrients. Islets of Langerhans in the Anx7(+/-) knockout mouse exhibit a profound reduction in ITPR3 protein expression, defective intracellular calcium signaling, and defective insulin secretion. Additional data presented here also show that mRNA for ITPR3 is virtually undetectable in isolated Anx7(+/-) islets. IP3Receptor type 3 (ITPR3) expression in islets of Langerhans is closely regulated by secretory stimuli, and it has been suggested that the level of the ITPR3 expression controls the ability of the islets to respond to nutritional signals. We report that although control islets respond to glucose in vitro by a transient increment in ITPR3 mRNA, the islets from the Anx7(+/-) mouse remain low. We therefore hypothesized that the Anx7/IP3 Receptor(3)/Ca(2+) signaling pathway plays a role in beta cell responses to glucose, and that in the absence of the Anx7/ITPR3 signaling system, the islets would be unable to discriminate between fed or fasted states in vivo. MATERIALS AND METHODS: To test this hypothesis, we subjected Anx7(+/-) and control mice to either food and water ad libidum or to an overnight fast with access to water only. We then isolated the respective islets and compared nutrient-dependent changes in global gene expression under the four conditions using genome-based microarray technology. RESULTS: Anx7 protein expression in these islets is only about 50% of control levels in normal littermate controls, and IPTR3 message and protein are virtually zero. cDNA microarray analyses show that in control animals gene expression is significantly affected by the fasting state. Many of the affected genes have historical relevance to development and differentiation of islets. These include preproglucagon, APOJ, cadherin2, phosphoglucoisomerase, oncostatin M, PAX6, HGF, and cytokeratin 18. However, there are also many other nutritionally sensitive genes in control islets that are principally associated with cell division and DNA repair. The latter genes have not specifically been associated with islet physiology in the past. By contrast, Anx7(+/-) mouse islets exhibit a greatly reduced ability to discriminate genomically between fed and fasted states for all classes of identified genes. Many of the validated genes are specific to islets in comparison to liver tissue examined. Real-time quantitative RT-PCR analysis of islets from Anx7 heterozygous mice and littermate controls revealed remarkable down-regulation in PTEN, Glut-2, PDX-1, IGF-1, and Neuro D1 expression, but not in liver. CONCLUSIONS: We conclude that reduced gene dosage in the Anx7(+/-) islet, with concomitant loss of ITPR3 expression and consequent defects in Ca(2+) signaling, may substantially contribute to the mechanism of the loss of genomic discrimination, in vivo, between the fed and fasted states. We believe that the requirement for complete Anx7 gene dosage and IPTR3 expression in islets of Langerhans will prove to be of fundamental importance for understanding the mechanism of nutritional sensing in health and disease.     

MYOZ2通过负调控TCAP的表达促进C3H10T1/2细胞成脂分化

本研究旨在明确钙调磷酸酶肌小节结合蛋白2(myozenin2,MYOZ2)的组织表达特性,阐明其对C3H10T1/2细胞成脂分化的影响及可能的作用机制.采集180日龄马身猪、60日龄ICR小鼠、35日龄罗斯肉鸡和12月龄小尾寒羊背最长肌、皮下脂肪和肝组织,检测MYOZ2基因mRNA表达.结果 显示,MYOZ2基因在所检...     

Overexpression of lncRNA SLC26A4-AS1 inhibits papillary thyroid carcinoma progression through recruiting ETS1 to promote ITPR1-mediated autophagy

Papillary thyroid carcinoma (PTC), accounting for approximately 85% cases of thyroid cancer, is a common endocrine tumour with a relatively low mortality but an alarmingly high rate of recurrence or persistence. Long non-coding RNAs (lncRNAs) is emerging as a critical player modulating diverse cellular mechanisms correlated with the progression of various cancers, including PTC. Herein, we aimed to investigate the role of lncRNA SLC26A4-AS1 in regulating autophagy and tumour growth during PTC progression. Initially, ITPR1 was identified by bioinformatics analysis as a differentially expressed gene. Then, Western blot and RT-qPCR were conducted to determine the expression of ITPR1 and SLC26A4-AS1 in PTC tissues and cells, both of which were found to be poorly expressed in PTC tissues and cells. Then, we constructed ITPR1-overexpressing cells and revealed that ITPR1 overexpression could trigger the autophagy of PTC cells. Further, we performed a series of gain- and loss-of function experiments. The results suggested that silencing of SLC26A4-AS1 led to declined ITPR1 level, up-regulation of ETS1 promoted ITPR1 expression, and either ETS1 knockdown or autophagy inhibitor Bafilomycin A1 could mitigate the promoting effects of SLC26A4-AS1 overexpression on PTC cell autophagy. In vivo experiments also revealed that SLC26A4-AS1 overexpression suppressed PTC tumour growth. In conclusion, our study elucidated that SLC26A4-AS1 overexpression promoted ITPR1 expression through recruiting ETS1 and thereby promotes autophagy, alleviating PTC progression. These finding provides insight into novel target therapy for the clinical treatment of PTC.     

Leptin介导JAK/STAT信号通路对猪皮下前脂肪细胞脂类代谢调控的研究

以猪原代皮下前脂肪细胞为研究材料,检测Leptin介导JAK/STAT信号通路中基因表达水平,旨在阐明Leptin介导JAK/STAT信号通路对脂肪代谢的分子机制.用0和100 ng/mL Leptin分别处理脂肪细胞48 h,油红O染色鉴定脂肪细胞,试剂盒测定细胞中甘油三酯和游离脂肪酸含量,Real-time PCR...     

p38 MAPK regulates calcium signal-mediated lipid accumulation through changing VDR expression in primary preadipocytes of mice

In the present study we have examined whether p38 mitogen activated protein kinase (p38 MAPK) signal pathway interacts with calcium signal on lipid accumulation in primary preadipocytes of mice. The primary preadipocytes were treated with p38 MAPK inhibitor SB203580, blockers and excitomotors of calcium channel for 24 h, respectively. Intracellular triglyceride (TG) content was measured by triglyceride kit and lipid accumulation was determined by Oil Red O staining. Meanwhile, the mRNA expressions of peroxisome proliferators-activated receptor gamma (PPARγ) gene, fatty acid synthetase (FAS) gene, lipoprotein lipase (LPL) gene, vitamin D receptor (VDR) gene and extracellular Ca²⁺-sensing receptor (CaSR) gene were analyzed with real-time PCR. The protein content and phosphorylation of VDR and p38 were tested with Western Blotting. The data showed that intracellular TG content and the mRNA expression levels of PPARγ, FAS, LPL in N group and L group as well as FAS, LPL in C group were increased significantly (P < 0.01) compared to the control. On the contrary, intracellular TG content and the mRNA expression levels of PPARγ, FAS in B group as well as intracellular TG content and PPARγ, FAS, LPL in SB group and B+SB group were decreased significantly (P < 0.01). VDR mRNA expression and protein content were decreased in B, C, and SB added groups (P < 0.01). In addition, p38 phosphorylation levels increased in N and L groups (P < 0.01) and decreased in SB added groups (P < 0.01). These findings suggest that p38 MAPK pathway through regulating VDR mRNA expression participates in mediation of calcium signal and affects calcium signal regulating lipid accumulation in mice preadipocytes through changing PPARγ, FAS and LPL mRNA expression. In addition, calcium signal have a feedback effect in phosphorylation of p38.     

Scavenger receptor BI facilitates hepatic very low density lipoprotein production in mice

Scavenger receptor BI (SR-BI) is a selective uptake receptor for HDL cholesterol but is also involved in the catabolism of apolipoprotein (apo)B-containing lipoproteins. However, plasma levels of apoB-containing lipoproteins increase following hepatic SR-BI overexpression, suggesting that SR-BI not solely mediates their catabolism. We therefore tested the hypothesis that hepatic SR-BI impacts on VLDL production. On day 7 following adenovirus (Ad)-mediated overexpression of SR-BI, VLDL-triglyceride and VLDL-apoB production rates were significantly increased (P < 0.001), whereas VLDL production was significantly lower in SR-BI knockout mice compared with controls (P < 0.05). In mice injected with AdSR-BI, hepatic cholesterol content increased (P < 0.001), microsomal triglyceride transfer protein activity was higher (P < 0.01) and expression of sterol-regulatory element binding protein (SREBP)2 and its target genes was decreased (P < 0.01). Conversely, in SR-BI knockout mice, microsomal triglyceride transfer protein activity was lower and expression of SREBP2 target genes was increased (P < 0.01). Finally, we demonstrate in vitro in isolated primary hepatocytes as well as in vivo that cholesterol derived from HDL and taken up via SR-BI into the liver can be resecreted within VLDL. These data indicate that hepatic SR-BI expression is linked to VLDL production, and within liver, a metabolic shunt might exist that delivers HDL cholesterol, at least in part, to a pool from which cholesterol is mobilized for VLDL production. These results might have implications for HDL-based therapies against atherosclerotic cardiovascular disease, especially with SR-BI as target.     

Adipophilin increases triglyceride storage in human macrophages by stimulation of biosynthesis and inhibition of beta-oxidation

Lipid accumulation alters macrophage biology and contributes to lipid retention within the vessel wall. In this study, we investigated the role of adipophilin on triglyceride accumulation and lipid-droplet formation in THP-1-derived macrophages (THP-1 macrophages). In the presence of acetylated low-density lipoprotein, macrophages infected with an adenovirus expressing human adipophilin showed a 31% increase in triglyceride content and a greater number of lipid droplets compared with control cells. Incubation of macrophages with very low-density lipoprotein (VLDL) dramatically increased cellular triglyceride content similarly in control and adipophilin-overexpressing cells. By itself, VLDL increased adipophilin expression, which explains the lack of effect of adipophilin overexpression on cellular triglyceride content in macrophages loaded with VLDL. The lipid-droplet content of macrophages was increased by overexpression of adipophilin and/or loading with VLDL. In contrast, inhibition of adipophilin expression using siRNA prevented lipid-droplet formation and significantly reduced intracellular triglyceride content. Using inhibitors of beta-oxidation and acyl-coenzyme A synthetase, results were obtained which suggest that adipophilin elevates cellular lipids by inhibition of beta-oxidation and stimulation of long-chain fatty acid incorporation into triglycerides. Adipophilin expression in THP-1 macrophages altered the cellular content of different lipids and enhanced the size of lipid droplets, consistent with a role for adipophilin in human foam cell formation.     

Expression and localization of Ca2+-ATPase in the uterus during the reproductive cycle of king quail (Coturnix chinensis) and zebra finch (Poephila guttata)

Calcium ATPase (Ca2+-ATPase) is a key enzyme that participates in the translocation of calcium in the uterus of oviparous amniotes during eggshell formation. We used Western blot and indirect immunofluorescence microscopy to determine expression and localisation of uterine Ca2+-ATPase during the reproductive cycle of king quail and zebra finch. The pattern of Ca2+-ATPase expression and localisation during the reproductive cycle was similar for both species. Immunoblots of uterine extracts from quail and finch indicated that Ca2+-ATPase expression is reduced in non-reproductive compared to reproductive females. Similarly, in non-reproductive females, weak apical immunofluorescent staining of Ca2+-ATPase is localised to epithelial cells in a small number of uterine tubular glands. A large increase in apical immunofluorescent staining of tubular gland epithelia occurs in both vitellogenic and reproductive females. The presence of Ca2+-ATPase on the apical surface of tubular gland epithelial cells suggests that the enzyme is involved in the translocation of calcium out of the tubular gland epithelia and into the concentrated fluid of the uterine lumen. Presence of Ca2+-ATPase in vitellogenic females indicates that the enzyme is expressed prior to the time of ovulation and eggshell calcification.     

Expression and localization of Ca2+-ATPase in the uterus during the reproductive cycle of king quail (Coturnix chinensis) and zebra finch (Poephila guttata).

Calcium ATPase (Ca2+-ATPase) is a key enzyme that participates in the translocation of calcium in the uterus of oviparous amniotes during eggshell formation. We used Western blot and indirect immunofluorescence microscopy to determine expression and localisation of uterine Ca2+-ATPase during the reproductive cycle of king quail and zebra finch. The pattern of Ca2+-ATPase expression and localisation during the reproductive cycle was similar for both species. Immunoblots of uterine extracts from quail and finch indicated that Ca2+-ATPase expression is reduced in non-reproductive compared to reproductive females. Similarly, in non-reproductive females, weak apical immunofluorescent staining of Ca2+-ATPase is localised to epithelial cells in a small number of uterine tubular glands. A large increase in apical immunofluorescent staining of tubular gland epithelia occurs in both vitellogenic and reproductive females. The presence of Ca2+-ATPase on the apical surface of tubular gland epithelial cells suggests that the enzyme is involved in the translocation of calcium out of the tubular gland epithelia and into the concentrated fluid of the uterine lumen. Presence of Ca2+-ATPase in vitellogenic females indicates that the enzyme is expressed prior to the time of ovulation and eggshell calcification.