饲料n-6/n-3多不饱和脂肪酸比例变化对小鼠血浆瘦素表达的影响

目的 探讨饮食n-6/n-3多不饱和脂肪酸(PUFAs)比例变化对小鼠瘦素和可溶性瘦素受体(sOB-R)表达水平的影响.方法 清洁级3～4周龄C57BL/6J雌性小鼠50只.随机分为5组,每组10只.实验组分别予4种含不同比例n-6/n-3 PUFAs的饲料(即20:1,10:1,5:1,1:1);对照组予n-3 PUFAs缺乏饲料,持续喂养3个月.各组饲料中脂肪所占比例(质量比)均为6%,其中n-6 PUFAs来源于葵花籽油,n-3 PUFAs来源于鱼油.采用ELISA检测各组小鼠血浆瘦素及sOB-R表达水平变化.采用SPSS 11.5软件进行统计学分析.结果 经不同比例n-6/n-3PUFAs饲料喂养3个月后,各组小鼠体质量无显著性差异(F:0.78 P=0.47).与n-3 PUFAs缺乏饲料组比较,n-6/n-3 PUFAs比值为1:1饲料喂养组小鼠血浆瘦素及sOB-R表达水平均显著降低(F=73.31 P=0,F=7.20 P=0.03);n-6/n-3 PUFAs比值为20:1、10:1、5:1饲料组小鼠血浆瘦素及sOB-R表达水平未见显著性变化.结论 饲料n-6/n-3 PUFAs比例变化会对小鼠瘦素和sOB-R的表达水平产生影响;鱼油n-3 PUFAs对瘦素及sOB-R表达的调节作用需要在其高水平的情况下才能表现出来.     

PI3K抑制剂LY294002对鼠前脂肪细胞分化和C/EBPα及PPARγ表达的影响

目的：研究磷脂酰肌醇激酶（PI3K）抑制剂LY294002对小鼠前脂肪细胞分化和CCAAT增强子结合蛋白α（C/EBPα）及过氧化物酶体增殖物激活受体γ（PPARγ）表达的影响,探讨胰岛素受体底物（IRSs）/PI3K信号通路在前脂肪细胞分化中的作用。方法：小鼠3T3-L1细胞分为实验组和对照组,实验组加入LY294002（25 μmol/L）,对照组加入同体积DMSO。应用0.5 mmol/L 3-异丁基-1-甲基黄嘌呤(IBMX)、10-6 mol/L地塞米松和5 μg/mL胰岛素诱导两组前脂肪细胞分化,在培养的0 d、2 d、4 d、8 d分别收集细胞,实时PCR法及Western blot法检测细胞中C/EBPα和PPARγ表达水平。培养8 d油红O染色,观察细胞分化情况。结果：小鼠3T3-L1细胞基础状态下两组C/EBPα及PPARγ表达差异无统计学意义（P＞0.05）;诱导分化时实验组C/EBPα及PPARγ表达低于对照组（分别P＜0.05,P＜0.01）。油红O染色示实验组细胞无明显脂滴。结论：PI3K抑制剂LY294002能抑制小鼠前脂肪细胞分化及C/EBPα和PPARγ的表达,提示IRSs/PI3K信号通路可通过调节PPARγ和C/EBPα的表达在3T3-L1前脂肪细胞的分化中发挥重要作用。     

胰岛素受体底物1基因沉默对3T3-L1细胞CCAAT增强子结合蛋白α、过氧化物酶体增殖物激活受体γ表达的影响

目的研究胰岛素受体底物1(IRS-1)沉默对3T3-L1前脂肪细胞分化关键分子CCAAT增强子结合蛋白α(C/EBPα)、过氧化物酶体增殖物激活受体γ(PPARγ)mRNA及蛋白表达的影响,并观察IRS-1沉默后前脂肪细胞分化为成熟脂肪细胞能力的改变,探讨胰岛素受体底物(IRSs)是否作为C/EBPα、PPARγ的上游调节信号在前脂肪细胞分化中起到重要的调节作用。方法合成4条IRS-1 shRNA,Western blotting筛选出沉默效率最高的1条。3T3-L1前脂肪细胞分为IRS-1沉默组和对照组。IRS-1沉默组细胞用沉默效率最高的IRS-1 shRNA质粒转染3T3-L1前脂肪细胞,沉默细胞中的IRS-1分子;对照组采用无意义IRS-1 shRNA质粒转染3T3-L1前脂肪细胞。转染24 h后诱导其分化成熟,分化72 h后检测促进前脂肪细胞分化的关键分子C/EBPα、PPARγ的表达。继续诱导分化至第7天,油红O染色观察IRS-1沉默后前脂肪细胞分化为成熟脂肪细胞的比例,检测其分化能力的改变。结果与对照组相比,IRS-1沉默组在诱导分化72 h后,Real-time PCR结果显示3T3-L1前脂肪细胞中C/EBPαmRNA、PPARγmRNA表达明显下降(Pa<0.05),Western blotting结果显示C/EBPα、PPARγ蛋白水平也同样显著下降(Pa<0.05);细胞继续分化至第7天,油红O染色显示,IRS-1沉默组前脂肪细胞分化成熟的比例与对照组比较显著降低。结论胰岛素可能通过IRS-1刺激C/EBPα、PPARγ的表达促进前脂肪组织的分化。     

生长激素促分泌素受体-1a基因在3T3-L1脂肪细胞诱导分化过程中的表达

目的 观察生长激素促分泌素受体-1a(GHSR-1a)基因在3T3一K1肪细胞诱导分化过程中不同时段表达水平的变化,探讨GHSR-1a基因在脂肪细胞分化中的作用.方法 体外培养3T3-L1前脂肪细胞,在诱导其向成熟脂肪细胞分化的不同时段(第0-8天).通过形态学观察、油红0染色测定脂肪细胞分化程度及脂滴积聚情况,化学比色法测定脂肪细胞三酰甘油(TG)总量,半定量反转录一聚合酶链反应(RT-PCR)技术检测脂肪细胞中GHSR-1a基因mRNA的表达水平.采用SPSS 12.0软件进行组间t检验.结果 3T3-L1前脂肪细胞在诱导分化前呈梭形的成纤维细胞形态,胞浆内无脂滴;诱导分化后细胞逐渐由梭形变为圆形,胞体变大变圆,胞浆内出现明显的脂滴.3T3-L1前脂肪细胞诱导分化第4天细胞内TG水平已明显升高,第8天TG水平升高更加明显,与前脂肪细胞相比均有显著统计学意义(Pa<0.01).同时GHSR-1a基因mRNA低表达于3T3-L1前脂肪细胞和分化第1天的脂肪细胞,随着3T3-L1脂肪细胞逐渐分化成熟,分化第4天和第8天GHSR-1a基因mRNA的表达逐渐增加,GHSR-1a基因的表达水平除在诱导分化第0-1天和第1-4天内差异无统计学意义(P＞0.05)外,其余各时间点表达水平比较均有统计学意义(Pa<0.05).结论 3T3-L1脂肪细胞分化过程中GHSR-1a基因表达逐渐上调,其表达变化与脂肪细胞分化、脂质积聚过程一致,可能参与了脂肪细胞分化过程.     

3T3-L1脂肪细胞分化中促酰化蛋白对脂肪因子肾上腺髓质素mRNA表达的影响

目的 研究3T3-L1脂肪细胞分化过程中脂肪因子肾上腺髓质索(AM)mRNA表达变化及促酰化蛋白(ASP)对细胞分化过程中AM mRNA表达的影响,探讨ASP在脂肪代谢中的作用.方法 1.应用反转录(RT) -PCR法检测诱导分化4个时点(0d、1d、2d、3d)的3T3-L1脂肪细胞中AM mRNA的表达水平;2.对诱导分化过程中不同时间点(0h、12 h、24h、48 h)的3T3-L1脂肪细胞给予适合浓度的ASP处理,并设立相应空白对照,应用RT-PCR法检测细胞中AM mRNA的表达.结果 (1)脂肪细胞诱导分化各时间点(0d、1d、2d、3 d)AM mRNA均明显表达,且表达水平呈时间依赖性递减;(2)0 h、12 h和24h3个时点ASP组细胞中AMmRNA表达水平较空白对照组均显著下降(P＜0.05,0.01),而48 h时间点ASP组细胞中AM mRNA表达水平与空白对照组比较差异无统计学意义(P＞0.05).结论 ASP促成脂作用可能与其调节脂肪细胞分化过程中脂肪因子AM mRNA表达密切相关.     

DHA与细胞免疫的调节

DHA(全顺式4,7,10,13,16,19-二十二碳六烯酸,22:6 n-3)是人体所需必需脂肪酸,经由α-亚麻酸脱饱和、并经碳链延长作用而生成的重要营养代谢物质,它属于人体内多不饱和脂肪酸(polyunsaturated fatty acids,PUFAs)的n-3型;而人体内PUFAs根据其不饱和双键的位置,被分为n-3型和n-6型PUFAs.自然界中DHA主要存在于陆地植物和海洋生物体内,近年来的研究发现,许多微生物的脂质中也含有DHA,特别是一些低等真菌中其含量较为丰富.     

PRNP基因在3T3-L1脂肪前体细胞分化过程中的表达及肿瘤坏死因子-α对其调节作用

目的 探讨3T3-L1脂肪前体细胞诱导分化过程中PRNP基因表达水平的变化及TNF-α对其调节作用.方法 体外培养3T3-L1前体脂肪细胞,胰岛素加地塞米松加1-甲基-3-异丁基黄嘌呤(MDI)方案诱导3T3-L1细胞分化成熟,并收集分化前、分化0～10 d各时段细胞,采用RT-PCR技术检测诱导分化不同时段3T3-L1细胞PRNP基因表达水平;同时在成功诱导3T3-L1细胞分化成熟的基础上,应用不同水平TNF-α(0.1、1.0、10.0 μg/L)干预分化后的成熟脂肪细胞(第10天),收集TNF-α刺激前(0 h)及刺激后0.5、2.0、6.0、12.0、24.0 h的脂肪细胞,通过RT-PCR测定TNF-α干预前后不同时间点脂肪细胞中PRNP基因表达水平.采用Excel软件进行统计学分析.结果 1.PRNP基因低表达于3T3-L1脂肪前体细胞中,随细胞分化成熟该基因表达水平逐渐上调,至分化第10天其表达水平最高.PRNP基因表达水平除在诱导分化前(第-1天)至第4天、第2～5天、第6～10天时段内无显著性差异外(Pa＞0.05),其余各时段间表达水平均有显著性差异(Pa＜0.05);2.在分化前的3T3-L1脂肪细胞和成熟脂肪细胞中,不同水平TNF-α(0.1、1.0、10.0 μg/L)均能在短时间内明显抑制PRNP基因mRNA的表达,且呈时间依赖性.结论 PRNP基因可能参与3T3-L1脂肪细胞分化及脂质积聚过程;不同水平重组TNF-α对成熟脂肪细胞的PRNP基因表达具有抑制作用,其抑制效应总体趋势上呈时间依赖性.     

NYGGF4基因表达沉默对3T3-L1脂肪细胞线粒体代谢的影响

目的 探讨NYGGF4(又称PID1)基因沉默对脂肪细胞线粒体代谢的影响.方法 以3T3-L1前体脂肪细胞为研究对象,体外培养稳定转染NYGGF4基因沉默载体(pGPU6/GFP/Neo-shRNA,NYGGF4-RNAi)的3T3-L1前体脂肪细胞,以转染空载体(pG-PU6/GFP/Neo-shRNA,NCshRNA)的3T3-L1前体脂肪细胞作为对照,经1-甲基-3异丁基黄嘌呤加地塞米松、胰岛素方案诱导分化为成熟脂肪细胞,采用实时定量RCR技术检测成熟脂肪细胞中线粒体代谢酶指标:己糖激酶、乙酰辅酶A羧化酶柠檬酸合成酶、肉毒碱棕榈酰转移酶1、细胞色素C基因表达水平.结果 NYGGF4表达沉默与NYGGF4基因过表达相比,可以部分逆转3T3-L1脂肪细胞中已糖激酶、乙酰辅酶A羧化酶、柠檬酸合成酶、肉毒碱棕榈酰转移酶1、细胞色素C基因表达水平,差异均有统计学意义.结论 NYGGF4基因过表达,下调3T3-L1脂肪细胞中定位于线粒体的代谢关键酶,提示NYGGF4基因的动态平衡对维持3T3-L1脂肪细胞的线粒体代谢具有重要作用.     

ω-3多不饱和脂肪酸对脓毒症大鼠淋巴细胞凋亡率的影响

目的探讨ω-3多不饱和脂肪酸对脓毒症大鼠胸腺和脾脏淋巴细胞凋亡的影响。方法将80只7~8周龄雌性Sprague-Dawley大鼠随机分成模型组、传统脂肪乳剂治疗组(每日0.1 g/kg)、ω-3PUFAs低剂量治疗组(每日0.1 g/kg)、ω-3PUFAs中剂量治疗组(每日0.2 g/kg)和ω-3PUFAs高剂量治疗组(每日0.3 g/kg)。采用盲肠结扎穿孔法建立脓毒症大鼠模型,造模后各治疗组分别给予脂肪乳剂和不同剂量ω-3PUFAs葡萄糖稀释液尾静脉注射给药,模型组予等量葡萄糖注射液尾静脉注射。按处死时间将各组大鼠随机分为24 h和72 h两个亚组,每组8只大鼠。采用苏木精-伊红染色观察各组大鼠胸腺和脾脏的病理改变;采用TUNEL法检测各组大鼠脾脏和胸腺淋巴细胞凋亡指数。结果不同剂量ω-3PUFAs组大鼠胸腺小叶结构完整,皮髓质结构清晰,模型组和传统脂肪乳剂治疗组皮髓质界限不清,淋巴细胞明显减少;ω-3PUFAs组脾脏红髓和白髓结构尚能保持,可见脾小结存在,而模型组和传统脂肪乳剂治疗组红髓和白髓结构紊乱,脾小结明显缩小或消失。不同剂量ω-3PUFAs组24 h和72 h胸腺和脾脏淋巴细胞凋亡率均较模型组、传统脂肪乳剂治疗组显著降低(P0.01),ω-3PUFAs高剂量治疗组24 h和72 h脾脏淋巴细胞凋亡率均较ω-3PUFAs低剂量治疗组显著降低(P0.05)。结论ω-3多不饱和脂肪酸有减轻脓毒症大鼠胸腺和脾脏淋巴细胞凋亡的作用,并且可能与剂量相关。     

肥胖相关新基因NYGGF4对3T3-L1脂肪前体细胞的增殖调节作用

目的 观察NYGGF4基因过表达对3T3-L1脂肪前体细胞增殖的影响,探讨该基因在肥胖发生发展中的作用.方法 采用反转录聚合酶链反应(RT-PCR)法扩增NYGGF4开放阅读框(ORF)的全长,通过双酶切连接法构建NYGGF4-pcDNA3.1真核表达载体.采用脂体法转染3T3-L1前体脂肪细胞,以pcDNA3.1空载和未转染细胞为对照.遗传霉素(G418)压力筛选稳定转染细胞株,PCR鉴定转染细胞的NYGGF4的mRNA水平.采用四甲基偶氮唑蓝(MTT)检测稳定转染细胞株连续7 d的生长状态,以转染空载和未转染正常3T3-L1为对照,绘制生长曲线.采用SPSS 10.0软件进行统计学分析.结果 1.稳定转染细胞株有NYGGF4基因的表达,而pcDNA3.1空载未见人源性NYGGF4的表达;2.转染NYGGF4基因的3T3-L1细胞增殖速度明显较未转染和转染空载的3T3-L1细胞加快.结论 新基因NYGGF4可明显促进3T3-L1脂肪前体细胞的增殖,可能影响肥胖的发生发展.     

3-HYDROXY-3-METHYLGLUTARYL COENZYME A LYASE DEFICIENCY

ABSTRACT. Shilkin, R., Wilson, G. and Owles, E. (Princess Margaret Hospital for Children, Perth, Western Australia). 3-Hydroxy-3-methylglutaryl Coenzyme A lyase deficiency: follow-up of first described case. Acta Paediatr Scand, 70:265, 1981. –We report the progress of a child with a defect in leucine metabolism due to a deficiency of 3-hydroxy-3-methylglutaryl Coenzyme A lyase activity. This child was reported briefly in 1976 when the abnormality was first suspected at which time he was 7 months old. He is now aged 4 years 7 months and appears to be well and developing satisfactorily. His diet has been difficult to control and the biochemical defect is extremely sensitive to small amounts of leucine in the diet.     

3-hydroxy-3-methylglutaraciduria (case report of a female Turkish sisters with 3-hydroxy-3- methylglutaryl-Coenzyme A lyase deficiency

3-Hydroxy-3-methylglutaric aciduria is a rare inborn error of metabolism, caused by reduced enzyme activity of the intramitochondrial 3-hydroxy-3-methylglutaryl-CoA lyase. We describe two turkish sisters with this disease. In the older sister clinical symptoms with lethargy, convulsions, metabolic acidosis, hypoglycemia and hyperammonemia lead to the diagnosis. The younger sister was diagnosed prenatally. The clinical course of our patients is compared with those reported in the literature with respect to clinical symptoms, differential diagnosis and therapeutic regimens.     

3-Methylglutaconic and 3-methylglutaric aciduria in a patient with suspected 3-methylglutaconyl-CoA hydratase deficiency

A girl suffering from marked muscular hypotonia, severe statomotor and mental retardation, bilateral optic atrophy with chorioretinal degeneration, convulsions and a moderate compensated metabolic acidosis is described. Screening for metabolic disorders revealed massive 3-methylglutaconic with 3-methylglutaric aciduria leading to the tentative diagnosis of 3-methylglutaconyl-CoA hydratase deficiency. Metabolite excretion was correlated with variation of leucine intake. 3-methyl-3-hydroxyglutaryl-CoA lyase activity in cultured fibroblasts was normal. The suspected metabolic defect was not demonstrable in cultured skin fibroblasts, however.Abbreviation MSUD Maple syrup urine disease     

X-linked severe combined immunodeficiency with γδT cells

A patient with X-linked severe combined immunodeficiency (X-SCID) was found to have a deletion mutation of a four base pair in the transmembrane domain of the IL-2 receptor γ chain gene, a subunit shared by the receptors for IL-4, IL-7, IL-9, and IL-15 (common γ chain; γc). He had very few αβT cells but had a considerable number of γδT cells in his peripheral blood. Fluorescence in situ hybridization (FISH) analysis showed that the γδT cells in his peripheral blood were not of maternal origin. He had received a Bacillus Calmette-Guerin (BCG) vaccination before recognition of the disease, and the BCG infection remained quiescent with no reaction for 19 months. After successful bone marrow transplantation, the site of the BCG vaccination showed a reaction, and live BCG were detected. It is useful to consider the relationship between the existence of γδT cells and BCG in this case, and it is suggested that γδT cells may be, in a given situation, less dependent on the γc chain than are αβT cells.     

Gastrografin aspiration in a 3 3/4-year-old girl

A case of Gastrografin aspiration in a 3 3/4-year-old child admitted for multiple trauma is described.     

迟发型3-羟基-3-甲基戊二酸尿症导致脑白质病

3-羟基-3-甲基戊二酸尿症是一种罕见的有机酸代谢病,病因为常染色体隐性遗传所导致的3-羟基-3-甲基戊二酰辅酶A裂解酶缺陷。患者通常在新生儿期至婴幼儿期发病。本研究报告1例晚发型3-羟基3-甲基戊二酸尿症导致的脑白质病。患儿为7岁男孩,急性起病,表现为头痛、困倦、呕吐, 进行性加重,一般化验发现肝损害、酮症、白细胞减少,脑磁共振扫描示双侧脑白质对称弥漫性病变。血液羟异戊酰肉碱、乙酰肉碱显著增高。尿液3-羟基-3-甲基戊二酸显著增高,3-甲基戊烯二酸、3-羟基戊二酸、甲基巴豆酰甘氨酸增高。经静脉滴注葡萄糖和左旋肉碱症状缓解。维持治疗半年后复诊,尿3-羟基3-甲基戊二酸降低,全身情况良好。     

A case of biotin-responsive 3-methylcrotonylglycin- and 3-hydroxyisovaleric aciduria

During selective screening for organic acidurias, a 10-week-old girl with muscular hypotonia and recurrent fits was shown to be excreting 3-methylcrotonylglycin and 3-hydroxyisovaleric acid. Besides these metabolites of leucine the presence of small but pathological amounts of propionic and methylcitric acids were demonstrable in her urine, pointing to a defect in the metabolism of biotin.On treatment with biotin (2×5 mg/day) the convulsions stopped at once, her clinical condition improved gradually, and the abnormal metabolites disappeared from the urine. Within 6 weeks the child was discharged in a good general condition without apparent signs of neurological damage.