鸡PPARs基因组织表达特性的研究

以8周龄Arber Acres(AA)肉鸡为研究材料,采用RT-PCR和Northern blot方法对鸡PPAR基因组织表达特点进行了研究。RT-PCR半定量检测显示,在检测的10种组织中除胸部肌肉组织外,PPAR-α基因在其他9种组织中都表达,其中较高表达于脑、肺脏、肾脏、心脏和小肠,中等程度表达于胃、肝脏和脂肪,较低表达于脾脏。PPAR-γ基因除了在肝脏和肌肉中没有检测到外,在其他8种组织都有表达,其中高表达于脂肪,其次是脑和肾脏,低量表达于脾脏、心脏、肺脏、胃和小肠。Northern blot检测显示,PPAR-α基因在心脏、肝脏、肾脏和胃这4种组织表达,其中在肝脏杂交信号最强;PPAR-γ基因只在脂肪和肾脏表达,其中在脂肪组织有强的杂交信号。以上结果表明,鸡PPARS基因的组织表达特点同啮齿动物和人基本一致,但也有其自身的特殊性,即PPAR-α基因不在肌肉组织中表达,PPAR-γ基因在肾脏中有高表达。PPAR基因与鸡的多种组织的生长和发育有关。     

ADRP基因在宗地花猪及其杂交猪不同组织中的表达分析

为研究ADRP基因在宗地花猪及其二元杂交猪不同组织中的表达规律,探讨该基因与脂肪含量的关系,试验以宗地花猪及其二元杂交猪为材料,采用实时荧光定量PCR技术,检测ADRP基因在心脏、肝脏、脾脏、肺脏、肾脏、小肠、背最长肌及脂肪中的相对表达量。结果表明,ADRP基因在宗地花猪及其杂交猪各组织中均有表达,不同组织之间及不同猪种之间的表达存在差异,表达丰度关系宗地花猪的为小肠脂肪肺脏肝脏心脏背最长肌脾脏肾脏,宗江二元猪的为脂肪小肠脾脏肺脏肾脏肝脏背最长肌心脏;显著性检验结果显示,宗江二元猪肝脏、脾脏、肺、小肠、背最长肌中ADRP基因的相对表达量显著高于宗地花猪的(p0.01),心脏、肾脏和脂肪组织中的表达量显著低于宗地花猪的(p0.01)。结果提示,ADRP基因在宗地花猪及其二元杂交猪不同组织中的差异表达可能与脂肪沉积有关。     

猪SOCS-3 cDNA序列克隆及在各组织中表达

 根据大鼠细胞因子信号转导抑制因子（suppressor of cytokine signaling,SOCS)-3设计并合成一对引物,从八眉猪皮下脂肪组织提取总mRNA,RT-PCR扩增获得猪SOCS-3 cDNA;利用半定量（semi-quantitative, SQ）RT-PCR技术检测大白猪各组织中SOCS-3 mRNA的表达量.扩增获得SOCS-3 基因GenBank 登陆号DQ644577,用CDD软件进行SOCS-3 基因结构分析,该序列具有SOCS-3 特有的保守结构域SH2和SOCS-box. 用Clustal W软件进行同源性分析发现,猪SOCS-3 基因与人、大鼠和小鼠的同源性分别为92%、88%和88%.SQ RT-PCR组织表达检测表明：SOCS-3 基因在心脏、肝脏、脾脏、肺脏、肾脏、肌肉、皮下脂肪和内脏脂肪中均有表达,其中肺脏和脾脏的表达丰度最高,肝脏和内脏脂肪中的表达量最低.     

广西巴马小型猪SLC30-A8基因克隆及组织表达差异分析

本研究的目的是克隆广西巴马小型猪SLC30-A8基因,并确定各组织的表达量。利用RT-PCR的方法扩增并克隆SLC30-A8基因;荧光定量PCR检测SLC30-A8基因在12月龄广西巴马小型猪心脏、肝脏、脾脏、肺脏、肾脏、胰腺、小肠和脂肪中的表达。结果显示,成功克隆广西巴马小型猪SLC30-A8基因CDS区全长1 110 bp,荧光定量PCR结果显示SLC30-A8基因在12月龄广西巴马小型猪胰腺组织中表达量最高,其次是心脏、脾脏,在肺脏、小肠以及脂肪组织中几乎不表达。本研究成功克隆了广西巴马小型猪SLC30-A8基因并确定在胰腺组织中表达最高。该研究将为下一步研究SLC30-A8基因在糖尿病的发生过程中对糖代谢的作用奠定基础。     

猪Musclin基因的组织表达特征及其与生脂基因的相关性分析

根据人、大鼠、小鼠Musclin基因序列设计引物,从大白猪肌肉组织提取总mRNA, R T-PCR扩增猪Musclin cDNA;利用半定量(Semi Quantitative, SQ) RT-PCR检测猪Musclin mRNA在不同组织、以及不同生长阶段和不同品种猪脂肪与肌肉组织中的表达量,研究不同品种猪皮下脂肪组织Musclin基因与脂肪酸合成酶(FAS)、过氧化物酶体增殖物激活受体γ (PP ARγ)、甘油三酯水解酶(TGH)基因mRNA表达量的相关性.结果表明:猪Musclin基因片段与其他物种的同源性达59%以上, 该基因在内脏脂肪和皮下脂肪表达丰度最高,脾脏表达量最低(P<0.05);随月龄增长猪肌肉和皮下脂肪组织中Musclin mRNA表达量呈显著下降( P<0.05) ;瘦肉型猪肌肉和皮下组织Musclin mRNA表达显著高于脂肪型猪(P<0.05);皮下脂肪组织中该基因mRNA表达与FAS、PPARγ分别呈显著负相关和正相关(P<0.05),与TGH相关性不显著 (P>0.05).因此,猪Musclin基因除在肌肉中表达外,在脂肪等其他组织也能表达 ;该基因的表达与猪生长阶段、品种有关,且与生脂基因FAS、PPARγ密切相关.推测该基因可能在脂代谢中起到一定的作用 [动物学报 54(3):453-459,2008].     

高原鼠兔ob基因的组织表达特征

ob基因编码的leptin蛋白在调节生物体能量平衡中起到重要作用。本研究应用Taqman探针real time PCR技术对高原鼠兔ob基因的组织分布进行检测。通过提取不同组织总RNA,经DNase I消化后,用随机引物进行反转录合成cDNA,采用特异性Taqman探针和引物分别对ob基因及β-actin基因进行实时定量PCR扩增,对不同组织中ob基因和β-actin基因的初始拷贝数之比进行比较。结果表明ob基因在脑、心脏、肺、肝脏、脾脏、肾脏、骨骼肌、脂肪组织中均有表达,其中以白色脂肪组织中ob基因表达量最高,其次为心脏和肺,表达量最低的是肝脏和肾脏。     

鸡PPARγ基因的表达特性及其对脂肪细胞增殖分化的影响

为分析鸡PPARγ基因的组织表达特性及其在脂肪细胞增殖和分化过程中的功能,文章以东北农业大学高、低腹脂双向选择品系肉鸡为实验材料,利用Western blotting方法,检测PPARγ基因的组织表达特性及其在高、低脂系肉鸡腹部脂肪组织间的表达差异;采用RNAi技术,在鸡原代脂肪细胞中抑制PPARγ基因的表达后,通过MTT和油红O提取比色的方法,研究鸡PPARγ基因对脂肪细胞增殖和分化的调控作用;利用Real-timePCR和Western blotting技术,分析PPARγ基因表达下调后,其他脂肪细胞分化转录因子以及与脂肪细胞分化相关的重要基因的表达变化情况。结果表明,PPARγ基因在7周龄高脂系肉鸡腹部脂肪组织、肌胃、脾脏、肾脏组织中表达量较高,在心脏中表达量较低,在肝脏、胸肌、腿肌、十二指肠中未检测到表达信号;与高脂系相比,PPARγ基因在5和7周龄低脂系肉鸡腹部脂肪组织中的表达量较低(P<0.05);PPARγ基因的表达量下降后,鸡脂肪细胞的增殖能力增强,分化能力减弱;同时,C/EBPα、SREBP1、A-FABP、Perilipin1、LPL、IGFBP-2基因的表达量均下降(P<0.05)。由此可见,PPARγ基因的表达可能与肉鸡腹部脂肪的沉积有一定的关系,该基因可能是调控鸡脂肪细胞增殖与分化的关键因子。     

猪ANK1新的可变剪接体功能预测、组织表达谱分析与真核表达载体构建

为进一步了解猪ANK1基因,本研究以广西巴马猪作为实验对象,分析ANK1基因结构与功能,并利用实时荧光定量PCR方法,分析ANK1基因在广西巴马小型猪11个不同组织(心脏,肝脏,脾脏,肺脏,肾脏,大肠,小肠,胰腺,皮下脂,胃,背最长肌)中的表达水平。组织表达谱分析结果表明ANK1基因在11个组织中均有表达,mRNA表达量表现为:胰腺心脏背最长肌胃大肠肝脏肾脏小肠脾脏肺脏脂肪。ANK1基因编码的蛋白通过生物信息学分析表明,编码156个氨基酸,存在跨膜结构和跨膜信号肽,分子量为17 799.27,理论等电点6.31,α螺旋和无规则卷曲所占比例比较大。通过酶切技术成功获得p EGFP-ANK1重组质粒并转染到C2C12细胞中,于48 h拍照发现细胞发出荧光,说明ANK1基因真核表达载体构建成功。     

Sirt3在不同热量饮食下的大鼠脂肪组织中的表达

Sirt3是一种NAD+依赖性组蛋白去乙酰化酶,在肾脏、棕色脂肪、心脏和其它代谢活跃的组织中高表达. 多项研究表明,Sirt3在细胞能量代谢、衰老、肿瘤发生等方面起着重要的作用.为探讨Sirt3基因在不同热量饮食下的SD大鼠脂肪组织中的表达差异和意义,将60只3周龄,重量(60±5) g的断乳SD雄性大鼠,随机分为热量限制组、自由摄食组、高热量组,其中高热量组用于构建肥胖SD大鼠模型,喂养16周后处死,分别用Western印迹和real-time RT-PCR方法检测各组大鼠肾周脂肪组织中Sirt3基因的蛋白和mRNA的表达量.Western印迹检测与real-time RT-PCR检测结果一致: Sirt3基因的蛋白和mRNA在肥胖大鼠脂肪组织中的表达低于自由摄食组（P<0.05）,在热量限制组大鼠脂肪中的表达高于自由摄食组(P<0.05).研究结果说明,Sirt3基因在不同热量饮食下的SD大鼠脂肪组织中的表达有差异,可能与能量代谢及肥胖有着密切关系.     

小鼠不同组织及缺氧损伤后的大鼠心肌细胞中RIP3的表达

目的:检测小鼠组织中受体相互作用丝氨酸/苏氨酸蛋白激酶家族(RIPs)表达谱,并检测RIP3在大鼠心肌细胞缺氧损伤后的表达。方法:①采用荧光实时定量PCR分别检测RIPs家族基因在小鼠组织(心、肝、肺、肾、脑、小肠、骨骼肌、脾和主动脉)中的mRNA表达谱,并采用Western blot进一步检测RIP3在小鼠组织的蛋白表达谱。②将培养的大鼠心肌细胞分为缺氧组和对照组,缺氧组置于缺氧环境中培养48 h,采用western blot检测其中RIP3的表达变化。结果:①mRNA水平:RIP1 mRNA在脑组织中表达最高,心脏、肺、肾、骨骼肌较低;RIP2在心脏和肺表达量较其他组织高;RIP3在肠中表达较其他组织高出4倍以上,脑组织中未检测到RIP3表达;RIP4的表达以肺最高,而骨骼肌、脑和血管中表达量低。②蛋白水平:在小鼠组织中,RIP3表达以脑、骨骼肌中最高,心脏、肝、肺中表达较低。③培养的大鼠心肌细胞中,缺氧组心肌细胞的RIP3表达量显著高于对照组(P0.05)。结论:RIPs在小鼠组织中呈现差异表达,而在培养的大鼠心肌细胞缺氧损伤后RIP3表达升高。     

Molecular cloning and expression of bovine (Bos taurus) leptin receptor isoform mRNAs

We characterized Bos taurus leptin receptor (Ob-R) isoform mRNAs as well as their expression in different tissues, including some adipose depots (perirenal, subcutaneous and intermuscular adipose tissues). Based on the GenBank database sequences of the bovine partial Ob-R, primers were designed to amplify cDNAs of bovine Ob-R isoforms. The full-length cDNAs of bovine the Ob-R isoforms were cloned by combination with 3'-and 5'-RACE. Three bovine Ob-R isoform cDNAs were cloned and the sequence analyses revealed that these cDNAs were bovine Ob-R isoforms, i.e., the long form (Ob-Rb), the middle form (Ob-Ra) and the short form (Ob-Rc). The open reading frames of Ob-Ra, Ob-Rb and Ob-Rc gene were 2688, 3498 and 2673 bp, respectively. The deduced amino acid sequences suggested that the isoforms were single transmembrane proteins, and differed in the C-terminal amino acid sequences. The amino acid sequence of these bovine Ob-R isoforms showed 73-75% identity compared with the corresponding mouse isoforms. The tissue-specific expression of the bovine Ob-R isoforms were measured by semi-quantitative RT-PCR. Expression of Ob-Rb was highest in liver, heart, spleen and kidney, with lower expression in lung and testis, and slight expression in muscle. Ob-Ra was highly expressed in liver and spleen, whereas moderate expression was observed in heart, testis, and muscle, and its expression was the lowest in lung and kidney. Ob-Rc mRNA was expressed in the liver, heart, testis, kidney and muscle, but not in the lung and spleen. In adipose tissues, higher expression of Ob-Ra and Ob-Rb mRNA was observed in intermuscular adipose tissue than in subcutaneous or perirenal adipose tissues. Ob-Ra mRNA level was positively correlated with Ob-Rb mRNA level in the adipose tissues (r=0.81, P<0.05). The results demonstrated that each Ob-R isoform mRNA was differentially expressed in various tissues of cattle, which may be involved in the difference of peripheral actions for leptin.     

PPARγ与c/EBPα基因在苏太猪不同组织中的表达水平探究

为探究PPARγ与c/EBPα基因在苏太猪不同组织中的表达与脂肪沉积的关系,本实验以10月龄苏太猪为研究对象,运用实时荧光定量PCR (q RT-PCR)技术检测PPARγ与c/EBPα基因mRNA在苏太猪心、肝、脾、肺、肾、胃、背最长肌和皮下脂肪8个组织中的表达水平。结果表明,PPARγ与c/EBPα基因在苏太猪的8个组织中均有不同程度的表达,其中,PPARγ基因在苏太猪脾脏组织中的表达量最高,皮下脂肪中的表达水平仅次于脾;以背最长肌中PPARγ基因的相对表达量作对比,背最长肌与脾、肺和皮下脂肪的相对表达差异极显著(p<0.01),其余为差异不显著(p>0.05),表达量高低顺序为脾>皮下脂肪>肺>心>胃>肾>肝>背最长肌;c/EBPα基因在苏太猪的皮下脂肪的表达量最高,以背最长肌中c/EBPα基因的相对表达量作对比,在肝、脾、皮下脂肪组织中表达差异极显著(p<0.01),肺的相对表达差异显著(p<0.05),其余组织中差异不显著(p>0.05),表达量的高低顺序为皮下脂肪>肝>脾>肺>肾>心>胃>背最长肌。两基因在各组织中表达趋势趋于一致。试验结果表明PPARγ和c/EBPα基因可能对猪脂肪沉积有重要影响。     

Distribution and quantification of β-3 adrenergic receptor in tissues of sheep

The β-3 adrenergic receptor (ADRB3) is a G-protein coupled receptor involved in regulating lipolysis, as part of homeostatic regulation. In this study, South African Mutton Merino and Shanxi Dam Line were used to study the distribution and quantification of ADRB3 in adipose (subcutaneous, omental, retroperitoneal, mesenteric and perirenal fat) and non-adipose (heart, liver, spleen, lung and kidney) tissues of sheep. The protein was determined by immunohistochemical technique and by mRNA abundance via real-time polymerase chain reaction. ADRB3 was detected in all studied tissues with abundance in adipose tissues higher than in non-adipose tissues (P < 0.001). For adipose tissues, greater expression was found in deep deposits such as great omental and retroperitoneal fat than in subcutaneous fat (P < 0.05). Significant differences (P < 0.05) both for mRNA and for protein expression also existed between the two sheep flocks. These findings are consistent with the known function of ADRB3 in mediating lipolysis and homeostasis in adipose tissues.     

流行性出血热病毒感染NIH裸鼠免疫组织化学研究

流行性出血热病毒（ＥｐｉｄｅｍｉｃＨｅｍｏｒｒｈａｇｉｃＦｅｖｅｒｖｉｒｕｓ,ＥＨＦＶ）感染ＮＩＨ裸鼠后,濒死状态取材,应用免疫组织化学方法（ＡＢＣ）检测各组织中的特异性病毒抗原。结果表明,ＮＩＨ裸鼠对ＥＨＦＶ感染敏感,感染后其脑、肺、肝、肾和心脏组织实质细胞浆内均可检出特异性抗原。     

不同日龄大白猪视黄酸受体α基因组织表达

研究采用RT-PCR方法对大白猪的视黄酸受体α基因在1日龄、90日龄、180日龄、270日龄和360日龄的心、肝、胃、脾、肾、肺、大肠、小肠、肌肉、子宫、卵巢共11个组织的表达情况进行了研究。结果表明,RARαmRNA在肝、脾、肾、大肠、小肠、子宫和卵巢中持续表达,其中脾、大肠和小肠是持续高表达;180日龄时,所有组织的RARαmRNA的表达量普遍降低;360日龄时,所检的11个组织均高水平表达该基因。     

Differential expression of phospholipases D1 and D2 in mouse tissues

The differential expression of phospholipase D (PLD) isozymes, which include PLD1 and PLD2, was examined in various murine tissues, including the cerebrum, cerebellum, heart, lung, liver, spleen, stomach, pancreas, ileum, colon, adrenal gland, kidneys, testes, ovaries, and uterus. In Western blot analysis, only PLD1 was detected in the heart and ovary, while only PLD2 was detected in the pancreas and ileum. Both PLD1 and PLD2 were strongly expressed in the cerebrum, cerebellum, and lung, and both were also expressed in the liver, spleen, stomach, colon, kidney, testes, and uterus. Immunohistochemistry showed intense PLD immunostaining in the cerebrum, cerebellum, lungs, intestines, and testis, and weak PLD immunostaining in the liver, kidneys, spleen, and heart. These findings suggest that PLD1 and PLD2 are differentially expressed in the various organs of mice, and that each PLD isozyme plays a distinct role in each organ.     

PP-1催化亚基（PP-1c）在成体小白鼠不同组织器官中的分化表达与定位

为了确定蛋白磷酸酶-1(protein phosphatase-1)的催化亚基(PP 1c)在小白鼠不同器官组织(肌肉、卵巢、肾、胃、 脾、大脑、心、肝、肺及乳腺)中的表达模式,运用RT-PCR、Western 印迹及荧光免疫组织化学技术等实验手段进行了检测 和分析.结果表明,在mRNA水平, PP-1c在大脑中表达最高,卵巢及肺中表达次之,在肌肉、肾、心、肝中表达较低,在胃 和乳腺中表达最低;在蛋白质水平,肝中表达最高,肾、大脑、肺和乳腺中表达较高,而肌肉、卵巢、心和脾中表达相对较 低,胃中表达最低.免疫荧光组织化学实验结果显示,PP 1c的表达也具有明显的组织特异性和细胞特异性.这些结果为进一 步探讨PP 1在哺乳动物不同组织器官中的功能提供了重要的实验依据.     

Strategies for maintaining Na⁺ balance in zebrafish (Danio rerio) during prolonged exposure to acidic water

Teleost fish store lipids among several tissues primarily as triacylglycerol (TG). Upon metabolic demand, stored TGs are hydrolyzed by hormone-sensitive lipase (HSL). In this study, two distinct cDNAs encoding HSL were isolated, cloned, and sequenced from adipose tissue of rainbow trout. The full-length cDNAs, designated HSL1 and HSL2, were 2562-bp and 2887-bp in length, respectively, and share 82% nucleotide identity. Phylogentic analysis suggests that the two HSLs derive from paralogous genes that may have arisen during a teleost-specific genome duplication event. Quantitative real-time PCR revealed that HSL1 and HSL2 were differentially expressed, both in terms of distribution among tissues as well as in terms of abundance within selected tissues of juvenile trout. HSL1 and HSL2 mRNAs were detected in the brain, spleen, pancreas, kidney, gill, intestine, heart, and white muscle, but were most abundant in the red muscle, liver, and adipose tissue. HSL1 mRNA was more abundant than HSL2 mRNA in the adipose tissue, whereas HSL2 mRNA was more abundant than HSL1 mRNA in the liver. Short term fasting (4 weeks) increased HSL1 and HSL2 mRNA expression in the adipose tissue, but only HSL1 mRNA levels increased in the liver and the red muscle. During a prolonged fast (6 weeks), there was continued elevation of HSL1 and HSL2 mRNA levels in the liver and muscle; HSL mRNA expression in mesenteric fat declined, coincident with depletion of mesenteric fat mass. Refeeding fish reduced HSL expression to levels seen in continuously fed fish. These findings indicate that the pattern of HSL expression is consistent with the diverse lipid storage pattern of fish and suggest that distinct mechanisms serve to regulate differential expression of the two HSLs in tissues and during a progressive fast.