过表达Grx1抑制HEK293T细胞中H2O2诱导的p38MAPK信号通路

谷氧还蛋白1（glutaredoxin1,Grx1）是细胞内一种重要的巯基 二硫键氧化还原酶,在细胞内氧化还原状态的调控及抵抗氧化应激损伤过程中发挥重要作用.为进一步探讨Grx1的抗氧化机制,本实验将重组质粒pcDNA3.1(+)-hGrx1瞬时转染HEK293T细胞,经RT-PCR和Western印迹验证,细胞转染后实现了Grx1的过表达;以不同浓度H₂O₂为损伤因素,建立细胞氧化应激模型,检测过表达Grx1后细胞存活率,丙二醛（MDA）含量,超氧化物歧化酶（SOD）活力和乳酸脱氢酶（LDH）漏出率的变化,观察过表达Grx1后细胞的抗氧化能力;用终浓度100 μmol/LH₂O₂作用于细胞,利用Western 印迹检测120 min内HEK293T细胞中p38MAPK磷酸化水平.实验结果表明,HEK293T细胞过表达Grx1后,缓解了细胞的氧化应激损伤;转染空载体组细胞p38MAPK磷酸化水平在H₂O₂刺激后5 min开始升高,15 min达到最高值,并可维持至120 min左右;而过表达Grx1组细胞p38MAPK磷酸化水平在H₂O₂刺激后各时间段没有明显改变,提示Grx1通过抑制H₂O₂诱导的p38MAPK信号通路激活发挥其抗氧化作用.     

番茄红素对血管内皮细胞氧化应激损伤的作用及机制研究

目的:观察番茄红素(lycopene,LYC)对于血管内皮细胞功能的作用,探讨其作用机制。方法:人脐静脉内皮细胞(HUVECs)处理实验分组:对照组,H2O2组,H2O2+LYC组(1、2、4、8μmolL-1)。MTT法检测HUVECs存活率;免疫印迹法(Western blot)检测p38MAPK蛋白磷酸化水平、抗凋亡蛋白B淋巴细胞/白血病-2(bcl-2)及线粒体凋亡通路相关蛋白bax的表达;细胞黏附能力测定和伤口愈合实验检测HUVECs粘附率和迁移率;TUNEL法检测HUVECs凋亡率;ELASA法测定HUVECs内活性氧(ROS),超氧化物歧化酶(SOD),乳酸盐脱氢酶(LDH)释放量和caspase-3的活性。结果:H2O2损伤后HUVECs存活率显著降低(P0.01),凋亡率显著增加(P0.01),黏附和迁移能力显著降低(P0.01),bax和p-p38MAPK的表达上调,bcl-2的表达下调,并且ROS、LDH的释放和caspase-3的活性增加(P0.01),SOD的释放减少。而LYC的预处理可以明显逆转H2O2以上作用。结论:H2O2氧化应激损伤中,LYC保护内皮细胞可能与其抗过氧化损伤细胞凋亡,抑制异常的p38MAPK信号通路有关。     

高氧胁迫下Peroxiredoxin2蛋白的细胞保护作用的研究

衰老和凋亡是细胞的两个重要生理过程,一直以来都是细胞生物学领域研究的热点。Peroxiredoxin 2(Prdx2)蛋白是过氧化物酶的其中一个亚型,分布于细胞质中。为了研究它在高氧条件诱导的细胞衰老及凋亡中的保护作用,我们分别将大鼠来源的Prdx2基因转染进人间充质干细胞(Human mesenchymal stem cells,hMSCs)和HEK293T细胞中,并建立了稳定表达Prdx2蛋白的HEK293T细胞系,利用SA-β-gal染色(Senescence-Associated β-Galactosidase Assay)、TUNEL染色及磷酸化p53蛋白的免疫印迹来检测高氧处理后细胞的衰老和凋亡情况。实验结果表明,高氧处理细胞后,转染了Prdx2的hMSCs和HEK293T细胞其衰老和凋亡率与对照组相比都有较为明显的减少,暗示Prdx2蛋白在细胞抵抗氧化损伤中发挥了重要作用。     

早老素通过下调CDK4使HEK293T细胞周期阻滞

早老素（progerin）的累积导致儿童早老症（Hutchinson Gilford progeria syndrome, HGPS）的发生,并与正常衰老相关。早老素能使细胞内稳态失衡但分子机制仍有待深入研究。本研究旨在探讨早老素导入人胚胎肾293T细胞（human embryo kidney 293T cell, HEK293T）后细胞增殖、周期变化的分子机制。形态学观察发现过表达早老素的HEK293T细胞密度下降,(57±2.47)%细胞核形态皱缩。细胞增殖和周期实验证明早老素使细胞增殖减慢,发生G1/S期阻滞,G1细胞从 (42.3±1.31)%升至(47.2±1.26)%,而S期细胞从 (43.1±1.36)%降至 (38.5±1.42)%。Western印迹结果显示早老素的高表达引起p21蛋白表达上调（103.2±1.49）%,CDK4下调（63±1.52）%,而p53、ATM、CyclinE1以及p16等蛋白质水平均不变;HEK293T细胞中早老素的过表达导致γ H2AX水平下调（53±1.36）%,H2O2处理后变化趋势不变。我们的研究结果提示,早老素通过上调p21和下调CDK4使细胞发生周期阻滞,不能增加HEK293T细胞的损伤及衰老。     

低氧预处理通过上调钙网蛋白表达减轻大鼠心肌细胞氧化应激损伤

本文旨在探讨钙网蛋(calreticulin,CRT)是否参与低氧预处理(hypoxic preconditioning,HPC)对心肌细胞氧化应激损伤的保护及其信号转导过程.将原代培养的Sprague.Dawley乳鼠心肌细胞随机分为8组:氧化应激(H2O2)组、短暂低氧(HPC)组、HPC H202组、SB203580(p38 MAPK特异性抑制剂) HPC H2O2组、干扰心肌细胞CRT表达的反义寡核苷酸(antiscnse oligodeoxynucleotides,AS)组、AS H2O2组、AS HPC H202组和对照组,以细胞存活率、乳酸脱氢酶(1actate dehydrogenase,LDH)漏出及流式细胞术检测细胞损伤情况;采用RT-PCR和Western blot分别检测CRT表达和p38MAPK磷酸化水平.结果表明:(1)HPC可减轻氧化应激损伤,与H202组比较,HPC H2O2组细胞存活率增高18.0%,细胞凋亡率和LDH漏出分别降低19.4%和53.0%(均P<0.05);HPC前以SB203580预孵育可消除HPC保护作用,与HPC H202组相比,SB203580 HPC H2O2组细胞凋亡率和LDH漏出分别增高13.1%和96.0%,存活率降低7.3%(均P<0.05);(2)氧化应激明显上调CRT表达(H202组较对照组高7.1倍,P<0.05);HPC也诱导CRT表达上调(HPC组较对照组高2.4倍,P<0.05),但上调程度较H2O2组低59%(P<0.05);即HPC可减轻氧化应激诱导的CRT过表达:(3)AS干扰CRT表达后,HPC保护作用降低,相关性分析显示HPC诱导的CRT适度表达与细胞存活率呈正相关(r=0.8023,P<0.05);(4)HPC前SB203580预孵育可抑制CRT表达上调(分别较HPC H2O2组和HPC组低75%和53%,均P<0.05).上述结果提示,HPC可能通过p38 MAPK信号途径诱导CRT表达上调,减轻心肌细胞氧化应激损伤.     

Smad7基因对胞外信号调节激酶磷酸化水平的调控

研究在BEP2D细胞中,作为Smads蛋白家族的抑制分子,Smad7对胞外信号调节的蛋白激酶(ERK1／2或p44／42)磷酸化水平的调控。将Smad7真核表达载体或人工合成的Smad7-siRNA转染BEP2D细胞,TGF—β刺激,通过Western印迹检测Smad7对p44／42蛋白磷酸化的影响。结果在永生化BEP2D细胞中,TGF-β1刺激后5min开始,可以检测到磷酸化的p44／42;到60min达到高峰,之后逐渐降低。细胞转染Smad7,TGF-β作用60rain后,p44／42磷酸化水平明显增高;而转染Smad7-SiRNA,TGF-β作用60min后,p44／42磷酸化水平显降低。p44／42蛋白水平基本上不受TGF—β1刺激及Smad7表达水平的影响。以上结果说明,在BEP2D细胞中,Smad7可参与TGF—β对ERK／MAPK通路的活化作用。     

α1, 2-岩藻糖转移酶基因转导对人卵巢癌细胞RMG-I p38MAPK信号通路介导的凋亡的影响

以α1,2-岩藻糖转移酶基因转染前后卵巢癌细胞RMG-I、RMG-I-H为细胞模型,用细胞免疫荧光方法检测转染前后细胞p38MAPK和p-p38MAPK的细胞内定位,RT-PCR和Western blot方法从mRNA和蛋白质两个水平检测转染前后细胞p38MAPK表达的变化;以兔抗人IgG抗体处理组为对照,分别利用RT-PCR和Western blot方法检测Lewisy单克隆抗体处理前后RMG-I-H细胞p38MAPK mRNA和蛋白质表达水平的变化;以0.1%DMSO为对照,用流式细胞仪(FCM)检测p38MAPK特异性抑制剂SB203580处理后RMG-I-H凋亡比率的变化,并利用RT-PCR和Western blot方法检测caspase-3的mRNA和蛋白质水平的变化;用RT-PCR方法检测卡铂和SB203580处理后p38MAPK及caspase-3表达的变化.结果表明,RMG-I与RMG-I-H的p38MAPK蛋白主要定位在细胞质,p-p38MAPK蛋白定位在细胞核,转染后p38MAPK的mRNA水平明显高于转染前(P<0.05);Lewisy单克隆抗体处理后RMG-I-H细胞p38MAPK m...     

伯氏肩孔南极鱼dusp1基因在冷应激中的功能研究

为了研究极地鱼类双特异性磷酸酶1 (dual-specificity phosphatase 1, dusp1)基因在低温胁迫下的作用,实验采用RT-PCR技术从Trematomus bernacchii中克隆获得了编码区含有1128个核苷酸的dusp1同源基因,可编码376个氨基酸残基。将其通过同源重组的方法构建真核表达载体pcDNA3.1-dusp1并转染至人胚肾293T(HEK293T)细胞中,同时以pcDNA3.1空载质粒作为对照。使用荧光探针DCFH-DA检测了细胞活性氧(Reactive oxygen species assay, ROS)含量,采用流式细胞术检测了低温胁迫下细胞的存活率,采用Western Blot检测了P38/MAPK的磷酸化水平和RT-qPCR技术分析了半胱氨酸天冬氨酸蛋白酶3(caspase-3)的mRNA表达水平。结果表明,伯氏肩孔南极鱼dusp1基因能在293T细胞中大量表达,并定位于细胞核;在低温胁迫下,与对照组相比,伯氏肩孔南极鱼dusp1基因的过表达能显著减少细胞ROS的含量和细胞凋亡率,并抑制促凋亡基因P38/MAPK的过度磷酸化和凋亡效...     

PARP-1对高糖诱导的心肌细胞增殖的影响及机制研究

目的:研究PARP-1对高糖诱导的心肌细胞增殖的影响及可能机制。方法:用高糖处理H9C2细胞,qRT-PCR和Western blot检测细胞中PARP-1 m RNA和蛋白水平。H9C2细胞转染PARP-1 si RNA和si RNA control,q RT-PCR和Western blot检测细胞中PARP-1 m RNA和蛋白水平。用高糖处理转染PARP-1 si RNA后的H9C2细胞,CCK-8检测细胞增殖情况,硫代巴比妥酸法检测丙二醛(MDA)水平,黄嘌呤氧化酶法检测超氧化物歧化酶(SOD)水平,Western blot检测增殖细胞核抗原(PCNA)、p38丝裂原活化蛋白激酶(p38MAPK)、磷酸化的p38MAPK(p-p38MAPK)蛋白的表达。结果:高糖诱导的H9C2细胞中PARP-1 m RNA和蛋白水平明显高于正常培养的H9C2细胞(P0.05)。PARP-1 si RNA能够明显下调H9C2细胞中PARP-1 m RNA和蛋白水平。高糖处理后H9C2细胞存活率明显降低,细胞中MDA水平升高,细胞中SOD水平降低,细胞内的PCNA水平降低,p38MAPK磷酸化水平升高,与正常培养的H9C2细胞相比,差异均具有统计学意义(P0.05)。用高糖培养下调PARP-1的H9C2细胞,细胞存活率有所升高,细胞中MDA水平降低,细胞中SOD水平也升高,细胞中PCNA水平升高,细胞中p38MAPK磷酸化水平降低,与单纯高糖培养的细胞相比,差异均具有统计学意义(P0.05)。结论:PARP-1在高糖诱导的心肌细胞中表达上调,可能通过激活p38MAPK信号途径,增加细胞脂质氧化应激抑制心肌细胞增殖。     

结核分枝杆菌38kD蛋白真核表达载体的构建及其在HEK293T细胞中的分泌性表达

目的:构建含结核分枝杆菌38kD蛋白基因的真核表达载体并转染HEK293T细胞,高效表达分泌性38kD蛋白。方法:设计合成的结核分枝杆菌38kD基因被克隆到T载体,然后亚克隆到真核表达载体pcDNA3.0,经酶切鉴定正确后,PEI转染法导入293T细胞,换不含血清的DMEM培养基培养3 d后收集细胞及上清,采用Western印迹检测38kD蛋白的表达。结果:酶切结果显示,获得正确的含38kD基因的重组表达载体;Western印迹结果显示表达载体导入293T细胞中后能在细胞及上清中检测到38kD蛋白表达。结论:构建了含重组结核分枝杆菌38kD蛋白基因的真核表达载体pcDNA3.0-38kD,该载体可在哺乳动物细胞HEK293T中高效分泌性表达38kD蛋白,为结核病诊断试剂盒研发奠定了基础。     

BCL2-CISD2

CISD2, an ER BCL2-associated autophagy regulator also known as NAF-1, is responsible for the human degenerative disorder Wolfram Syndrome 2. In order to interrogate the physiological role of CISD2 we generated and characterized the Cisd2 gene deletion in mice. Cisd2 null mice manifest significant degeneration in skeletal muscle tissues, which is accompanied with augmented autophagy, dysregulated Ca²⁺ homeostasis and elongated mitochondria. Our findings describe a novel role for BCL2-CISD2 in the homeostatic maintenance of skeletal muscle. It remains to be elucidated how and if the antagonism of the BECN1 autophagy-initiating complex and modulation of ER Ca²⁺ homeostasis by BCL2-CISD2 are interconnected.     

Synthesis of [11-2H2], [8-2H2], [7-2H2], [6-2H2], [5-2H2], [4-2H2] and [3-2H2] cis-9-octadecenoates

Deuterated oleates have been synthesized by semihydrogenation of acetylenic intermediates. [11-²H₂]Oleate was prepared by two-carbon chain extension of the C₁₆ alcohol obtained from [1-²H₂]octyl bromide and 7-octyn-1-ol. [8-²H₂] and [7-²H₂]oleates were both prepared from dimethyl suberate, tetradeutero intermediate C₁₆ alcohols were synthesized from [1,8-²H₄] and [2,7-²H₄]octane diols by monobromination, conversion to deuterated 9-decyn-1-ols and reaction with octyl bromide. Oxidation gave [8-²H₂]-9-octadecynoate and [2,7-²H₂]-9-octadecynoate, after semihydrogenation of the latter, deuterons at C-2 were removed by exchange with aqueous alkali. [6-²H₂] and [5-²H₂]oleates were obtained from methyl 5-tetradecynoate, semihydrogenation, deuterium exchange at C-2 and two malonate extensions gave [6-²H₂]oleate; reduction with lithium aluminum deuteride, two malonate extensions and semihydrogenation gave the [5-²H₂] ester. [4-²H₂] and [3-²H₂]oleates were both obtained from methyl 7-cis-hexadecenoate, exchange of the α protons and chain extension gave the [4-²H₂] ester and reduction with lithium aluminum deuteride and chain extension gave the [3-²H₂] ester.     

Synthesis and physicochemical properties of 2'-deoxy-2',2'-difluoro-beta-D-ribofuranosyl and 2'-deoxy-2',2'-difluoro-alpha-D-ribofuranosyl oligonucleotides

We present procedures for nucleoside and oligonucleotide synthesis, binding affinity (Tm) and structural analysis (CD spectra) of 2'-deoxy-2',2'-difluoro-alpha-D-ribofuranosyl and 2'-deoxy-2',2'-difluoro-beta-D-ribofuranosyl oligothymidylates. Possible reasons for the thermal instability of duplexes formed between these compounds and RNA or DNA targets are discussed.     

Stereospecific 2'-amination and 2'-chlorination of adenosine by Actinomadura in the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin

2'-Amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin (2'-CldCF) are two nucleoside antibiotics produced by Actinomadura. The biosynthesis of these two nucleoside antibiotics has been studied by the addition of [U-14C]adenosine with or without unlabeled adenine to cultures of Actinomadura. By this experimental approach, it is possible to demonstrate that adenosine is the direct precursor for the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-CldCF. These conclusions are based on the observation that the percentage distribution of 14C in the aglyconic and pentofuranosyl moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were similar to the distribution of 14C in the adenine and ribosyl moieties of the [U-14C]adenosine (i.e., 48:52) added to cultures of Actinomadura. Experimentally, the percentage distribution of 14C in the (i) adenine:2-amino-2-deoxy-beta-D-ribofuranose of 2'-amino-2'-deoxyadenosine is 51:49; (ii) 8-(R)-3,6,7,8-tetrahydroimidazo[4,5-d]-[1,3-diazepin-8-o1]:2 -chloro-2- beta-D-ribofuranose of 2'-CldCF is 45:55; and (iii) adenine:ribose of the adenosine isolated from the RNA of Actinomadura is 42:58. Further proof that adenosine is the direct precursor for the biosynthesis 2'-amino-2'-deoxyadenosine and 2'-CldCF was demonstrated by the addition of 75 mumol of unlabeled adenine together with [U-14C]adenosine to nucleoside-producing cultures of Actinomadura. The percentage distribution of 14C in the aglycon and the sugar moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were 46:54 and 47:53, respectively; the percentage distribution of 14C in the adenine and ribose moieties of the adenosine isolated from the RNA of Actinomadura was 51:49. These data show that the hydroxyl on C-2' of the ribosyl moiety of adenosine undergoes a replacement by a 2'-amino or a 2'-chloro group to form 2'-amino-2'-deoxyadenosine or 2'-CldCF with retention of stereconfiguration at C-2'. Finally, Actinomadura can utilize inorganic chloride from the medium as demonstrated by the isolation of [36Cl]2'-CldCF following the addition of [36Cl]chloride to the culture medium. Mechanisms for the regioselective modification of the C-2' hydroxyl group and stereospecific insertion of the amino and chloro groups are discussed.     

Stereospecific formation of alpha-hydroxycarboxylato oxo peroxo complexes of vanadium(V). Crystal structure of (NBu4)2[V2O2(O2)2(L-lact)2] x 2H2O and (NBu4)2[V2O2(O2)2(D-lact)(L-lact)] x 2H2O

An overview of structurally characterized alpha-hydroxycarboxylatodioxo- and alpha-hydroxycarboxylatooxoperoxovanadates(V) is presented and the geometric parameters of the V2O2 bridging core are discussed. The first case of a stereospecific formation of oxoperoxovanadates(V) is reported: The crystal structures of the isomeric compounds (NBu4)2[V2O2(O2)2(L-lact)2] x 2H2O and (NBu4)2[V2O2(O2)2(D-lact)(L-lact)] x 2H2O (lact = C3H4O3(2-), the anion of the lactic acid) differ mainly in the arrangement of the V2O2 core and in mutual orientation of the V=O bonds. The complexes with achiral ligands adopt the same structural type as the complexes formed from a racemic mixture of a chiral ligand, while the structure obtained using an enantiopure L,L-hydroxycarboxylate is different.     

Stereoselective Synthesis of 2-Amino-2-deoxy-d-arabinose and 2-Deoxy-d-ribose

An efficient method for the stereoselective synthesis of 2-amino-2-deoxy-d-arabinose and 2-deoxy-d-ribose is described.

The key step in this method was accomplished by the nucleophilic addition of methyl isocyanoacetate to 2,3-O-isopropylidene-d-glyceraldehyde with high erythro-selectivity (nearly 100%).

Subsequent intermolecular cyclization predominantly gave the desired oxazoline derivative (trans-form), in which two new chiral centers were formed. The oxazoline derivative was efficiently converted to both 2-amino-2-deoxy-d-arabinose and 2-deoxy-d-ribose.