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.     

NYGGF4基因过表达对成熟脂肪细胞线粒体形态及动力学的影响

目的 探讨NYGGF4基因过表达对成熟3T3-L1脂肪细胞线粒体形态及动力学的影响.方法 以3T3-L1前体脂肪细胞为载体,建立NYGGF4基因稳定过表达细胞株,以空载质粒转染的细胞为对照,将前体脂肪细胞诱导分化为成熟脂肪细胞.采用透射电镜观察成熟脂肪细胞的线粒体形态,采用荧光定量PCR技术检测成熟脂肪细胞中线粒体融合基因(Mfn)1 mRNA、Mfn2 mRNA、线粒体动态相关基因(Drp)1 mRNA的表达水平.采用Western blot方法检测Mfn1蛋白、Mfn2蛋白、Drp1蛋白的表达水平.结果 1.电镜观察发现NYGGF4基因过表达的成熟脂肪细胞线粒体体积变小、数量明显减少,线粒体嵴断裂、减少、消失,部分线粒体肿胀,甚至呈空泡状;对照组成熟脂肪细胞的线粒体形态基本正常,线粒体嵴清晰可见,线粒体无明显肿胀、皱缩.2.NYGGF4基因过表达成熟脂肪细胞Mfn1 mRNA及Mfn1蛋白表达水平均显著高于对照组.3.NYGGF4基因过表达成熟脂肪细胞的Mfn2 mRNA、Drp1 mRNA及相应蛋白表达水平与对照组比较差异均无统计学意义.结论 在3T3-L1成熟脂肪细胞中,NYGGF4基因过表达可导致线粒体形态发生变化、数量减少,同时上调Mfn1 mRNA和Mfn1蛋白表达水平,提示NYGGF4基因在成熟脂肪细胞中过表达能影响细胞线粒体形态及动力学.     

NYGGF4基因过表达对脂肪细胞胰岛素敏感性及分泌功能的影响

目的：观察肥胖相关新基因NYGGF4过表达对脂肪细胞的胰岛素敏感性及分泌功能的影响。方法：体外培养稳定转染NYGGF4基因（NYGGF4-pcDNA3.1）的3T3-L1前体脂肪细胞,以转染空载体（pcDNA 3.1）的3T3-L1细胞为对照,胰岛素加地塞米松加1-甲基-3-异丁基黄嘌呤(MDI)方案诱导细胞分化成熟,液闪仪测定3H标记的葡萄糖摄取率,Western blot检测葡萄糖转运体4（GLUT4）的表达及转位;采用ELISA双抗体夹心法检测培养上清中TNF-α、IL-6、脂联素、抵抗素4种细胞因子的水平。结果：NYGGF4过表达显著降低胰岛素刺激的葡萄糖摄取率（P<0.05）。NYGGF4过表达对总的GLUT4的表达量没有影响,但明显降低胰岛素刺激的GLUT4由细胞浆向细胞膜的转位（P<0.05）。过表达NYGGF4的脂肪细胞其培养上清中TNF-α、IL-6、脂联素及抵抗素的分泌水平与对照组相比差异无显著性（P>0.05）。结论：NYGGF4基因过表达通过下调胰岛素刺激的GLUT4的转位而减少脂肪细胞的葡萄糖摄取率,提示脂肪细胞对胰岛素的敏感性降低,而对脂肪细胞的分泌功能无明显影响。［中国当代儿科杂志,2009,11（10）：846-849］     

Resistin基因对胰岛β细胞RINm5F增殖的影响

目的：Resistin被认为是联系肥胖与2型糖尿病的脂肪因子。本研究旨在探讨resistin基因过表达对胰岛β细胞株RINm5F增殖的影响。方法：体外培养RINm5F大鼠胰岛β细胞,通过pcDNA3.1-resistin真核表达质粒转染RINm5F细胞,筛选稳定表达株。采用四甲基偶氮唑盐（MTT）法检测细胞增殖,采用RT-PCR法验证Resistin mRNA水平及检测SOCS-3的mRNA水平,Western blot法检测Akt的总蛋白和磷酸化水平。结果：过表达resistin的RINm5F细胞增殖速度显著低于正常对照组（P<0.05）。过表达resistin的RINm5F细胞中Akt磷酸化水平为正常对照组的0.6倍(P<0.05),而SOCS-3 mRNA水平为正常对照组的3.2倍(P<0.05)。结论：Resistin基因过表达减缓了胰岛β细胞RINm5F的增殖,其机制可能与上调了SOCS-3从而导致Akt下调有关。［中国当代儿科杂志,2010,12（1）：43-46］     

鞘氨醇激酶基因在3T3-L1脂肪细胞诱导分化中的表达水平

目的 探讨鞘氨醇激酶基因(SPHK）在3T3-L1脂肪细胞诱导分化中表达水平的变化。方法采用细胞培养和RT-PCR技术检测细胞分化不同阶段脂肪细胞中SPHK基因表达水平。结果1．SPHK基因在3T3-L1脂肪前体细胞诱导分化初期表达呈明显上调趋势；2．随着脂肪前体细胞分化成熟，该基因表达水平明显低于诱导分化初期的基因表达水平。结论 SPHK基因可能参与脂肪细胞分化的调控过程，与肥胖发生有一定联系。     

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.     

解偶联蛋白4基因在3T3-L1脂肪细胞诱导分化中的表达水平

目的　观察 3T3 L1脂肪前体细胞诱导分化过程中解偶联蛋白 4 (UCP4 )基因表达水平变化 ,探讨UCP4基因与肥胖发生之间的关系。方法　体外培养 3T3 L1细胞 ,诱导细胞分化 ,采用RT PCR技术在细胞分化成熟的不同时段检测脂肪细胞中UCP4基因mRNA表达水平。结果　UCP4基因高表达于 3T3 L1脂肪前体细胞中 ,随细胞分化成熟该基因表达水平渐下调。UCP4基因表达水平在诱导分化前 (d - 1)至d0、d0～ 3、d4～6、d7～ 8、d9～ 10内无显著性差异 (P >0 .0 5 ) ,余各时段表达水平均有显著差异 (P均 <0 .0 1)。结论　UCP4基因与肥胖发生相关 ,其在 3T3 L1细胞分化过程中表达逐渐下调可能有利于脂肪细胞的脂质积聚     

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。     

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.     

LYRM1基因沉默对成熟脂肪细胞线粒体形态及其相关基因的影响

目的 探讨LYRM1基因沉默对成熟脂肪细胞线粒体形态及相关基因的影响.方法 以3T3-L1前体脂肪细胞为工具细胞,建立LYRM1基因沉默细胞株,以转染空载质粒的3T3-L1脂肪细胞作为阴性对照,将前体脂肪细胞诱导分化为成熟脂肪细胞,采用油红O染色观察脂肪细胞分化状态,采用透射电镜观察成熟脂肪细胞的线粒体形态,采用荧光定量PCR技术检测成熟脂肪细胞中线粒体融合基因(Mfn) 1/2 mRNA、线粒体动态相关基因(Drp)1 mRNA、线粒体分裂基因(Fis)l mRNA的表达水平.结果 1.油红O染色诱导分化第8天的脂肪细胞,发现85％以上脂肪细胞已分化为成熟脂肪细胞,细胞形态大而圆,胞质含大量被油红O染成亮红色的脂滴,比较发现2组细胞无论脂滴的大小、数量均无显著差异.2.透射电镜观察发现2组细胞的线粒体形态基本正常,线粒体无明显肿胀、皱缩,线粒体嵴清晰可见,2组细胞间线粒体数目无明显差异.3.LYRM1基因沉默成熟脂肪细胞的Mfn2 mRNA表达水平显著高于对照组成熟脂肪细胞.4.LYRM1基因沉默成熟脂肪细胞的Mfnl mRNA、Drpl mRNA和Fisl mRNA表达水平与对照组成熟脂肪细胞的表达水平无明显差异.结论 LYRM1基因沉默能够引起成熟脂肪细胞Mfn2 mRNA表达水平升高,对线粒体形态结构并无显著影响,提示LYRM1基因沉默可部分影响成熟脂肪细胞线粒体形态相关基因.     

Familial partial trisomy of the long arm of chromosome 3 (3q).

A case of partial trisomy of the long arm of chromosome 3 (3q21 leads to qter) is described. The clinical findings are compared with those in 5 previously reported cases. There is hirsutism and characteristic facial dysmorphism, the common features of which are a square-shaped face, prominent nasal bridge, everted nostrils, hypertelorism, and palate abnormalities; occurring less often are abnormalities of vertebrae, thorax, and digits, or cardiovascular, urinogenital, and central nervous system. New features noted in this present case are absence of right eye from orbit and spina bifida. The spectrum of this syndrome is discussed, with possible relation to the degree of trisomy. The present case is the 6th to be reported with partial trisomy of the long arm of chromosome 3.     

Expression of homeotic genes Hoxa3, Hoxb3, Hoxd3 and Hoxc4 is decreased in the lungs but not in the hearts of adriamycin-exposed mice

Exposure of rat and mouse embryos to adriamycin (doxorubicin chlorhydrate) induces esophageal atresia (EA) and VACTERL association. Sonic hedgehog (Shh) and Gli2/Gli3 pathways are involved in these conditions and knockout mice for homeotic Hox genes Hoxa3, Hoxb3, Hoxc3, Hoxc4 and Hoxa5 show phenotypes with some of the associated VACTERL features. This study aims at evaluating the possible influence of Hoxa3, Hoxb3, Hoxd3 and Hoxc4 as upstream regulators of this complex signalling. Pregnant mice were exposed either to 4 mg/kg of adriamycin (EA group) or vehicle (controls) on embryonic days 7.5 and 8.5. Embryos were recovered at four endpoints (E12.5–E15.5) and randomly assigned for immunohistochemical or molecular biology studies. Lungs and hearts were separately harvested and processed for Hoxa3, Hoxb3, Hoxd3 and Hoxc4 quantitative RT-PCR measurements. Antibodies for Hoxa3, Hoxb3 and Hoxd3 proteins were used for immunohistochemical studies. RT-PCR studies showed a drastic and statistically significant decrease of the four genes in the lungs of EA mice when compared to controls, with a slight recovery from E15.5. Hearts of both groups showed a similar expression of all the genes throughout gestation. Control embryos expressed the hox3 paralogous genes in heart, skin, foregut derivatives and their surrounding mesoderm through E12.5–E15.5 whereas adriamycin-exposed embryos showed a severe decrease in expression of these three proteins in the same tissues but not in the heart. Adriamycin drastically reduced the expression of Hoxa3, Hoxb3, Hoxd3 and Hoxc4 in mice embryonic lungs. Their expression in the heart did not seem to be influenced by adriamycin in this experimental setting. Supported by #03/0892 FIS (Fondo de Investigación Sanitaria) and FMMM grants and research fellowship from the Hospital Universitario La Paz.     

T-Lymphocytes bearing the γδ T-cell receptor in cutaneous lesions of langerhans' cell histiocytosis

The aim of this study was to investigate the regional importance of γδ T cells in cutaneous lesions of Langerhans' cell histiocytosis. Six cases of Langerhans' cell histiocytosis were investigated by immunohistochemical techniques (alkaline phosphatase-antialkaline phosphatase complex and indirect immunoperoxidase). Increase of γδ T cells was observed in 3 cases of Langerhans' cell histiocytosis. In these cases up to 30% of CD3⁺ cells stained with an anti-TCR γδ monoclonal antibodies and in two of them γδ T cells showed a marked epidermotropism. In the specimens of the remaining three cases γδ T cells were found in an overall percentage of 5% of CD3⁺ cells, but in two cases a significant increase of epidermal γδ T cells was observed. The finding of numerous γδ T cells in Langerhans' cell histiocytosis is provocative and supports the suggestion of a functional relationship between γδ T cells and LCH cells. © 1993 Wiley-Liss, Inc.     

Partial deletion of the short arm of chromosome 3

A case of deletion of the short arm of chromosome 3 (46,XY,del(3)(p253) is described. The patient is a youth of 18 years in an institution for the mentally retarded. Phenotypically, he presents congenital heart disease, hypertelorism, ptosis, epicanthus, blepharophimosis, strabismus, nystagmus, synophrys, low-set ears, frequent infections, epilepsy (abnormal EEG and grand mal seizures), rocker bottom feet, flat occiput and muscular hypotonia. The parents are healthy and with normal karyotypes. A silent allele in the GPT system was found in the mother, the propositus and 4 of the 5 siblings.     

Trisomy for the distal part of the short arm of chromosome 3

A new case is presented of partial trisomy 3p in a one-year-old mentally retarded female infant with characteristic craniofacial dysmorphism and rare-faction of the stroma of the iris. The partial trisomy resulted from paternal balanced translocation t(3;6)(p25;p25). A review of the literature revealed that 1. both sexes are equally affected; 2. holoprosencephaly, found in 4 of 45 cases, may be considered the major and most severe anomaly of this syndrome; 3. the life-span of partial trisomy 3p is shorter than generally believed as most patients with severe malformations probably die before karyotype studies are initiated.     

MR imaging of the neonatal brain at 3 Tesla

3 Telsa MR scanners are now becoming more widely available and 3 Telsa is likely to become the filed strength of choice for clinical imaging of the brain. The neonatal brain can be safely and successfully imaged at 3 Telsa. The improved signal to noise afforded by a higher field strength may be used to improve image quality or shorten acquisition times. This may be exploited for conventional T1 and T2 weighted imaging and also for advanced techniques such as diffusion tensor imaging, angiography and functional magnetic resonance studies.