槲皮素对节律钟基因的调控研究*

目的:探讨槲皮素对节律钟基因表达的影响。方法:通过50%的马血清刺激诱导人骨肉瘤U2OS细胞同步化,利用槲皮素处理同步化后的U2OS细胞。进一步利用荧光定量PCR检测节律钟关键基因的变化。利用western blot检测槲皮素对组蛋白乙酰化的影响,并且通过试剂盒测定槲皮素对细胞内氧化型烟酰胺腺嘌呤二核苷酸(NAD+)的影响。通过尼克酰胺和槲皮素同时处理U2OS细胞,利用荧光定量PCR检测节律钟关键基因的变化。结果:U2OS细胞经过槲皮素处理后节律钟关键基因芳烃受体核转位蛋白3(brain and muscle Arnt-like protein-1,BMAL1),节律周期蛋白2(Period 2, PER2),孤儿核受体alpha(REV-ERBα)和蓝光受体蛋白1(Cryptochrome 1,CRY1)在转录水平的表达水平有明显的升高。槲皮素处理可以显著降低U2OS细胞的组蛋白乙酰化的水平,并且显著升高U2OS细胞内的氧化型烟酰胺腺嘌呤二核苷酸的水平。进一步研究发现,尼克酰胺(Nicotinamide,NAM)处理完全抑制了槲皮素对节律基因的影响。结论:槲皮素显著地激活了节律钟关键基因的mRNA表达水平,槲皮素对于节律基因的调控依赖于Sirtuins的活性,其机制可能是由于槲皮素增加了细胞内的氧化型烟酰胺腺嘌呤二核苷酸的水平所导致。     

拟南芥中光信号系统中关键基因CRY1、CRY2和COP1启动子的表达模式分析

通过构建表达光信号系统关键基因CRY1、CRY2和COP1启动子与GUS融合基因的拟南芥转基因植株,并对转基因植株进行GUS组织化学染色的结果表明,CRY1、CRY2和COP1的表达模式不受光条件的调控,并且在各器官有广泛的表达。分别分析CRY1基因启动子在cop1突变体以及COP1基因启动子在cry1突变体遗传背景中表达模式的结果表明,CRY1和COP1在转录水平上不存在明显的相互调控关系。     

Toll样受体2与巨噬细胞极化在胰岛素抵抗中的作用

胰岛素由胰岛β细胞分泌,经胰岛素信号通路发挥作用。当机体肥胖或其他原因导致胰岛素信号通路受阻时,引起体内胰岛素抵抗(insulin resistance, IR),胰岛素抵抗与低度炎症关系密切。促炎因子,例如肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)、白细胞介素-1β(interleukin-1β, IL-1β)、白细胞介素-6(interleukin-6, IL-6)等可抑制胰岛素受体底物(insulin receptor substrate, IRS)酪氨酸磷酸化,发生丝氨酸磷酸化,导致胰岛素受体细胞或靶器官对葡萄糖的摄取和利用下降。Toll样受体2(Toll-like receptor 2, TLR2)是一种重要的模式识别受体,可与TLR1或TLR6结合形成二聚体,与炎症和胰岛素信号通路关系密切,TLR2通过髓系分化因子88(myeloid differentiation factor 88, MyD88)依赖途径激活核因子-κB(nuclear factor, NF-κB)和激活蛋白1(activator protein 1, AP-1),上调促炎基因的转录。巨噬细胞是天然免疫系统中重要一员,可参于体内促炎因子和抗炎因子的调节。TLR2于巨噬细胞表面表达。在脂肪酸(fatty acids)的诱导下,TLR2通过上调促炎基因使巨噬细胞向M1表型极化,M1表型巨噬细胞分泌促炎因子,下调胰岛素靶器官对胰岛素的敏感性。本文拟对TLR2基因和巨噬细胞极化对胰岛素抵抗的影响,以及三者的相关性做一简要综述,从分子水平探讨胰岛素抵抗的发生机制,为胰岛素抵抗的相关研究提供理论参考。     

时钟节律与细胞周期

昼夜节律和细胞周期是生命有机体中两种主要的节律性、周期性的活动,参与机体代谢与生理节律．在分子水平上,它们的周期性活动是由一种周期性振荡的网络构成的,这种网络由一系列节律性表达的蛋白所形成．研究发现,多种节律因子通过调节周期蛋白的表达影响细胞周期进程,如G ₁-S期,REV-ERBa抑制p21促进细胞进程,RORα激活p21抑制细胞进程,DEC1抑制cyclinD1,CLOCK/BMAL1负调控c-Myc;G ₂-M期,BMAL1/CLOCK、BMAL1/NPAS2或Cry1作用于Wee1抑制或激活G_2-M期进程．此外,昼夜节律钟蛋白也参与了DNA损伤修复及细胞死亡的过程：Per1、Tim分别作用于ATM、ATR,因而促进细胞周期停滞,p53缺失的细胞中敲除Cry促进细胞凋亡过程,抑制了肿瘤的形成,DEC1以p53依赖的方式促细胞衰老等．同时,节律因子的紊乱引起多种疾病的产生．因此,阐明昼夜节律对细胞周期及死亡的影响,将为肿瘤的治疗提供分子理论基础．     

人外周血淋巴细胞核心钟基因Clock和Bmal1的昼夜节律性表达

探讨自然光制下正常成年人外周血淋巴细胞钟基因Clock和Bmal1的昼夜节律性表达。用实时定量RT-PCR方法,测定不同昼夜时点(ZT)受试者外周血淋巴细胞总RNA中核心钟基因Clock和Bmal1的mRNA表达量,通过余弦法和Clock Lab软件获取节律参数,并经振幅检验分析是否存在昼夜节律。结果发现正常成年人外周血淋巴细胞钟基因Clock和Bmal1的mRNA表达呈昼夜节律性振荡(P0.05),Clock的峰时和谷时分别位于ZT13和ZT1,Bmal1的峰时和谷时分别位于ZT12和ZT24;两个基因在所检测的各个昼夜时点中表达水平均有明显差异(P0.05),Bmal1的表达水平较Clock降低;二者表达的峰值相位、振幅、峰时和谷时相一致(P0.05),但Bmal1转录的中值水平以及峰时mRNA水平和谷时mRNA水平降低(P0.05)。提示正常成年人外周血淋巴细胞钟基因Clock和Bmal1的表达存在同步化的昼夜节律性转录特征。     

节律基因CRY1在肝癌中的表达及对肝癌细胞生长的作用

目的:探讨节律基因CRY1(cryptochrome circadian clock 1)在肝癌组织中的表达情况及对肝癌细胞生长的作用。方法:利用实时荧光定量PCR、Western blotting及免疫组织化学染色法,检测CRY1在肝癌组织及细胞中的表达;利用肝癌公共数据信息,分析和验证CRY1在肝癌组织中的表达;真核表达质粒将肝癌细胞CRY1过表达后,采用MTS和Annexin V-FITC/PI双染法,分别检测CRY1对细胞增殖和凋亡的影响。结果 CRY1在癌组织中阳性率为57.5%(23/40),癌旁组织中阳性率为87.5%(35/40),癌中CRY1表达低于癌旁的占80%(32/40),与癌旁组织比较,CRY1蛋白在肝癌组织中的表达明显降低,差异有统计学意义(t=7.53,P0.001)。CRY1在3个肝癌公共数据信息显示,与对应癌旁组织相比,其在癌组织中表达水平显著降低,差异均具有统计学意义(TCGA:413.62 vs 335.17,P=0.0155;GSE22058:397.67 vs 360.08,P=0.0207;GSE25097:1.627 vs 1.433,P0.0001)。同时CRY1在被检测的4株肝癌细胞系中表达也显著低于永生化的正常肝细胞。与对照组比,过表达CRY1组细胞增殖能力明显降低,两组间差异有统计学意义(1.100 vs 0.662,P0.0001);与对照组比,过表达CRY1组细胞凋亡显著增加(凋亡细胞百分比:7.9 vs 17.5,P=0.0014)。结论:节律基因CRY1在肝癌中低表达,同时CRY1抑制肝癌细胞增殖和促进细胞凋亡。     

乙型肝炎病毒表面抗原对细胞脂质合成作用的研究

乙型肝炎病毒(hepatitis B virus,HBV)合成的蛋白调节细胞脂质代谢的研究不断被报道,但乙型肝炎病毒表面抗原(hepatitis B virus surface antigen,HBsAg)与脂质代谢的相互调控研究较少,且机制尚不明确。本研究通过对细胞转录组学的分析,揭示HBsAg对脂质代谢的调控机制。选用稳定表达HBsAg的细胞系HepG2-S-G2与其对照细胞系HepG2-neo-F4进行转录组学分析。利用定量聚合酶链反应(polymerase chain reaction,PCR)、蛋白质印迹法(Western blot,WB)分别检测重要差异基因OXCT1和CYP4F3在mRNA水平和蛋白水平的表达差异。为验证HBsAg促进脂质合成上调的表型,对两种细胞系进行油红O染色并检测细胞脂肪酸、总胆固醇水平。进一步对稳定转染HBV的细胞系HepG2.2.15进行降脂处理,以观察细胞上清液中HBsAg与脂质合成之间是否存在相互调控。结果显示,参与脂质代谢的差异基因发生显著变化,提示HBsAg引起了宿主细胞脂质合成途径的上调和消耗途径下调。定量PCR结果显示,相对于HepG2-neo-F4细胞,HepG2-S-G2细胞的3-酮酸辅酶A转移酶1(3-oxoacid CoA-transferase 1,OXCT1)mRNA水平升高约9倍,与转录组测序结果基本一致;CYP4F3基因在HepG2-S-G2细胞中转录相对下调。 WB结果显示,OXCT1和CYP4F3蛋白表达均出现相应的显著上调或下调,并且趋势与转录组分析一致。油红O染色以及细胞脂肪酸、总胆固醇水平检测结果证实HepG2-S-G2细胞中脂滴更明显,且游离脂肪酸和总胆固醇均显著升高。降脂处理结果显示细胞上清液中HBsAg显著降低。上述结果表明,HBsAg可上调脂质代谢、促进脂质合成,提示降脂可能成为抑制HBsAg的潜在有效途径。     

白细胞介素22对T细胞介导的小鼠肝损伤的治疗作用及初步机制探讨

目的研究人白介素22（IL-22）对T细胞介导的肝损伤小鼠的治疗作用。方法利用刀豆蛋白A（ConA）建立T细胞介导的肝损伤小鼠模型,检测静脉注射IL-22对肝损伤小鼠血清丙氨酸转氨酶（ALT）、天门冬氨酸转氨酶（AST）活性的影响,同时取小鼠肝组织进行病理学检查。并采用半定量RT-PCR方法检测IL-22刺激HepG2和LO2细胞后,对c-myc及Bcl-2基因转录表达水平的影响。结果IL-22明显降低ConA致小鼠急性肝损伤血清ALT、AST值的升高,减轻ConA对肝组织的病理损伤。体外检测IL-22对HepG2和LO2细胞表达c-myc及Bcl-2基因转录水平有促进作用。结论IL-22对T细胞介导的肝损伤小鼠模型具有治疗作用,该作用可能是通过IL-22促进肝细胞的抗凋亡因子的表达实现的。     

RNA干扰技术抑制内源性绿色荧光蛋白的表达

目的建立稳定表达绿色荧光蛋白(GFP)的细胞株;构建短发夹RNA(shRNA)表达质粒并观察其对内源性GFP的抑制作用。方法转染pEGFP-N1至HepG2细胞,利用G418筛选获得稳定表达GFP的细胞株(HepG2.GFP);设计合成针对GFP基因的siRNA对应的DNA片段,插入转录载体pTZU6 1,构建shRNA表达载体pSHGFP,转染HepG2.GFP,荧光显微镜观察细胞荧光强度,以western blot检测GFP蛋白水平,以RT-PCR检测mRNA水平。结果利用PCR方法从HepG2.GFP细胞基因组DNA中检测到GFP基因;pSHGFP能够显著抑制该细胞中GFP的表达。结论GFP基因成功整合至HepG2细胞基因组中,pSHGFP能够显著抑制内源性GFP的表达,该系统能够用于RNA干扰机制等研究中。     

Sal B对肝癌细胞株HepG2的促凋亡作用

目的揭示丹酚酸B(Salvianolic Acid B,Sal B)对肝癌细胞株HepG2的杀伤作用。方法用不同浓度的丹酚酸B处理HepG2细胞,37℃培养24h。用RT-PCR检测促凋亡基因Bax的转录水平,并用流式细胞术检测细胞凋亡的水平。结果①100μmol/L、50μmol/L、25μmol/L等浓度的Sal B处理都能使HepG2细胞促凋亡基因bax的转录水平升高,其中100μmol/L处理组最为明显。②不同浓度的Sal B处理都能使HepG2细胞发生凋亡,其中100μmol/L处理组最为明显。结论 Sal B有促进HepG2细胞凋亡的作用。     

酚酸类物质对三七幼苗的化感影响

该文研究了不同浓度的阿魏酸、对香豆酸、丁香酸、对羟基苯甲酸、香草酸5种酚酸类物质对三七幼苗生长和生理的影响。结果表明:处理后,三七幼苗的苗高、根长、可溶性蛋白质含量、根系活力、CAT以及POD活性均有所降低。其中,阿魏酸各处理组幼苗的苗高及POD活性均显著降低,50、100 mg·L~(-1)的对香豆酸以及100 mg·L~(-1)的香草酸处理组幼苗苗高也分别比对照显著降低16.19%、16.67%和29.29%;对香豆酸、丁香酸以及对羟基苯甲酸各处理组幼苗根长均显著低于对照;香草酸处理组幼苗的根系活力也显著低于对照,且幼苗的CAT活性在10、50、100 mg·L~(-1)丁香酸、对羟基苯甲酸以及香草酸处理下也达到了显著降低水平。此外,1 mg·L~(-1)阿魏酸以及100 mg·L~(-1)香草酸处理组幼苗的叶绿素含量也均显著降低;中高浓度的阿魏酸、对香豆酸、丁香酸、对羟基苯甲酸增加了三七幼苗的MDA含量,而香草酸在0.1、1、10、100 mg·L~(-1)浓度下显著降低幼苗的MDA含量;丁香酸、香草酸、对羟基苯甲酸以及中高浓度的对香豆酸增加了三七幼苗的SOD活性,且香草酸各处理组均达到了显著性水平。综上结果表明,5种酚酸类物质对三七幼苗均具有一定的化感抑制作用,但各酚酸物质的作用方式及强度并不完全一致,阿魏酸的化感影响较大,这为进一步研究三七的化感自毒作用提供了一定的理论参考。     

Effects of flaxseed,raw soybeans and calcium salts of fatty acids on apparent total tract digestibility,energy balance and milk fatty acid profile of transition cows

Oilseeds offer some protection to the access of ruminal microorganisms and may be an alternative to calcium salts of fatty acids (FA), which are not fully inert in the ruminal environment. This study aimed to evaluate the effects of different sources of FA supplementation on apparent total tract nutrient digestibility, milk yield and composition, and energy balance (EB) of cows during the transition period and early lactation. We compared diets rich in C18:2 and C18:3 FA. Multiparous Holstein cows were randomly assigned to receive one of the four diets: control (n=11); whole flaxseed (WF, n=10), 60 and 80 g/kg (diet dry matter (DM) basis) of WF during the prepartum and postpartum periods, respectively; whole raw soybeans (WS, n=10), 120 and 160 g/kg (diet DM basis) of WS during the prepartum and postpartum periods, respectively; and calcium salts of unsaturated fatty acids (CSFA, n=11), 24 and 32 g/kg (diet DM basis) of CSFA during the prepartum and postpartum periods, respectively. Dry cows fed WF had higher DM and net energy of lactation (NE_L) intake than those fed WS or CSFA. The FA supplementation did not alter DM and NDF apparent total tract digestibility, dry cows fed WF exhibited greater NDF total tract digestion than cows fed WS or CSFA. Feeding WS instead of CSFA did not alter NE_L intake and total tract digestion of nutrients, but increased milk fat yield and concentration. Calculated efficiency of milk yield was not altered by diets. FA supplementation increased EB during the postpartum period. Experimental diets increased long-chain FA (saturated and unsaturated FA) in milk. In addition, cows fed WS and CSFA had higher C18:1 trans-11 FA and C18:2 cis, and lower C18:3 FA in milk than those fed WF. Furthermore, cows fed CSFA had higher C18:1 trans-11 and cis-9, trans-11 FA than cows fed WS. Although supplemental C18:2 and C18:3 FA did not influence the milk yield of cows, they positively affected EB and increased unsaturated long-chain FA in milk fat.     

Identification of glutamine chain lengths of endogenous Folypoly-γ-glytamates in rat tissues

A simplified procedure for the determination of the glutamate chain lengths of endogenous tissue folate is described.Natural pteroylpoly-γ-glutamates in tissue extracts, irrespective of their one-carbon moiety, were converted by a reductive cleavage procedure to a homologous series of p-aminobenzoylpoly-γ-glutamates, differing only in glutamate chain length. Desalting and concentration of the extracts were achieved by absorbing the derivatives on active charcoal followed by their elution with ethanol: ammonia. Aminobenzoylpolyglutamates were separated by DEAE- cellulose chromatography and quantitated by a colorimetric procedure for primary aromatic amines.The major endogenous folates in rat liver and kidney were pteroylpentaglutamate derivatives with smaller amounts of pteroyltetra- and hexaglutamate.     

氨基酸L-和D-异构体对离体小麦胚植株生长的影响

5种氨基酸L-和D-异构体对离体小麦胚植株生长的影响结果表明,在1~7 mmol.L-1浓度范围内,脯氨酸异构体均不抑制植株生长;缬氨酸和蛋氨酸的L-型严重抑制生长,D-型不抑制或轻微抑制;丙氨酸则相反,L-型无抑制作用,D-型是严重抑制类型;丝氨酸的L-型轻微抑制生长,D-型严重抑制生长。生长结果也表明,DL-异构体的抑制作用介于D-型和L-型之间。     

Separation and simultaneous determination of nalidixic acid, hydroxynalidixic acid and carboxynalidixic acid in serum and urine by micellar electrokinetic capillary chromatography

Separation in capillary electrophoresis is governed by various factors, including buffer type, buffer concentration, pH, temperature, voltage and micelles. Through proper adjustment of these parameters, nalidixic acid and its two major metabolites, 7-hydroxynalidixic and 7-carboxynalidixic, could be separated by micellar electrokinetic capillary chromatography using an electrophoretic electrolyte consisting of 50 mM borate buffer (pH 9) containing 25 mM sodium dodecyl sulphate and 10% acetonitrile. A linear relationship between concentration and peak area for each compound was obtained in the concentration range 0.15–100 μg ml⁻¹, with a correlation coefficient greater than 0.999 and detection limits in the 0.2–0.7 ng ml⁻¹ range. Intra- and inter-day precision values of about 0.8–1.2% RSD (n=11) and 1.3–2.0% RSD (n=30), respectively, were obtained. The method has been applied to the analysis of nalidixic acid and its two major metabolites in serum and urine with limits of sensitivity lower than 0.8 ng ml⁻¹.     

氨基酸分析仪测定饲料添加剂——叶酸的方法

用６ｍｏｌ／Ｌ盐酸于１１０℃条件下水解饲料添加剂——叶酸,使之游离出谷氨酸,用氢氧化钠中和调节ｐＨ到２,氨基酸分析仪测定谷氨酸含量,经与标准叶酸水解样品比较,计算出叶酸的纯度。该方法重现性好,变异系数ＣＶ＝０．０８％,平均回收率为９８．３４％,浓度与峰面积呈线性相关,相关系数ｒ＝０．９９８７,可随氨基酸分析同时进行,不需改变任何分析条件。     

Characterization of Transgenic Rice Plants Expressing an Arabidopsis FAD7

Fatty acid -3 desaturase (FAD) is the key enzyme catalyzing the formation of trienoic fatty acids. We utilized an Arabidopsis FAD7 gene and the seven independent transgenic rice plants harbouring 1 to 3 copies of this gene were generated. The expression of FAD7 mRNA was different among independent transgenic lines regardless of the copy number. The total linolenic acid (18:3) contents reduced by about 7 – 32 % in transgenic rice plants but the linoleic acid (18:2) content increased accordingly. With or without wounding treatments, the jasmonate content was higher in transgenic lines than in wild-type rice plant. The transgenic lines overproducing jasmonate also showed increased expression of PR1b mRNA and allene oxide synthase inresponse to wounding.     

Achievements and challenges of sialic acid research

Sialic acids are one of the most important molecules of life, since they occupy the terminal position on macromolecules and cell membranes and are involved in many biological and pathological phenomena. The structures of sialic acids, comprising a family of over 40 neuraminic acid derivatives, have been elucidated. However, many aspects of the regulation of their metabolism at the enzyme and gene levels, as well as of their functions remain mysterious. Sialic acids play a dual role, not only are they indispensable for the protection to and adaptation of life, but are also utilised by life-threatening infectious microorganisms. In this article the present state of knowledge in sialobiology, with an emphasis on my personal experience in this research area, is outlined including a discussion of necessary future work in this fascinating field of cell biology.