Characteristic of β-glucosidase from Sicilian blood oranges in relation to anthocyanin degradation

华中科技大学2002年硕士研究生入学考试试题一、选择题1.膜蛋白的功能不包括（D）A.作为信号受体B.作为离子通道C.作为酶D.储藏能量2.蛋白聚糖中的糖包括（C）A.肽聚糖B.几丁质C.透明质酸D.葡聚糖3.蛋白质中提供糖基化位点的侧链来自下列哪一种氨基酸残基（C）A.甘氨酸B.天冬氨酸C.丝氨酸D.色氨酸4.鞘氨醇不是下列什么物质的组成成分（B）A.神经酰胺B.磷脂酰乙醇胺C.神经节苷脂5.在pH7 条件下电泳，下列什么物质会向阴极移动（B）A.糖胺聚糖B.磷脂酰胆碱C.多聚谷氨酸D.多聚腺苷酸6.动植物来源的脂肪酸多是（D）A.奇数脂肪酸B.20C-30C之间C.反式脂肪酸D.偶数脂肪酸7.下列哪一种试剂可以用于标记蛋白质的N端（B）A.甲醛B.丹磺酰氯C.二异苯基磷酸P385D.TPCK P3688.属于同一蛋白质家族成员，其氨基酸序列中的哪些氨基酸出现差异可能性最小（A）A.形成功能部位（活性部位）的氨基酸B.和底物专一性或配基专一性有关的氨基酸C.与种属来源有关的氨基酸9.认为肽键是平面结构的重要依据是（D）A.肽键的键长0.132nm，等于碳碳双键之长B.肽键的键长0.132nm，等于碳氮双键之长C.肽键的键长0.132nm，介于碳碳双键和碳碳单键之间D.肽键的键长0.132nm，介于碳氮双键和碳氮单键之间10.不属于还原糖的糖有（A）A.蔗糖B.麦芽糖C.葡萄糖D.乳糖11.提取细菌膜蛋白时使用去污剂（表面活性剂）的作用是（B）A.破坏二硫键B.破坏疏水作用，使非极性基团暴露于水中C.破坏离子键D.去掉细胞壁12.以下结合蛋白质多肽链与非肽链部分不以共价键相连的是（A）A.脂蛋白B.胶原蛋白，一种糖蛋白C.琥珀酸脱氢酶，一种含有FAD的黄素蛋白13.蛋白质不具有下述哪种功能（F）A.提供能量B.运输C.防御D.加速化学反应E.调节F.储存遗传信息14.下列哪种肽段易于形成α -螺旋（C）A.多聚甘氨酸B.多聚脯氨酸C.多聚（丙氨酸-缬氨酸）15.如果胰蛋白酶不能消化某种蛋白质，说明该蛋白质在组成上不含（C）A.甘氨酸B.缬氨酸C.精氨酸D.苯丙氨酸16.β转角中肽链主链出现（C）度回折A.80B.60C.180D.15017.判断2,3-二磷酸甘油酸是血红蛋白别构调节物的证据可能是该物质（A）A.使血红蛋白的氧合曲线更加S 型B.使血红蛋白的氧合曲线变成双曲线型C.自身与血红蛋白结合曲线为S 型18.NAD+/NADH+H+辅酶在反应中转移的电子应表示成（C）A.H-B.HC.2H19.阳光维生素的意思是阳光的能量使动物的某种维生素原变为维生素，这种维生素原是（B）A.β -胡萝卜素B.7-脱氢胆固醇C.视紫红质D.色氨酸20.TPCK 抑制胰凝乳蛋白酶的根本原因是（C）A.TPCK类似酶的底物B.TPCK与酶的丝氨酸残基反应，使该丝氨酸不能作为广义酸碱催化剂C.TPCK与酶的组氨酸残基反应，使该组氨酸不能作为广义酸碱催化剂D.TPCK改变了酶的一级结构21.作为典型催化剂的酶具有下列哪一种能量效应（B）A.增加活化能B.降低活化能C.提高产物能量水平D.降低反应物能量水平22.修饰碱基主要存在于下列哪种核酸中（C）A.rRNAB.mRNAC.tRNAD.核DNAE.线粒体DNA23.在溴化乙锭-氯化铯密度梯度离心时，相同分子量的DNA，沉降最快的是（A）A.闭环DNAB.开环DNAC.双链线形DNA24.DNA的Tm不与什么有关（A）A.DNA的分子量B.DNA的GC含量C.DNA的纯度D.缓冲液组成25.ACTH 作用肾上腺时，导致的细胞响应包括（D）A.受体获得酪氨酸激酶活性B.受体激活引起细胞膜钾离子通道改变C.G蛋白激活，抑制腺苷酸环化酶D.cAMP与钙离子浓度升高26.生物膜中最丰富的脂类是（A）A.磷脂B.胆固醇C.糖脂D.三酰甘油27.Ribozyme是（B）A.蛋白质B.核酸C.糖D.脂28.胰岛素与受体结合后，受体细胞质区域（D）A.具有丝氨酸/苏氨酸激酶活性B.结合G蛋白C.激活磷脂酶D.具有酪氨酸激酶活性29.被丝氨酸蛋白激酶作用的蛋白质将（B）A.去掉磷酸丝氨酸上的磷酸基团B.从ATP 获得一个磷酸基团，使丝氨酸磷酸化C.从GTP 获得一个磷酸基团，使丝氨酸磷酸化D.水解ATP而获得磷酸基团30.限制性内切酶EcoRⅠ作用对象是（D）A.蛋白质B.RNAC.单链DNAD.双链DNA二、分析题1、某同学在提取酶的过程中发现酶已经失活，一个原因是由于操作不当引起酶的变性，请问有哪些因素会引起变性？还有什么因素可能引起失活？如果认为酶的失活不是由于变性引起的，请提出一种推测，并设计一个实验路线予以证明。

福建农林大学半期考试试卷参考答案2009——2010学年第学期课程名称：分子生物学考试时间120分钟专业年级班学号姓名一、名词解释（每小题2分，共30分）1、Splitting gene：真核基因的编码序列被非编码序列所隔断的现象。

2、Semi-discontinuous replication：DNA复制时，一条链的合成是连续的，另一条链的合成是不连续的。

3、Restricition endonuclease：能识别特异碱基序列，并在特定位点切断核酸双链的核酸内切酶。

4、Nucleosome：构成真核生物染色质的基本结构单位，是DNA和蛋白质构成的紧密结构形式。

其核心为H2A、H2B、H3、H4各2分子组成八聚体，DNA在其表面上缠绕1.75圈，约146bp，H1结合在连接相邻核小体之间的DNA上。

核小体DNA的长度平均为200bp。

5、Operon：原核基因表达调控的单位，包括启动子、操纵基因和一系列功能相关的结构基因。

6、Frameshift mutations：DNA分子中插入或缺失一个或少数几个核苷酸，使整个阅读框改变而引起的突变。

7、Induction：培养基中添加了某些物质（常常是底物），导致某些酶的合成。

8、Synonymous codon：编码同一氨基酸的密码子。

9、cis-acting element：顺式作用元件，是同一DNA分子中具有转录调节功能的特异DNA 序列。

按功能特性，真核基因顺式作用元件包括启动子、增强子及沉默子等。

10、Ribosome binding site：核糖体结合位点，由Shine和Dalgarno发现原核生物起始密码子上游8~13个nt的保守多嘌呤序列，与核糖体16S小亚基的特定序列碱基配对，相对于起始密码子正确定位核糖体，保证翻译的准确。

11、Exon：外显子，在mRNA剪接后仍会被保存下来，并可在蛋白质生物合成过程中被表达为蛋白质。

12、Leucine zipper：亮氨酸拉链，是含有4个或5个亮氨酸残基, 彼此之间精确的相距7个氨基酸残基。

螺旋体螺旋体(Spirochete)是一群细长、柔软、弯曲呈螺旋状、运动活泼的革兰氏阴性原核细胞型微生物。

分类学上归于细菌范畴。

Representation of general spirochete morphology with a pair ofendoflagella inserted in the opposite poles, lying beneath the outer membrane and within the periplasmic space. In some spirochetes, the free ends overlap, as shown here.分类：根据抗原、螺旋的大小、数目与规则程度及两螺旋间距离。

１、疏螺旋体属：螺旋稀疏、不规则，呈波纹状回归热螺旋体Borrelia has 3-10 loose, irregualr coils.２、密螺旋体属：螺旋细密、规则梅毒螺旋体（雅司、品他）Treponema has 8-20 evenly spaced coils.３、钩端螺旋体属：螺旋更细密、规则，一端或两端弯曲呈钩状钩端螺旋体Leptospira has numerous fine, regularcoils and one or both curved ends.三属螺旋体形态模式图梅毒螺旋体蒋介石与第三任妻子陈洁如合影于黄浦军校。

在蒋与陈蜜月旅行之后，陈洁如发现她身上出了疹子。

“试用各种油膏涂抹，不但无效，而且越来越糟。

后来，突然发现腿上也出现疹块，手腕按脉处也出现两块红疤。

它们虽不痒，但看起来很不好，我很发愁，我一生从未得过这种病。

”单采花，男，某厂产品推销员，经常出差去南方。

不久前，他在旅途中与一位浓妆艳抹的女性亲热过一次，事后追悔莫及。

３周后阴茎冠状沟外发生无痛性硬结，触之硬感如软骨，逐渐扩大，约３－４天前表面发生糜烂、破溃，并无疼痛。

双侧腹股沟淋巴结肿大如蚕豆大小，不与皮肤粘连，无红肿热痛表现。

刺梨多糖提取物对小鼠Ⅱ型糖尿病的干预研究作者：伍勇韦艾骥杨堃李金桂董思思刘大利侯春兰刘红玲程驰薛飞龙刘松青来源：《广西植物》2023年第11期摘要：为探究刺梨多糖（Rosa roxbunghii Tratt. polysaccharide， RRTP）与刺梨不溶性膳食纤维（Rosa roxbunghii Tratt. insoluble dietary fiber， RTIDF）在降血糖功能上是否存在协同作用，该研究对RRTP和RTIDF进行提取、分离和纯化，通过体外实验与体内小鼠Ⅱ型糖尿病的干预实验，测定其抗氧化、降血糖活性，分析体内降血糖功能与小鼠肠道菌群结构关系。

结果表明：（1）体外实验发现，RRTP有较好的自由基清除力，能对α-葡萄糖苷酶、α-淀粉酶起显著抑制作用，IC50值分别为0.293、4.251 mg·mL-1，而RTIDF仅表现对α-淀粉酶活性具有一定的抑制作用。

（2）刺梨多糖提取物干预模型小鼠后，逆转了肥胖小鼠继续消瘦的趋势，与CK组（生理盐水）相比，RTIDF和RRTP+RTIDF干预的小鼠的血糖水平显著下调，血清中过氧化氢酶（CAT）活力明显增强，RRTP+RTIDF优于RTIDF。

（3）RRTP和RTIDF 干预可降低肝脏的炎症因子，缓解细胞肿胀程度，增加盲肠的吸收细胞数目，恢复肠壁黏膜层。

（4）分析肠道菌群发现，RRTP+RTIDF可降低拟杆菌门与厚壁菌门的比例，增加醋酸杆菌属等有益菌的种群丰度，RTIDF对于种群的调节作用更加显著。

综上认为，RRTP与RTIDF 在糖尿病小鼠的血糖干预中具有一定的协同作用，或可共同作为改善Ⅱ型糖尿病的干预剂。

关键词：刺梨多糖，膳食纤维素，分离纯化，Ⅱ型糖尿病，血糖干预中图分类号： Q946 文献标识码： A 文章编号： 1000-3142（2023）11-2120-11Intervention study of Rosa roxbunghii polysaccharide xtracts on Type Ⅱ diabetes in miceWU Yong WEI Aiji YANG Kun LI Jingui DONG Sisi LIU Dali HOU ChunlanLIU Hongling CHENG Chi XUE Feilong LIU Songqing1，2*（ 1. College of Chemistry and Life Sciences， Chengdu Normal University， Chengdu 611130， China; 2. Sichuan Provincial Key Laboratoryfor Development and Utilization of Characteristic Horticultural Biological Resources， Chengdu 611130， China ）Abstract： To investigate the synergistic effect of Rosa roxbunghii Tratt. polysaccharide （RRTP） and Rosa roxbunghii Tratt. insoluble dietary fiber（RTIDF） on hypoglycemic function. In this study， RRTP and RTIDF were extracted， isolated and purified， the antioxidant and hypoglycemic activities of polysaccharides were measured by in vitro experiment and the relationship between hypoglycemic function and intestinal microbial community structure in mice was analyzed in vivo intervention experiment of Type Ⅱ diabetes in mice. The results were as follows：（1） RRTP had a good free radical scavenging ability in vitro，and could significantly inhibit α-glucosidase and α-amylase activities with IC50 values of 0.293 and 4.251 mg·mL- respectively. RTIDF only showed certain inhibitory activity on α-amylase activity. （2） After the intervention of the extracts in the model mice， the tendency of the obese mice to continue to lose weight was reversed. Compared withCK group （physiological saline）， the blood glucose level of RTIDF and RRTP+RTIDF mice were significantly down-regulated， and the activity of CAT enzyme in serum was significantly enhanced. RRTP+RTIDF group was superior to RTIDF group. （3） The extract intervention could reduce the inflammatory factors in the liver， relieve the degree of cell swelling， increase the number of absorbing cells in the cecum， and restore the intestinal wall mucosal layer. （4） Further analysis of intestinal microbial community showed that RTIDF+RRTP could reduce the proportion of Bacteroidetes and Firmicutes， increase the abundance of beneficial bacteria such as Acetobacter，but RTIDF had more significant regulation effect on the population. Therefore， based on in vitro hypoglycemic simulation and in vivo intervention results， RRTP and RTIDF have a certain synergistic effect on glucose intervention in diabetic mice， it may be used together as an intervention to improve Type Ⅱ diabetes.Key words： Rosa roxbunghii Tratt. polysaccharide， dietary cellulose， isolation and purification， Type Ⅱ diabetes， blood glucose interventionⅡ型糖尿病作为现代人群高发的代谢性疾病，严重威胁着人类的生命健康。

Chapter 10qRT-PCR of Small RNAsErika Varkonyi-Gasic and Roger P. HellensAbstractPlant small RNAs are a class of 19- to 25-nucleotide (nt) RNA molecules that are essential for genome stability, development and differentiation, disease, cellular communication, signaling, and adaptive responses to biotic and abiotic stress. Small RNAs comprise two major RNA classes, short interfering RNAs (siRNAs) and microRNAs (miRNAs). Efficient and reliable detection and quantification of small RNA expression has become an essential step in understanding their roles in specific cells and tissues. Here we provide protocols for the detection of miRNAs by stem-loop RT-PCR. This method enables fast and reliable miRNA expression profiling from as little as 20 pg of total RNA extracted from plant tissue and is suitable for high-throughput miRNA expression analysis. In addition, this method can be used to detect other classes of small RNAs, provided the sequence is known and their GC contents are similar to those specific for miRNAs.Key words: Small RNA, miRNA, RT, Stem-loop RT, qPCR, SYBR Green I assay, UPL probe assay 1. I ntroductionSmall RNAs are 19–25 nucleotide long noncoding RNA moleculesthat include short interfering RNAs (siRNAs), implicated in post-transcriptional and transcriptional gene silencing (1), and microR-NAs (miRNAs), implicated in processes ranging from developmentalpatterning to stress responses (2–5). While siRNAs arise from longdouble-stranded RNA precursors, miRNAs are derived from largerprecursors with a characteristic hairpin secondary structure. Similarto siRNAs that target perfect complementary sequences, plant miR-NAs repress gene expression by acting on near-perfect complemen-tary sequences in target mRNAs to guide cleavage and translationalrepression (6–9), or on DNA to guide chromatin modification (10).The majority of plant miRNA targets are developmentally importanttranscription factors (11, 12) and stress-regulated genes (13, 14).Igor KovaIchuk and Franz Zemp (eds.), Plant Epigenetics: Methods and Protocols,Methods in Molecular Biology,vol. 631,DOI 10.1007/978-1-60761-646-7_10, © Springer Science + Business Media, LLC 2010109110Varkonyi-Gasic and HellensDue to the miRNA action, these targets are either eliminated completely during cell-fate changes (12, 15, 16), or are reduced to appropriate levels of expression in tissues, where both the miRNA and the target mRNA are co-expressed (17, 18). In addition, a pos-sible long-distance signaling role was proposed for some miRNAs (19, 20), in contrast to miRNAs with demonstrated cell-autonomous expression and effects (21, 22).This complexity in miRNA modes of action demonstrates that reliable detection and quantification of miRNA expression in spe-cific tissues is an essential first step for better understanding of miRNA-mediated gene regulation. Although miRNAs represent a relatively abundant class of transcripts, their expression levels can vary dramatically between cells and tissues and they often escape detection by conventional technologies such as cloning, northern hybridization, and microarray analysis because of low abundance combined with high complexity of the small RNA population in plants (11, 23). High sensitivity and specificity of reverse transcription-polymerase chain reaction (RT-PCR) detec-tion methods provide a superior detection and quantification method over the conventional technologies. Stem-loop reverse transcription primers were shown to provide better specificity and sensitivity than linear primers (24), and a pulsed reverse transcrip-tion (RT) reaction further increases the sensitivity of miRNA detection (25). These features were utilized to derive a two-step miRNA detection method. First, the stem-loop RT primer is hybridized to the miRNA molecule and then reverse transcribed in a pulsed RT reaction. Next, the RT product is amplified using a miRNA-specific forward primer and the universal reverse primer. The product can be visualized by gel-electrophoresis upon a set number of PCR cycles or monitored in real-time using a SYBR Green I assay or a UPL probe assay that involves a dual labeled hydrolysis probe to increase specificity (Fig. 1).In addition to expression analysis of endogenous miRNAs, this method is amenable for the detection and quantification of other small RNAs, including artificial miRNAs and synthetic siRNAs.1. Plant tissue collected into liquid nitrogen and handled accord-ing to standard practices to prevent degradation of RNA.1. TRIzol reagent for isolation of total RNA (Invitrogen, Carlsbad, CA) (see Note 1).2. Solutions listed in the TRIzol protocol: chloroform, isopro-panol, 75% ethanol, water to resuspend the RNA pellet (see Note 2).2. M aterials2.1. Plant Material 2.2. Isolation and Gel-Electrophoresis of RNA111qRT -PCR of Small RNAs3. 12.3 M formaldehyde-containing 1% agarose gel. CAUTION: Formaldehyde is toxic through skin contact and inhalation of vapours. Manipulations involving formaldehyde should be done in a chemical fume hood.5’-UGACAGAAGAGAGUGAGCAC-3’5’-UGACAGAAGAGAGUGAGCAC GTTGGCTCTGGTGC 3’-CTCGTG CAaccgagacCACG A G G G T C C G A G GT A T T C 5’-GTTGGCTCTGGTGC 3’-CTCGTG CAaccgagacCACG A G G G T C C G A G G T A T TC5’-UGACAGAAGAGAGUGAGCACGTTGGCTCTGGTGC 3’-ACTGTCTTCTCTCACTCGTG CAaccgagacCACG A G G G T C CG A G G T A T T C CAaccgagacCACGCTTATGGAGCCTGGGACGTGGTCTCGGTTG-5’5’-GCGGCGG TGACAGAAGAGAGT-3’miR156stem-loop RT primer forward primer 5’-GCGGCGG TGACAGAAGAGAGT-3’3’-ACTGTCTTCTCTCACTCGTG CAaccgagacCACGCTTATGGAGCCTGGGACGTGGTCTCGGTTG-5’3’-ACTGTCTTCTCTCACTCGTG CAaccgagacCACGCTTATGGAGCCTGGGACGTGGTCTCGGTTG-5’5’-GCGGCGG TGACAGAAGAGAGTGAGCAC GTTGGCTCTGGTGCGAATACCTCGGACCCTGCACCAGAGCCAAC-3’by the quenching label 5’-GCGGCGG TGACAGAAGAGAGTGAGCAC GTTGGCTCTGGTGCGAATACCTCGGACCCTGCACCAGAGCCAAC-3’3’-CGCCGCC ACTGTCTTCTCTCACTCGTG CAaccgagacCACGCTTATGGAGCCTGGGACGTG-5’5’-tggctctg-3’+5’-GTGCAGGGTCCGAGGT-3’3’-ACTGTCTTCTCTCACTCGTG CAaccgagacCACGCTTATGGAGCCTGGGACGTGGTCTCGGTTG-5’5’-GCGGCGG TGACAGAAGAGAGTGAGCAC GTTGGCTCTGGTGCGAATACCTCGGACCCTGCACCAGAGCCAAC-3’3’-TGGAGCCTGGGACGTG-5’5’-GCGGCGG TGACAGAAGAGAGTGAGCAC GTTGGCTCTGGTGCGAATACCTCGGACCCTGCACCAGAGCCAAC-3’3’-CGCCGCC F 5’-tggctctg-3’Q 3’-CGCCGCC “unquenched”signal universal reverse primerSYBR Green I21435687910RTPCR UPLprobe SYBR Green I Fig. 1. Schematic showing the primer design and RT-qPCR process using the example of miR156. A stem-loop RT primer binds to the 3¢ portion of the miRNA, initiating reverse transcription. Then, the RT product is amplified using a miRNA specific forward primer and the universal reverse primer. Quantification is achieved either through SYBR Green I incorporation during amplification, or by the fluorescence generated upon cleavage of the UPL probe. Sequences related to miR156 are presented in grey. Sequences related to UPL probe #21 are in lower case. (1) Annealing, (2) Pulsed RT, (3) Denaturation,(4) Annealing, (5) Extension, (6) Denaturation, (7) Annealing, (8) Extension, (9) Hybridisation, (10) Cleavage112Varkonyi-Gasic and Hellens4. 10× MOPS buffer: 0.4 M MOPS, pH 7.0, 0.1 M sodium acetate, 0.01 M EDTA.5. Formaldehyde Load Dye (Ambion, Austin, TX).6. Ethidium bromide to final 10 m g/ml. CAUTION: Ethidium bromide is a strong mutagen and should be handled with extreme care .7. Molecular weight markers, e.g. 0.5–10 Kb RNA Ladder (Invitrogen). 1. Stem-loop RT primers. Prepare 100 m M stocks for long-term storage and 1 m M dilutions for immediate use. 2. 10 mM dNTP mix. Prepare by mixing dATP, dCTP, dGTP, and dCTP stock solutions, aliquot out and store at −20°C. 3. Reverse transcriptase, e.g. SuperScript III RT, 200 units/m l that is supplied with the First-Strand buffer for cDNA synthesis and 0.1 M DTT (Invitrogen). 4. RNase inhibitor such as RNaseOUT, 40 units/m l (Invitrogen). 5. Nuclease free water, e.g. UltraPure DEPC-treated Water (Invitrogen). 1. LightCycler FastStart SYBR Green I master mix (Roche Diagnostics, Mannheim, Germany), prepared according to manufacturer’s instructions. 2. Universal reverse primer. Prepare 100 m M stock for long-term storage and 10 m M dilution for immediate use. 3. Forward miRNA-specific primer. Prepare 100 m M stock for long-term storage and 10 m M dilution for im mediate use. 4. 10 mM dNTP mix as above. 5. Nuclease free water. 1. LightCycler TaqMan master mix (Roche Diagnostics) prepared according to manufacturer’s instructions. 2. UPL probe #21 prepared as 10 m M stock (Roche Diagnostics). 3. Universal reverse oligo. Prepare 100 m M stock for long-term storage and 10 m M dilution for immediate use. 4. Forward miRNA-specific oligonucleotide. Prepare 100 m M stock for long-term storage and 10 m M dilution for immediate use. 5. 10 mM dNTP mix as above. 6. Nuclease free water. 1. Standard laboratory equipment for isolation of RNA (fume hood, centrifuge, tubes, pipettes, and tips).2.3. Stem-LoopedPulsed ReverseTranscription2.4. qPCR2.4.1. miRNA SYBR GreenAssay2.4.2. miRNA UPL ProbeAssay2.5. Equipment113qRT -PCR of Small RNAs 2. A spectrophotometer for quantification of RNA, e.g. NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE) (see Note 3).3. Standard gel electrophoresis equipment (casting trays, gel tanks, power supply, UV transilluminator).4. A thermal cycler for pulsed reverse transcription. Our reverse transcription reactions and end-point PCR analyses were per-formed on the Mastercycler (Eppendorf, Hamburg, Germany).5. A real-time thermal cycler for qPCRs. All our real-time PCR anal-yses were performed on LightCycler 1.5 (Roche Diagnostics).The primers are designed according to Chen et al. (24) with some modifications (26) (Fig. 1). The stem-loop RT primers have a universal backbone and a specific extension. The universal back-bone sequence is as follows:5¢-GTTGGCTCTG GTGCAGGGTCCGAGGT ATTCGCACcagagccaAC-3¢.This backbone sequence can form a stem-loop structure because of the complementarity between the nucleotides in the 5¢ and 3¢ end; it includes the reverse complement of the UPL probe #21 (in lower case) and the universal reverse primer site in the loop region (in bold).The specificity of a stem-loop RT primer to an individual miRNA is conferred by a six-nucleotide extension at the 3¢ end; this extension is a reverse complement of the last six-nucleotides at the 3¢ end of the miRNA. In an miR156 example, the miRNA sequence is as follows (last six nucleotides are underlined):5¢-UGACAGAAGAGAGUGAGCAC-3¢.Thus, the miR156 stem-loop RT primer sequence is as fol-lows (last six nucleotides that provide specificity are underlined):5¢-GTTGGCTCTG GTGCAGGGTCCGAGGT ATTC GCACcagagccaACGTGCTC-3¢.Forward primers are specific to the miRNA sequence but exclude the last six nucleotides at the 3¢ end of the miRNA. A 5¢ extension of 5–7 nucleotides is added to each forward primer to increase the length and the melting temperature; these sequences were chosen randomly and are relatively GC-rich, bringing the GC content of the forward primer to 50–60%. In an miR156 example, the forward primer sequence is as follows (the GC-rich 5¢ extension is underlined):5¢-GCGGCGGTGACAGAAGAGAGT-3¢.3. M ethods3.1. Primer Design114Varkonyi-Gasic and HellensWe provide an example of a method for isolation, quantification, and evaluation of RNA. Other methods may be used (see Notes 1–3). 1. Isolate RNA from the plant tissue snap-frozen in liquid nitrogen using the TRIzol reagent, according to manufacturer’s instructions. 2. Determine concentration by spectrophotometric analysis. Use an aliquot (200 ng–1 m g) to assess quality by gel electro-phoresis. Store the remaining RNA on ice or at −20°C. 3. Determine RNA quality by gel-electrophoresis. Prepare the gel by heating 1 g agarose in 72 ml water until dissolved, and then cool slightly. Add 10 ml 10× MOPS running buffer and mix. Add 18 ml 37% formaldehyde (12.3 M). If required, top up with water to 100 ml. Pour the gel and wait until set. 4. Assemble the gel in the tank. Add 1× MOPS running buffer to cover the gel by a few millimetres. 5. Prepare the RNA sample by adding 3× volumes Formaldehyde Load Dye to 200 ng–1 m g RNA. Add ethidium bromide to the Formaldehyde Load Dye at a final concentration of 10 µg/ml. 6. Prepare the molecular weight marker in the same manner. 7. Heat denature samples at 65°C for 5–15 min. Load the gel and electrophorese at 5–6 V/cm. 8. Stop the run when the bromophenol blue dye has migrated as far as 70% of the length of the gel. 9. Visualize the RNA on a UV transilluminator. High quality RNA will have clearly visible rRNA bands. 10. Adjust RNA concentration with nuclease free water to 20 ng/m l.The most reproducible results are obtained with 2–20 ng of total RNA per reaction, but abundant miRNAs can be detected from as little as 20 pg of total RNA. The protocol is designed to evalu-ate expression of a specific miRNA in a large number of samples or expression of a large number of miRNAs in one sample. If test-ing many RNA samples for one miRNA, prepare a “no RNA” master mix; if testing for many different miRNAs in one sample, prepare a “no RT primer” master mix. Include 10% excess to cover pipetting errors. At least three replicates per RT reaction are recommended. Also prepare “minus RT” controls by omit-ting reverse transcriptase from the reactions and “no template” controls by adding nuclease-free water in place of RNA. It is important to keep the reactions on ice and work in the cold room if handling large number of samples. 1. Prepare the “no RNA” master mix by scaling the volumes for an individual RT reaction to the desired number of RT reactions. Prepare an individual reaction by adding the following compo-nents to a nuclease-free microcentrifuge tube:3.2. Isolation andGel-Electrophoresisof RNA3.3. Stem-Loop PulsedReverse TranscriptionProtocol3.3.1. RT Reaction WhenTesting Many RNASamples for One miRNA115qRT -PCR of Small RNAs 0.5 m l 10 mM dNTP mix,11.15 m l nuclease-free water and1 m l of appropriate stem-loop RT primer (1 m M). 2. Heat mixture to 65°C for 5 min and incubate on ice for2 min. 3. Centrifuge briefly to bring solution to the bottom of the tube. 4. Add the following:4 m l 5× First-Strand buffer,2 m l 0.1 M DTT,0.1 m l RNaseOUT (40 units/m l) and 0.25 m l SuperScript III RT (200 units/m l). 5. Mix gently and centrifuge to bring solution to the bottom of the tube. 6. Assemble the RT reaction by aliquoting 19 m l of the “no RNA” master mix and adding 1 m l RNA template (see Note 4). 7. Mix gently and centrifuge to bring solution to the bottom of the tube. 1. Prepare the “no RT primer” master mix by scaling the volumes for an individual RT reaction to the desired number of RT reactions. Prepare an individual reaction by adding the follow-ing components to a nuclease-free microcentrifuge tube:0.5 m l 10 mM dNTP mix,11.15 m l nuclease-free water and 1 m l of appropriate RNA template (see Note 4). 2. Add the following:4 ml 5× First-Strand buffer,2 ml 0.1 M DTT,0.1 ml RNaseOUT (40 units/ml) and 0.25 ml SuperScript III RT (200 units/m l). 3. Mix gently and centrifuge to bring solution to the bottom of the tube. 4. Assemble the RT reaction by aliquoting 19 m l of the “no RT primer” master mix and adding 1 m l of appropriate stem-loop RT primer (1 m M) previously denatured by heating to 65°C for 5 min. 5. Mix gently and centrifuge to bring solution to the bottom of the tube. 1. Load thermal cycler and incubate for 30 min at 16°C, followed by pulsed RT of 60 cycles at 30°C for 30 s, 42°C for 30 s and 50°C for 1 s.2. Incubate at 85°C for 5 min to inactivate the reverse transcriptase.3.3.2. RT Reaction WhenTesting One RNA Samplefor Many miRNAs3.3.3. Pulsed RT Reaction116Varkonyi-Gasic and HellensProtocols are provided for the SYBR Green I Assay and the UPL probe assay. SYBR Green I assay provides good specificity, if the number of PCR cycles is limited to the maximum of 35 to mini-mize nonspecific amplification. At this number of cycles, highly and moderately abundant miRNAs can be easily quantified (Fig. 2). For miRNA sequences that are expressed at low levels or when a particular set of primers produces background amplification, the UPL probe assay provides higher specificity (Fig. 3).3.4. qPCR Fig. 2. The sensitivity of the stem-loop RT-PCR assay. (a ) RT-PCR analysis of miR159 expression visualized on agarose gel stained with ethidium bromide. Very little non-specific amplification was detected with negative control reactions (−RT, minus RT, and NTC, “no template” control) at 35 cycles. The amount of RNA used for reverse transcrip-tion reactions are indicated on the top. PCR cycle numbers are indicated on the left. Size markers are indicated on the right. (b ) qPCR analysis of the same sample using the SYBR Green I assay at 35 cycles117qRT -PCR of Small RNAs 1. Prepare 5× LightCycler FastStart SYBR Green I master mix(Roche Diagnostics) according to manufacturer’s instructions.2. Prepare a PCR master mix by scaling the volumes listed below to the desired number of amplification reactions. Include 10% excess to cover pipetting errors. For a single reaction, add the following components to a nuclease-free microcentrifuge tube:12 m l nuclease-free water4 m l SYBR Green I master mix 3.4.1. miRNA SYBR Green IAssay Fig. 3. Improved specificity of the miRNA UPL probe assay. (a ) SYBR Green I assay PCR for miR166. Negative control reactions (−RT, minus RT, and NTC, “no template” control) produced detectable amplicons after 40 cycles. (b ) UPL probe assay PCR for miR166. No fluorescence was detected in the negative control reactions after 45 cycles. (c ) UPL probe assay amplification products for miR166 separated by gel electrophoresis on 4% agarose, showing specific and nonspecific amplification products above, below and in the size-range of specific products, obtained after 45 cycles of PCR. Arrowhead indi-cates the expected size of specific amplicons118Varkonyi-Gasic and Hellens1 m l forward (miRNA specific) primer (10 m M) and 1 m l reverse (universal) primer (10 m M) 3. Mix gently and centrifuge to bring solution to the bottom of the tube. 4. Store in cooling block or on ice. 5. Place required number of LightCycler capillaries in precooled centrifuge adapters. 6. Pipette 18 m l master mix into each LightCycler capillary. 7. Add2 m l RT product. 8. Seal each capillary with a stopper. 9. Place capillaries into the LightCycler carousel and spin in the carousel centrifuge. 10. Incubate the samples at 95°C for 5 min, followed by 35–40 cycles of 95°C for 5 s and 60°C for 10 s. 11. For melting curve analysis, denature samples at 95°C, then cool to 65°C at 20°C per second. Collect fluorescence signals at 530 nm wavelength continuously from 65°C to 95°C at 0.2°C per second. 1. Prepare 5× LightCycler TaqMan master mix (Roche Diagnostics)according to manufacturer’s instructions. 2. Prepare a PCR master mix by scaling the volumes listed below to the desired number of amplification reactions. Include 10% excess to cover pipetting errors. For a single reaction, add the following components to a nuclease-free microcentrifuge tube:11.8 m l nuclease-free water,4 m l TaqMan master mix,1 m l forward (miRNA specific) primer (10 m M) and1 m l reverse (universal) primer (10 m M) and0.2 m l UPL probe #21 (10 m M).3. Mix gently and centrifuge to bring solution to the bottom of the tube.4. Store in cooling block or on ice.5. Place required number of LightCycler capillaries in precooled centrifuge adapters.6. Pipette 18 m l master mix into each LightCycler capillary.7. Add 2 m l RT product.8. Seal each capillary with a stopper.9. Place capillaries into the LightCycler carousel and spin in the carousel centrifuge.10. Incubate samples at 95°C for 5 min, followed by 35–45 cycles of 95°C for 5 s and 60°C for 10 s.3.4.2. miRNA UPL ProbeAssay119qRT -PCR of Small RNAs The qPCR data can be analysed and presented as absolute or relative values. Relative quantification is the preferred method because it takes into account the potential errors due to variation in RNA input and RT efficiency. The most accurate method to correct these potential errors is normalization to endogenous control genes. An ideal endogenous control generally demonstrates gene expression that is relatively constant and highly abundant across tissues and cell-types. In addition, a suitable control for normalization of miRNA expression would have similar properties to miRNAs in terms of size and stability and would be amenable to the miRNA assay design. Some classes of small noncoding RNAs (ncRNAs) other than miRNAs are often expressed in an abundant and stable manner. Several human and mouse snRNAs and snoRNAs were tested across the range of tissues and experimental conditions and confirmed as suitable endogenous controls for quantification of miRNA expression levels (Applied Biosystems). No such analysis was performed with plant tissues yet, and a large-scale study is required to evaluate suitability of different plant ncRNAs for miRNA quantification. Therefore, plant researchers have to select a set of controls individually and screen under appropriate conditions or select a specific miRNA that demonstrates the least variability across tissues or experimental conditions under consideration. Either way, the consistency of expression should be confirmed under the specific conditions of the experiment (see Note 5).Here, we provide general instructions for data analysis using the LightCycler Software 4.05. If using a different instrument or software, refer to the appropriate instrument user manual for instructions on how to analyze data.1. This analysis is done after the SYBR Green I Assay to deter-mine that each of the primer pairs amplified a single predomi-nant product with a distinct melting temperature (Tm).2. Follow the instrument user manual for instructions forMelting Curve Analysis and Tm calling. 3. If a single melting peak is observed for a particular primer pair, itis likely that a single product with a distinct Tm was amplified. 4. Evaluate by gel-electrophoresis (see Note 6).1. Relative Quantification analysis compares two ratios: the ratioof the target gene to a reference gene sequence in an unknown sample is compared with the ratio of the same two sequences in a standard sample called a Calibrator. 2. To perform relative quantification with an external standard,prepare standard curves for the target and reference genes by serial dilutions of external standards with a known copy num-ber (see Note 7). Use at least three points or one point per log of concentration, whichever is greater. Always use a “no template” control.3.5. Data Analysis3.5.1. Melting Curve Analysis3.5.2. Relative Quantification120Varkonyi-Gasic and Hellens3. Prepare master mix and perform qPCR as described above.Use at least three replicates per standard dilution and “notemplate” control.4. Follow the instrument user manual for instructions for theStandard Method that will automatically calculate and displaythe amplification curves and the standard curve, crossingpoints, calculated concentrations, and statistics for replicates.5. Save as an external standard curve object.6. Perform Relative quantification, Calibrator normalized, with-out efficiency correction: select Relative Quantification –Monocolor Analysis, assign a “Target Calibrator” and a“Reference Calibrator” sample, assign appropriate pairs oftarget and reference samples and perform analysis followingthe instrument user manual (see Note 8).7. Perform efficiency correction by applying an external stan-dard curve.8. Download data and present as graphs or tables.4. N otes1. Whenever possible, we used the TRIzol Reagent for isolationof RNA because of its convenience, good RNA quality andspeed. Some plant tissues may not be amenable to isolation ofRNA by this method. Other methods for isolation of RNAmay be used; however, avoid RNA purification methods thatuse RNA-binding glass-fibre filters that do not recover smallRNA species quantitatively (e.g. Qiagen RNeasy mini andmidi kits). If unfamiliar with the method for isolation of RNA,subsequent isolation, quantification, and polyacrylamide gelelectrophoresis of the low molecular weight RNA fractioncan be used to evaluate its quantity and quality.2. RNA should be handled according to standard laboratorypractices to avoid RNase contamination. All buffers and solu-tions should be nuclease-free.3. Spectrophotometry followed by gel electrophoresis is still themost widely used method for assessing the RNA yield, purity,and quality. Fluorometry (e.g. RiboGreen, Molecular Probes)can also be used to determine yield, and microfluidic systems(such as Agilent’s bioanalyzer chips) can be used to deter-mine yield and quality.4. In our hands, both nondenatured RNA and RNA denaturedby incubation at 65°C for 5 min produced similar results.However, it has been suggested that denaturation of RNAmay reduce the yield of cDNA for some miRNAs.121qRT-PCR of Small RNAs5. In general, evaluation of endogenous controls involvesdemonstration of relatively abundant and relatively constantexpression levels across the tissues and environmental condi-tions, compared with the RNA input and expression of otherhousekeeping genes.6. Melting curve analysis needs to be combined with gel electro-phoresis. Due to the small size of the fragment, a primer-dimer product generated form the “minus RT” and “notemplate” controls often has a very similar Tm to that of theappropriate miRNA amplification fragment. 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β-葡萄糖苷酶的研究进展综述食品研究与开发2oo5.V oL26.No.6一葡萄糖苷酶的研究进展许晶张永忠孙艳梅东北农业大学应用化学系哈尔滨150030摘要:本文简述了B一葡萄糖苷酶的理化性质,催化反应机制,酶活性测定方法及其在食品工业中应用.关键词:B一葡萄糖苷酶;性质;反应机制;应用l83一==ISOMERESEARCHADV ANCEOFB—GLUCOSIDASEXUJingZHANGY ongzhongSUNY anmei DepartmentofAppliedChemistry,NortheastAgriculturalUniversity,Harbin,150030 Abstract:Thearticlebrieflydiscussedthephysicalandchemicalproperties,thecatalyticmec hanism,methodsofenzymeactivitydeterminationof13-glucosidaseanditsapplicationinfoodindust ry.Keywords:13-glucosidase;characteristic;reactionmechanism;applicationB一葡萄糖苷酶(.beta.一Glucosidase)系统名称为B—D一葡萄糖苷葡萄糖水解酶(.beta.一D—glucosideglucohydrolase;EC3.2.1.21).1837年,被Liebig和Wohler首次在苦杏仁中发现,后被发现存在于自然界许多植物中,还存在于一些酵母,曲霉菌,木酶菌及细菌体内[1].它起初引起人们的注意是因为它参与了纤维素材料的生物转化.B一葡萄糖苷酶是纤维素酶系中的一个组分,它主要作用于B一(1,4)糖苷国家"十五"重大科技专项"农产品深加工技术与设备研究开发"项目编号:2001BA501A02B作者简介:许晶,女,1979年9月出生,理学学士;助教,在读硕士, 研究方向:食品化学专业.键,还能作用于B一(1,1),(1,2),(1,3),(1,6)糖苷键.对于低聚葡萄糖聚合度越小,它的水解能力越强[.多年来,许多学者分别从苦杏仁,葡萄,刀豆,玉米,黑樱桃,水稻,大豆中分离纯化了B一葡萄糖苷酶[.现将B一葡萄糖苷酶的理化性质,催化反应机制,酶活性测定方法及其在食品工业中应用简述如下.113一葡萄糖苷酶的理化性质1.1相对分子量B一葡萄糖苷酶的相对分子质量一般在40000-250000之间[4].不同来源的B一葡萄糖苷酶的相对分子量由于其结构和组成不同而差异很大.例如,的营养保健食品,因而博得了产地群众的青睐.胡颓子果实,根,叶药用,收敛止泻,镇咳解毒.常见临床配方:①治疗风湿性关节炎疼痛,胡颓子根100g,黄酒6OraL,猪脚250g,加水煮1时许,取汤一碗,连同猪脚一同服食.②治疗吐血,便血,咯血,月经过多,胡颓子根30-6Og,煎服.③治疗支气管哮喘,慢陛支气管炎,胡颓子叶15g,枇杷叶15g,水煎服.④治疗咳嗽,鲜胡颓子叶30g,水煎,加白糖少许服用.⑤治疗痢疾下血,用胡颓子15g,乌梅20g,水煎服.⑥治疗月经不调,血崩,用胡颓子15g,山萸肉20g,水煎服.除此之外,胡颓子的鲜花含芳香油,可作调香原料.茎皮纤维是造纸和制纤维板的原料.植株可作园林绿化树,配植于花丛或林缘,颇有特色.胡颓子树对多种有害气体抗性强,特别适于工厂污染区的绿化.胡颓子的野生资源非常丰富,耐干旱瘠薄,适应性很强,对土壤要求不严,常生于山坡疏林下或林缘灌丛的阴湿环境,也发现于向阳山坡或路旁.繁殖非常容易,结果早,加之营养价值高,特别是氨基酸,维生素C和矿质元素含量丰富,开发利用的前景非常广阔[1].参考文献:[1]刘孟军.中国野生果树[M].北京:中国农业出版社,1998[2]张福平等.粤东地区野果植物资源[J].中国野生植物资源.2003,22(3):13-16[3]中国科学院研究所.中国高等植物图鉴(1-5册及补编1—2册)[M].北京:科学出版社,1985收稿日期:2005一O1—25一==2DD5.V o1.26.J,fO.6食品研究与ic}发ServeW.M.Kengen等人研究的古细菌Pyrococcus furiosus分泌的B一葡萄糖苷酶的分子是由四个亚基构成的四聚体,其分子量在230000左右;而中国科学院微生物研究所的曾宇成等所测出的海枣曲霉的B一葡萄糖苷酶由两个亚基构成,分子量为200000左右;由Day&Withers等人从Agrobacterium 中分离出的野生性B一葡萄糖苷酶是一种二聚体,由两个分子量质量为50000的B一葡萄糖苷酶的亚基构成.有的菌株本身含有胞内和胞外B一葡萄糖苷酶,因此,有时来源于同一菌株的B一葡萄糖苷酶,是二种不同分子量酶的混合物.1.2等电点(pI),最适pH及pH稳定性大部分B一葡萄糖苷酶的pI都在酸性范围,并且变化不大,一般在3.5-5.5之间,但最适pH可以超过7.0,而且酸碱耐受性强[.如:Paavilainen等人从Alkalophilus中就分离出细胞外B一葡萄糖苷酶, 其最适pH就在6-9之间,而在pH4.0—10.2以外还具有一定的催化活性;中国台北学者李约昆(音译)等人从Flavobacteriummeningosepticum中分离出的B一葡萄糖苷酶其pI在9.0左右,最适pH是5.0E.1.3最适温度及热稳定性B一葡萄糖苷酶的最适温度在40一ll0℃之间都有分布.一般来说,来自古细菌的B一葡萄糖苷酶其热稳定性和最适温度要高于普通微生物来源的B一葡萄糖苷酶.如古细菌Pyrococcusfuriosus的B一葡萄糖苷酶其最适温度102—105oC,100℃时的半衰期为85h;而李约昆等人分离出的B一葡萄糖苷酶最适温度在50—55℃之间,在60℃下于磷酸盐缓冲液中,其活力在15min后只余1%.对于工业应用来说,酶的热稳定性越高越有利,因此,从嗜热细菌中分离出B一葡萄糖苷酶逐渐引了人们的兴趣.至于来自嗜热性微生物的B一葡萄糖苷酶为何具有如此强的耐热稳定性还未获得共识.据MichaelW.Bauer 等人对来自嗜热性和非嗜热性B一葡萄糖苷酶的分析认为,两者在相互演化过程中发生的酶修饰作用并不改变酶的活性中心,也不改变其专一性,只是将酶蛋白结构作部分调整以适应高温环境[63.2B一葡萄糖苷酶的催化反应机制2.1反应机制[]M.W.Bauer等人对分别来自嗜热菌Pyrococcus furiosus和非嗜热菌Agrobacterium的B一葡萄糖苷酶进行研究发现,两种来源的菌催化反应时按同一种机制进行,即在催化糖苷键的裂解反应时都遵循双取代反应机制.其反应方程式如下:EsEP第一步是酶与底物键合形成米氏复合物ES(反应速率K.和K一.);第二步是酶一底物中间体(E—S)的形成(反应速率K2);酶的亲核基团按酸催化机制进攻异头碳,形成共价的糖基酶中间体(E—S).在这一过程中,B一葡萄糖苷酶的活性中心可根据不同类型的底物而相应发生一定程度的结构变化,从而使B一葡萄糖苷酶可以和多种糖类底物结合,这一步决定了B一葡萄糖苷酶具有的底物专一性;第三步是中间体的水解:由水按碱催化机制对异头碳进攻,形成B一葡萄糖基产物并使酶回复其初始的质子化态.其中,糖苷基酶中间体的形成和水解过程经历了共价结构的氧碳鲼正离子过渡态.另外,B一葡萄糖苷酶在整个反应过程中其构型严格保持不变.2.2活性中心结构[]在多数B一葡萄糖苷酶中起催化作用的残基是二个谷氨基酸残基,其中,靠近N一端的谷氨酸起酸/碱作用,另一氨基酸起亲核试剂的作用.但Grabnitz等人研究发现来自Clostridiumthermoce1.1um的B一葡萄糖苷酶的活性部分在N一端的130个氨基酸区域,该区的个性特征是氨基酸序列中心基团His—Asn—Glu—Pro,存在于该区域的具有催化作用的残基是相隔35—55个氨基酸的His和Glu,其中质子化态的完全保持残基Hisl21作为质子供体与Glu166协同稳定氧碳鲼正离子.而高度保持的c一端附近的残基也许参与了酶与糖苷基底物的键合,其中在该区的一些微小差异与不同B一葡萄糖苷酶的底物特异性有关.2.3底物特异性[8]几乎所有的B一葡萄糖苷酶对底物的糖基部分结构的专一性较差,能袭解C一0糖苷键,c—S键,c—N键,C—F键等;有些对糖基部分的C和C:构形也不专一,能同时水解B一葡萄糖苷键和B一半乳糖苷糖,有些甚至c位的专一性也不高,能水解木糖.但在所有底物中,B一葡萄糖苷酶对纤维二糖的活性最强.2.4反应抑制剂[9]Kempton等人研究发现Agrobacterium13一葡萄糖苷酶的一系列有机物抑制剂都与底物和过渡态结构相似,并且所有的抑制剂直接与底物竞争.有相似的过渡态结构即意味着带有相同的正电荷和综述食品研究与拜发2oo5.V oL26.NO.6相似的半椅状结构,这些抑制剂能与酶键合得更为紧密.比如,最好的抑制剂是反应过程中有相似过渡态结构的gluconolactone和gluconopheylurethane 而不同位置正电荷(如1-dexynojirimycin)的抑制剂与酶的亲和力就相对较弱,非半椅状结构(如椅式构形的is0pr0pyl—p—D—thi0uc0pyran0side和船式构形的l,6一anhydro—p-glucopyranose)的抑制剂与酶的亲和力更弱.这些抑制剂都直接与底物有竞争作用.在无机抑制剂Ag对B一葡萄糖苷酶有强的抑制作用,Hg及4mol/L脲也有较强的抑制作用,而Cu",Pb",SDS及EDTA等常见抑制剂对该酶活力无明显影响.3B一葡萄糖苷酶活性的测定方法加]1.Bamsh和swiain法:它以水杨苷作底物,酶解产物用4一氨基安替比林作显色剂,使释放出来的水杨醇显色,再用分光光度法比色测定.2.荧光法:利用伞形酮(7一轻基香豆素)与4一甲基伞形酮具强烈荧光的特点,将它们生为无荧光的底物,以此测定.3.以对硝基苯基一p—D一葡糖苷(P—NPG)作为底物进行酶解,底物水解后释放出来的配基对硝基苯酚可直接在400~420am之间测定.4B一葡萄糖苷酶的应用B一葡萄糖苷酶能参与生物体的糖代谢["],对维持生物体的正常生理功能起着重要作用.如它在酶解纤维素时就起着至关重要的作用,它把由纤维素酶降解生成的纤维二糖和三糖转化成可发酵的葡萄糖.B一葡萄糖苷酶的生物学功能使它在生产中有着广泛的应用.4.1作为食品风味酶应用[12目前,B一葡萄糖苷酶的主要应用是在食品工业中.随着食品工业的发展,风味化学的研究也引人关注.近年来,人们着重研究水果风味物质在水果中存在的前体,包括一些二级代谢产物,如糖苷类物质.这一研究领域在风味研究中被认为"前体分析".法国的Cordonnier等人1974年首次提出葡萄中可能存在键合态的不挥发的萜烯类化合物.80年代澳大利亚的Williams等人对葡萄做了比较深人细致的工作,发现葡萄中的一些风味物质如萜烯醇和芳香醇等不但以游离的形式存在,而且还以糖苷键合态的形式存在,并指出,糖苷键合态化合物的含量大大超过其游离态的含量.Engel和Tressl等经研究指出,许多水果中含有单萜烯糖苷,C(13)降异185==I类萜糖苷,倍半萜烯糖苷以及其它的糖苷,几乎所有的天然糖苷是B一糖苷,所以可以利用B一葡萄糖苷酶水解水果中的风味前体物一糖苷,释放出挥发性糖苷配基,用以增强葡萄酒等果酒,果汁香气.因此B一葡萄糖苷酶作为水果风味增香酶最合适. Shoseyov等(1990)报道分离到一株产内切B一葡萄糖苷酶的黑曲霉,用该酶来处理玫瑰红葡萄酒及西番莲果汁,通过GC—MS分析及感官评定,结果表明:葡萄酒中的单萜,苹果醇,苯乙醛等风味物质有明显提高.J.L.Iborra等(1992)则研究了甜杏B一葡萄糖苷酶水解苦藏花素,产生藏花醛(可作食品风味添加剂)的过程.可以预见,随着研究的不断深人和发展,运用这一生物技术手段提高果制品的天然风味的研究有着广泛的应用前景.4.2在青梅脱苦中应用B一葡萄糖苷酶在青梅脱苦中也有重要作用[13].梅果中有人体所需的多种氨基酸和有机酸,但也含有大量的苦味物质,主要是苦杏仁苷.据方祖达和五德裕(1988)报道,约七分成熟的新鲜梅子含苦杏仁苷高达784ppm,若是九分成熟者约含260～270ppm,约为前者三分之一.由此可见,愈成熟的果实愈不苦,但成熟的梅子制得的果汁仍然苦涩难以接受,这就是梅子过去都没有被利用做成纯天然果汁饮料的最大原因.而B一葡萄糖苷酶却可以把苦杏仁苷分解为苯甲醛及氢氰酸和两分子葡萄糖,而使苦味大大减少.虽然分解出的氢氰酸有时可使人食后中毒,但氢氰酸可在加热过程中蒸发掉,而且据七十年代《美国农业联合会》报道,一定剂量的苯甲醛和氢氰酸混合物具有防癌效果.4.3在降解纤维素中应用B一葡萄糖苷酶的另一主要应用是用于降解纤维素[14,15].纤维素酶转化纤维成葡萄糖的过程细节和作用机理还不清楚或未有定论.一般认为由内切葡聚糖作用于微纤维的非结晶区,纤维二糖水解酶再从非还原端依次分解产生纤维二糖和三糖,后者再由B一葡萄糖苷酶水解成葡萄糖.纤维素是葡萄糖以B一1,4糖苷键结合的聚合物,为植物细胞壁的构成成分,占植物干重的1/2—1/3.全球一年间由光合作用生产的纤维素达1000亿吨,是最丰富的可再生资源.将植物纤维应用于发酵食品工业原料,对人类将是一个重大的贡献,可以使我们摆脱对谷物粮食的绝对依赖,缓和世界资源紧缺.4.4在水解大豆异黄酮中应用一,,1862oo5.V o1.26.NO.6食品研究与并发综述我国水果果皮的利用现状和前景鲍金勇赵国建杨公明1.华南农业大学食品学院广州510642;2.西北农林科技大学食品科学与工程学院杨凌712100摘要:本文简述了水果果皮的结构性质和我国水果果皮加工的现状,分析了水果果皮在利用中所存在的问题,并提出相应的解决措施.关键词:水果果皮;利用现状;展望THEUTILIZINGCURRENTSITUATIONOFCHINESEFRUITPEELANDTHEPROS PECTBAOJinyongZHAOGuojianYANGGongming1.CollegeofFoodScience,SouthChinaAgriculturalUniversity,Guangzhou,510642B一葡萄糖苷酶还可以用来水解大豆异黄酮,制备高活性的大豆异黄酮苷元产品[16].酶水解条件温和,多采用弱酸性的缓冲溶液,大豆异黄酮苷元不易变性,是工业上制备富含大豆异黄酮苷元产品的十分有前途的途径.不过目前高活性的大豆异黄酮糖苷水解酶还在研制阶段,也没有进行工业化.大豆自身含有的内源B一葡萄糖苷酶水解活性不强,水解效率只有22-29%[73.添加足量的高活性酶(如苦杏仁和酵母中提取B一葡萄糖苷酶[1)可使水解达到100%.4.5其它应用[1.6.2]B一葡萄糖苷酶还可应用于乳品工业来分解乳糖,与其它酶协同作用生产葡萄糖与单细胞蛋白.多酚化合物是一类较好的天然抗氧化剂,它们在植物体中主要是以糖苷的形式存在,而苷元(即游离多酚)的抗氧化活性和防癌功效比其糖苷大得多.应用B一葡萄糖苷酶催化水解这类糖苷是一类生产天然抗氧化剂的好方法.应用p一葡萄糖苷酶可以生产天然色素.如栀子蓝色素(Gardeniablue)就是以茜草科植物栀子(GardeniaJasminoidesEllis)为原料,通过p一葡萄糖苷酶的作用而产生的一种天然色素.通过基因突变改变p一葡萄糖苷酶的氨基酸序列中活性中心中的氨基酸残基,可使p一葡萄糖苷酶的催化水解作用变为催化糖苷键生成的酶.如Withers用基因突变的方法[191把Agrobacterium3-葡萄糖苷酶358位上的谷氨酸残基用一丙氨酸替代使其成为合成低聚糖的酶.参考文献:[1]李远华.B一葡萄糖苷酶的研究进展.安徽农业大学, 2002,29(4):421—425[2]DriskillL.E.;BauerM.W.;KellyR.M.BiotechnologyandBioengineering.1999,66(1),51～6o[3]孙艳梅,张永忠.大豆B一葡萄糖苷酶的提取及其酶学性质的研究.食品工业科技,2004(1)[4]MichaelW.Bauer,RobertM.Kelly.Biochemistry,1998,37: 17170～17178[5]Julieb.Kempton,StephenG.Withes.Biochemistry,1992,31: 9961—9969[6]彭喜春等.B一葡萄糖苷酶的研究现状及应用前景.江苏食品与发酵,2001(12)[7]NamehukM.N.,andWitherss.G..Biochemistry,1995,34, 16194～16202[8]YiQ.BiotechnolBioen,1998,6o(3):385-390[9]Pitsons.M..Enzyme&MicrobialTichnology,1997,210): 183—190[10]王华夫,游小青.茶叶中B一葡萄糖苷酶活性的测定.中国茶叶,1996(3):16—17[11]LiY aw—kuen.J.Chn.Chem.Soc.(Taipei),1998,269(26): 17537—17541[12]顾卫民.B一葡萄糖苷酶的特性及其在食品工业中的应用. 江苏食品与发酵,2003(1)[13]陶宁萍.苦杏仁苷酶在纯天然青梅果汁中的应用.南京农业大学硕士论文,1993[14]周晓云.酶技术.石油工业出版社,1995[15]陈驹声.酶制剂生产技术,1994[16]孙艳梅,张永忠.水解制备大豆异黄酮苷元研究进展.食品研究与开发,2002(3)[17]MatsuuraM.,ObataA.J.FoodSci.,1993.58(1),144—147[18]PandiatanN.,HettiarachchyN.JuZ…YFoodChem.and Toxicology.2000,65(3),403—407『19]22Withers,eta1.UnitedStatesPatent5716812 收稿日期:2005-03-07。

基因相关词汇专业英语翻译Aactivation domain 活化结构域adapters 连接物adenine 腺嘌呤adenosine 腺ADP (adenosine diphosphate) 腺二磷酸affinity column 亲和柱AFLP (amplified fragment length polymorphisms) 增值性断片长度多态现象agrobacterium 农杆菌属alanine 丙氨酸allele 等位基因amber mutation 琥珀型突变AMP (adenosine monophosphate) 腺一磷酸ampicillin 氨青霉素anchor primer 锚状引物annealing 退火annealing temperature 退火温度anticodon 反密码子AP-PCR (arbitrarily primed PCR) 任意引物聚合链反应arbitrary primer 任意引物ATP (adenosine triphosphate) 腺三磷酸autosome 常染色体Bbaculovirus 杆状病毒base pair 基对base sequence 基顺序beta-galactosidase β-半乳糖beta-glucuronidase β-葡糖醛酸糖bioluminescence 生物发光bioremediation 生物降解biotechnology 生物技术blotting 印迹法blue-white selection 蓝白斑筛选blunt end 平(整末)端Ccatalyst 催化剂cDNA library 反向转录DNA库centromere 着丝体centrosome 中心体chemiluminescence 化学发光chiasma 交叉chromomere 染色粒chromoplast 有色体chromosomal aberration 染色体畸变chromosomal duplication 染色体复制chromosomal fibre 染色体牵丝chromosome 染色体chromosome complement 染色体组chromosome map 染色体图chromosome mutation 染色体突变clone 克隆cloning 无性繁殖系化codon 密码子codon degeneracy 密码简并codon usage 密码子选择cohesive end 黏性末端complementary DNA (cDNA) 反向转录DNA complementary gene 互补基因consensus sequence 共有序列construct 组成cosmids 黏性质粒crossing over 互换cyclic AMP (cAMP) 环腺酸cytosine 胞嘧啶Ddark band 暗带deamination 脱氨基作用decarboxylation 脱羧基作用degenerate code 简并密码degenerate PCR 退化性聚合链反应dehydrogenase 脱氢denaturation 变性deoxyribonucleoside diphospahte 脱氧核糖核一磷酸deoxyribonucleoside monophospahte 脱氧核糖核二磷酸deoxyribonucleoside triphospahte 脱氧核糖核三磷酸deoxyribose 去(脱)氧核糖dicarboxylic acid 二羧酸digoxigenin 洋地黄毒diploid 二倍体DNA (deoxyribonucleic acid) 去(脱)氧核糖核酸DNA binding domain DNA结合性结构域DNA fingerprinting DNA指纹图谱DNA helicase DNA解螺旋DNA kinase DNA激DNA ligase DNA连接DNA polymer DNA聚合物DNA polymerase DNA聚合double helix 双螺旋double-strand 双链Eelectroporation 电穿孔endonuclease 内切核酸enhancer 增强子enterokinase 肠激episome 游离基因ethidium bromide 溴乙锭eukaryotic 真核生物的euploid 整倍体exonuclease 外切核酸expressed-sequence tags 表达的序列标记片段extron 外含子FF factor F因子FAD (flavine adenine dinucleotide) 黄素腺嘌呤二(双)核酸feedback control 反馈控制feedback inhibition 反馈抑制feedback mechanism 反馈机制first filial (F1) generation 第一子代FISH (fluoresence in situ hybridization) 荧光原位杂交forward mutation 正向突变F-pilus F纤毛functional complementation 功能性互补作用fusion protein 融合蛋白Ggel electrophoresis 凝胶电泳gene 基因gene cloning 基因克隆gene conversion 基因转变gene duplication 基因复制gene flow 基因流动gene gun 基因枪gene interaction 基因相互作用gene locus 基因位点gene mutation 基因突变gene regulation 基因调节gene segregation 基因分离gene therapy 基因治疗geneome 基因组/ 染色体组genetic map 基因图genetic modified foods (GM foods) 基因食物genetics 遗传学genetypic ratio 基因型比/ 基因型比值genome 基因组/ 染色体组genomic library 基因组文库genotype 基因型giant chromosome 巨染色体globulin 球蛋白glucose-6-phosphate dehydrogenase 6-磷酸葡萄糖脱氢GP (glycerate phosphate) 磷酸甘油酸脂GTP (guanine triphosphate) 鸟三磷酸guanine 鸟嘌呤Hhaploid 单倍体haploid generation 单倍世代heredity 遗传heterochromatin 异染色质Hfr strain 高频重组菌株holoenzyme 全homologous 同源的housekeeping gene 家务基因hybridization 杂交Iimmunoglobulin 免疫球蛋白in vitro 在体外/ 在试管内in vivio 在体内independent assortment 独立分配induced mutation 诱发性突变induction 诱导initiation codon 起始密码子inosine 次黄insert 插入片段insertional inactivation 插入失活interference 干扰intergenic 基因间的interphase 间期intragenic 基因内的intron 内含子inversion 倒位isocaudarner 同尾酸isoschizomer 同切点JKkanamycin 卡那毒素klenow fragment 克列诺夫片段Llac operon 乳糖操纵子ligase 连接ligation 连接作用light band 明带linker 连接体liposome 脂质体locus 位点Mmap distance 图距离map unit 图距单位mature transcript 成熟转录物metaphase 中期methylase 甲基化methylation 甲基化作用microarray 微列microinjection 微注射missense mutation 错差突变molecular genetics 分子遗传学monoploid 单倍体monosome 单染色体messenger RNA (mRNA) 信使RNAmultiple alleles 复(多)等位基因mutagen 诱变剂mutagenesis 诱变mutant 突变体mutant gene 突变基因mutant strain 突变株mutation 突变mutation rate 突变率muton 突变子NNAD (nicotinamide adenine dinucleotide) 烟醯胺腺嘌呤二核酸NADP (nicotinamide adenine dinucleotide phosphate) 烟醯胺腺嘌呤二核酸磷酸nicking activity 切割活性nonsense codon 无意义密码子nonsense mutation 无意义突变Northern blot Northern印迹法nuclear DNA 核DNAnuclear gene 核基因nuclease 核酸nucleic acid 核酸nucleoside 核nucleoside triphosphate 核三磷酸nucleotidase 核酸nucleotide 核酸nucleotide sequence 核酸序列Ooligonucleotide 寡核酸one gene one polypeptide hypothesis 一个基因一种学说operon 操纵子oxidative decarboxylation 氧化脱羧作用oxidative phosphorylation 氧化磷酸化作用PPCR (polymerase chain reaction) 聚合链反应peptidepeptide bond 键phagemids 噬菌粒phosphorylation 磷酸化作用physical map 物理图谱plasmid 质粒point mutation 点突变poly(A) tail poly(A)尾polymerase 聚合polyploid 多倍体positional cloning 位置性无性繁殖系化primary transcript 初级转录物primer 引物probe 探针prokaryotic 原核的promoter 启动子protease 蛋白purine 嘌呤pyrimidine 嘧啶QRrandom segregation 随机分离RAPD (rapid amplified polymorphic DNA) 快速扩增多态DNAreading frame 阅读码框recessive gene 隐性基因recombinant 重组体recombinant DNA technology 重组DNA技术recombination 重组regulator (gene) 调控基因replica 复制物/ 印模replica plating 复制平皿(板)培养法replication 复制replication origin 复制起点reporter gene 报道基因repression 阻遏repressor 阻遏物repressor gene 阻遏基因resistance strain 抗药性菌株restriction 限制作用restriction enzyme 限制性内切restriction mapping 限制性内切图谱retrovirus 反转录病毒reverse transcription 反转录作用RFLP (restricted fragment length polymorphisms) 限制性断片长度多态现象ribonucleotide 核糖核酸ribose 核糖ribosomal RNA (rRNA) 核糖体RNAribosome 核糖体RNA (ribonucleic acid) 核糖核酸RNA polymerase I RNA聚合IRNA polymerase II RNA聚合IIRNA polymerase III RNA聚合IIIR-plasmid R质粒/ 抗药性质粒Ssecond filial (F2) generation 第二子代self-ligation 自我连接作用shuttle vectors 穿梭载体sigma factor σ因子single nucleotide polymorphism 单核酸多态性single-stranded DNA 单链DNAsister chromatid 姊妹染色单体sister chromosome 姊妹染色体site-directed mutagenesis 定点诱变somatic cell 体细胞Southern blot Southern印迹法splice 拼接star activity 星号活性stationary phase 静止生长期sticky end 黏性末端stop codon 终止密码子structural gene 结构基因supernatant 上层清液supressor 抑制基因Ttelophase 末期template 模板terminator 终止子tetracycline 四环素thymine 胸腺嘧啶tissue culture 组织培养transcription 转录作用transfer RNA (tRNA) 转移RNA transformation 转化作用transgene 转基因translation 翻译/ 平移transmembrane 跨膜triplet 三联体triplet code 三联体密码triploid 三倍体UVvector 载体WWestern blot Western印迹法Aalternative splicing -- Eukaryotic genes are composed of exons and introns, the latter being removed by RNA splicing before transcribed mRNA leaves the nucleus. Commonly, a single gene can encode several different mRNA transcripts, caused by cell- or tissue-specific combination of different exons. This is known as alternative splicing.Annealing -- The time- and temperature-dependent process by which two complementary single-stranded polynucleotides associate to form a double helix (see also hybridization)Antisense strand -- the DNA strand of a gene which, during transcription, is used as a template by RNA polymerase to synthesize a complementary RNA strand.反股-- 意指一股DN**段为基因之所在，因此可用来当做模版使得RNA反转录脢在转录RNA时，可以合成和此DN**段完全结合的RN**段。

α helix α螺旋A helical secondary structure in proteins.Pl. α helices. 蛋白质中一种螺旋形的二级结构。

复数：α helices。

α-amanitin α鹅膏蕈碱A toxin that inhibits the three eukaryotic RNA polymerases to different extents. Name derives from mushroom of genus Amanita in which toxin is found. 一种能不同程度地抑制三种真核生物RNA聚合酶的毒素。

名称来自于产生此毒素的Amanita属蘑菇。

β-galactosidase β-半乳糖苷酶Enzyme that cleaves lactose into galactose and glucose. Name origin: the bond cut by this enzyme is called a β-galactosidic bond. 将乳糖分解为半乳糖和葡萄糖的酶。

名称来源：该酶切割的键称为β-半乳糖苷键。

β sheet β折叠A secondary structure in proteins, relatively flat and formed hydrogen bonding between two parallel or anti-parallel stretches of polypeptide. 蛋白质的一种二级结构，相对平坦，在两条平行的或反向平行的肽段之间形成氢键。

σ subunit σ亚基Component of prokaryotic RNA polymerase holoenzyme. Required for recognition of promoters. 原核生物RNA聚合酶全酶的组成成分。

㊃综述㊃通信作者:崔轶霞,E m a i l :y i x i a _c yx @126.c o m 类风湿关节炎合并骨质疏松症发病机制和相关治疗药物对骨质疏松症影响的研究进展邹 琳,崔轶霞,张娜娜,陈思荣(延安大学附属医院风湿免疫科,陕西延安716000) 摘 要:类风湿关节炎是一种常见而复杂的自身免疫性疾病,临床上表现为滑膜炎症㊁血管翳形成㊁软骨退化和局灶性骨侵蚀,可累及多个系统,伴有许多关节外共病,包括骨质疏松症㊂骨质疏松症导致的关节疼痛和畸形可引起机体相关功能障碍㊁运动缺乏,从而造成严重不良后果㊂本文对类风湿关节炎合并骨质疏松症的发病机制和类风湿关节炎相关治疗药物对骨质疏松症的影响的研究进展进行综述,旨在为临床诊治提供参考㊂关键词:关节炎,类风湿;骨质疏松;发病机制;药物影响中图分类号:R 593.22 文献标志码:A 文章编号:1004-583X (2023)03-0279-06d o i :10.3969/j.i s s n .1004-583X.2023.03.016 类风湿关节炎(r h e u m a t o i da r t h r i t i s ,R A )是一种以慢性㊁侵蚀性㊁对称性多关节炎为主要临床表现的自身免疫性疾病㊂流行病学研究显示,R A 可发生于任何年龄段,其发病率随着年龄的增加呈增长趋势,在40~50岁人群中好发,女性发病率约为男性的3~5倍,全球发病率为0.5%~1%[1]㊂骨质疏松症(o s t e o po r o s i s ,O P )是一种以骨量降低和骨组织微结构破坏为特征的代谢性骨病,为R A 常见的并发症之一㊂O P 给R A 患者带来严重不良预后,使其生活质量下降,从而大大提高了R A 的致残率和病死率㊂本文对R A 合并O P 的发病机制和R A 相关治疗药物对O P 的影响的研究进展进行综述,旨在为临床诊治提供参考㊂1 R A 合并O P 的发病机制1.1 信号转导通路1.1.1 无翅型MMT V 整合位点家族/β-连环蛋白(w i n g l e s st y p e MMT V i n t e g r a t i o n s i t ef a m i l y /β-c a t e n i n ,W n t /β-c a t e n i n )信号通路 W n t 信号通路在体内参与细胞的增殖㊁迁移,组织稳态㊁再生,胚胎发育等多种生物学过程㊂W n t 蛋白与卷曲蛋白受体及低密度脂蛋白受体相关蛋白5/6(l o w -d e n s i t y l i p o p r o t e i nr e c e p t o r -r e l a t e d p r o t e i n5/6,L R P 5/6)结合,触发经典W n t /β-c a t e n i n 通路,参与R A 的发病,使间充质前体细胞和骨软骨祖细胞分化为成骨细胞,参与成骨细胞的增殖和分化㊂β-c a t e n i n 转移到细胞核中,调节O s t e r i x 信号轴及R u n x 家族转录因子2(R u n x f a m i l y t r a n s c r i pt i o n f a c t o r 2,R u n x 2)的表达[2]㊂W n t 信号传导受到卷曲蛋白受体相关蛋白㊁W n t 抑制因子1㊁硬化素和D i c k k o p f (D K K )家族蛋白等抑制剂的调节㊂硬化素拮抗L R P 5/6下游信号传导,进而抑制W n t 信号传导,增强成骨细胞凋亡,D K K -1与L R P 5/6及单通道跨膜受体K r e m e n 1/2结合,导致复合物的内化,进而抑制W n t信号传导,下调骨保护素(o s t e o p r o t e ge r i n ,O P G )和上调核因子κB (n u c l e a rf a c t o rk a p p a -B ,N F -κB )受体活化因子配体(r e c e pt o r a c t i v a t o r o f N F -κB l i ga n d ,R A N K L )的表达[3]㊂1.1.2 骨形态发生蛋白(b o n e m o r p h o ge n e t i c pr o t e i n ,B M P )/果蝇与秀丽隐杆线虫蛋白同源物(h o m o l o g u e s o ft h e d r o s o p h i l a p r o t e i n ,m o t h e r s a g a i n s t d e c a p e n t a p l e gi c a n d t h e c a e n o r h a b d i t i s e l e ga n s p r o t e i n ,S m a d )信号通路 B M P 是转化生长因子-β(t r a n s f o r m i n gg r o w t hf a c t o r -β,T G F -β)家族中最大的亚类,可刺激软骨细胞的增殖和分化,S m a d 蛋白将T G F -β与其受体结合产生的信号从细胞质传递到细胞核,从而调节成骨细胞和破骨细胞分化,在骨骼发育㊁骨形成和骨稳态过程发挥作用[4]㊂B M P 以S m a d 1/5/8(B -S m a d s )为信号传导器,与细胞表面具有丝氨酸㊁苏氨酸激酶活性的受体B M P 受体Ⅰ/Ⅱ结合磷酸化,磷酸化的B -S m a d s 与S m a d 4形成复合物在细胞核中积累,与转录辅助因子或抑制因子结合后再与目标基因的调控元件结合进行转录调控[5]㊂经典B M P -S m a d 信号通路的每一步都受到正性或负性因子调控,骨形成蛋白和激活蛋白膜结合抑制物(b o n em o r p h o ge n e t i c p r o t e i na n d a c t i v a t i n g p r o t e i n m e m b r a n e b i n d i n g in h i b i t o r ,B AM B I )被称为T G F -β伪受体,与B M P 受体Ⅰ结构相似但缺乏丝氨酸/苏氨酸磷激酶信号传导结构域,㊃972㊃‘临床荟萃“ 2023年3月20日第38卷第3期 C l i n i c a l F o c u s ,M a r c h20,2023,V o l 38,N o .3Copyright ©博看网. All Rights Reserved.B AM B I与B M P受体Ⅱ结合后形成异构复合物,阻止B M P-S m a d信号传导[6]㊂1.1.3磷脂酰肌醇-3-激酶(p h o s p h a t i d y l i n o s i t o l-3-k i n a s e,P I3K)/蛋白激酶B(p r o t e i nk i n a s eB,A k t)信号通路 P I3K-A k t信号通路受生长因子和细胞因子的调节,通过与受体酪氨酸激酶和G-蛋白偶联受体结合后磷酸化胞内的酪氨酸残基发挥作用[7]㊂P I3K-A k t信号通路调控成骨细胞和破骨细胞的存活和分化,进而维持骨稳态,并且与调控成骨分化的通路有密切联系㊂W n t/β-c a t e n i n信号通路受到小鼠糖原合成酶激酶-3β形成的降解复合物的调节, A k t通过磷酸化小鼠糖原合成酶激酶-3β使其失活丧失蛋白功能而影响W n t/β-c a t e n i n通路[8]㊂此外, A k t可以负调控B M P-S m a d通路中S m a d,抑制活化的S m a d1和S m a d5在细胞核聚集,抑制B M P-S m a d 信号传导[9]㊂1.1.4J a n u s激酶信号转导和转录激活因子(J a n u s k i n a s e/s i g n a l t r a n s d u c e r a n d a c t i v a t o r o f t r a n s c r i p t i o n,J A K/S T A T)信号通路J A K/S T A T 信号通路是多种细胞因子㊁激素和生长因子的重要下游介质,在骨发育㊁代谢和愈合中起重要作用[10]㊂白细胞介素(i n t e r l e u k i n,I L)-6通过糖蛋白130激活糖蛋白130相关的J A K,从而诱导S T A T3激活的J A K/S T A T通路上调R A N K L,导致破骨细胞分化,骨吸收增强[11]㊂W a n g等[12]研究发现,降低I L-6水平抑制了J A K/S T A T信号通路相关基因的m R N A 水平,减少了S T A T1蛋白表达并增加了成骨分化,相反增强I L-6的表达增加了S T A T1蛋白表达及J A K/S T A T信号通路相关基因的m R N A水平,降低成骨细胞的分化㊂1.1.5神经源性位点缺口同源蛋白(n e u r o g e n i c l o c u sn o t c h h o m o l o gp r o t e i n,N o t c h)信号通路N o t c h信号通路主要由N o t c h受体㊁N o t c h配体㊁转录因子及N o t c h信号调节分子组成,也包括N o t c h 的4种受体蛋白(N o t c h-1㊁2㊁3㊁4)及5种配体蛋白(D e l t a-1㊁3㊁4,J a g g e d-1㊁2),在先天性和获得性免疫的识别㊁调节中发挥作用,与T细胞㊁B细胞及巨噬细胞等免疫细胞作用调节炎症反应,参与R A[13-14]㊂此外,N o t c h通路在调控与维持骨稳态方面发挥重要的作用,参与破骨细胞的分化㊁成熟,影响软骨形成㊁骨形成和骨吸收㊂然而,不同N o t c h受体-配体结合激活的N o t c h通路存在不同的调控作用,如N o t c h1/ J a g g e d1轴抑制破骨细胞生成,而N o t c h2/D e l t a1轴促进破骨细胞生成[15]㊂1.1.6 H e d g e h o g信号通路 H e d g e h o g信号通路由H e d g e h o g信号蛋白㊁P t c h e d㊁S m o o t h e n e d特异性受体㊁G l i蛋白和下游靶基因4部分构成,H e d g e h o g 信号蛋白分为S h h㊁I h h和D h h,其中S h h分布最广,阳性表达率高,相关研究最多[16]㊂经典的H e d g e h o g 信号通路参与R A的发病㊂有研究发现,R A患者的S h h水平与类风湿因子(r h e u m a t o i d f a c t o r,R F)及抗环瓜氨酸肽(c y c l i cc i t r u l l i n a t e d p e p t i d e,C C P)抗体呈正相关,S h h信号通过细胞外信号调节丝裂原活化蛋白激酶信号介导R A成纤维样滑膜细胞的增殖和迁移[17]㊂此外,H e d g e h o g信号通路在骨髓间充质干细胞向成骨细胞分化及调控成骨细胞的增殖㊁分化㊁活性中发挥重要作用[18]㊂1.1.7新兴的骨质重塑调节因素微小R N A (m i c r o R N A,m i R N A)是非编码的单链R N A分子,在骨细胞内发挥转录后的调控作用,长链非编码R N A和环状R N A参与骨代谢的调节[19]㊂一些m i R N A针对W n t信号级联的关键成分,通过靶向部分互补序列的m R N A来转录调节基因表达,导致W n t信号的减弱或增强,例如,m i R-37c㊁m i R-23a㊁m i R-30e通过靶向W n t受体或共同受体(W n t3㊁L R P5/6)使W n t信号传导减少,使成骨细胞分化减少,相反,m i R-27a㊁m i R-142㊁m i R-29a㊁m i R-218㊁m i R-98㊁m i R-335㊁m i R-542通过靶向卷曲蛋白受体相关蛋白㊁D K K-1㊁硬化素等W n t抑制剂和负调控因子诱导W n t信号传导和成骨分化[20]㊂此外,虽然2022年L i a n g等[21]针对m i R N A对骨细胞活动的影响进行了探讨,涉及W n t信号通路㊁R u n x2㊁B M P/ S m a d s通路㊁R A N K L通路等多条信号通路,虽然这些研究仅涉及动物模型和队列研究,转化为临床有待测试,却对发现O P的潜在治疗靶点具有重要意义㊂1.2炎症因子众所周知,炎症因子的过度表达可增加R A患者发生O P的风险,以肿瘤坏死因子-α(t u m o rn e c r o s i s f a c t o rα,T N F-α)㊁I L-6和I L-1为代表的促炎细胞因子诱导R A N K L的表达刺激破骨细胞分化,增强骨吸收,打破骨形成和骨吸收间的平衡㊂1.2.1 T N F-α T N F-α在R A中的骨吸收作用已经被证实,推测其可能通过上调破骨细胞前体N F-κB受体活化因子和巨噬细胞集落刺激因子受体的表达,进而促进破骨细胞增殖,增强破骨细胞活性,影响破骨细胞分化[22]㊂然而,T N F-α不是破骨细胞形成㊁发生侵蚀性关节炎和骨溶解所必须的,这些改变在T N F-α缺乏的条件下也可发生㊂Z o u等[23]认为, T N F-α是破骨细胞生成的自分泌因子,在R A N K L㊃082㊃‘临床荟萃“2023年3月20日第38卷第3期 C l i n i c a l F o c u s,M a r c h20,2023,V o l38,N o.3Copyright©博看网. All Rights Reserved.的诱导下,破骨细胞生成增加并且随着T N F-α的增加而增加㊂1.2.2I L-1和I L-6I L-1直接作用于破骨细胞表面的I L-1受体,激活N F-κB,在T N F-α诱导破骨细胞形成的过程中发挥桥梁作用,且通过作用于前列腺素E2的合成影响破骨细胞形成,参与多核破骨细胞形成[24]㊂I L-6在R A患者的滑膜中大量表达,诱导关节炎症和损伤,软骨退化,关节侵蚀和变窄,I L-6与I L-6受体结合由J A K/S T A T信号通路介导直接促进R A患者成纤维细胞样滑膜细胞中R A N K L的表达,并且是T N F-α和I L-17诱导R A N K L所必需的[25]㊂此外,I L-6水平与慢性滑膜炎和关节破坏的严重程度相关,且I L-6水平降低可作为患者良好预后的标志[26]㊂1.2.3自身抗体瓜氨酸化蛋白抗体(a n t i-c i t r u l l i n a t ed p r o te i na n t i b o d i e s,A C P A s)通过与细胞表面瓜氨酸波形蛋白结合或形成免疫复合物调节细胞功能,A C P A s存在于R A发病的早期,在R A患者血清和关节滑膜内与破骨细胞前体细胞结合,诱导T N F-α的表达和释放,刺激破骨细胞分化,直接诱导骨丢失[27]㊂与A C P A s阴性的R A患者相比, A C P A s阳性的R A患者关节周围骨的破坏程度更明显,骨小梁结构改变㊁体积变小㊁数量变少㊁网络不均匀性增加,并且A C P A s与R F产生叠加效应,与R A 侵蚀关节数目及严重程度有关,A C P A s和R F均阳性的R A患者关节侵蚀程度更重[28-29]㊂E n g d a h l 等[30]研究发现,瓜氨酸波形蛋白所诱发关节炎较甲基化牛血清白蛋白轻,但关节周围骨丢失更明显,可能与抗瓜氨酸波形蛋白作用于关节周围骨髓中破骨细胞前体和增加成熟破骨细胞的浸润而触发关节周围骨丢失有关,这解释了在R A患者早期炎症发作前为什么骨量已经改变,甚至在A C P A s阳性而未确诊的R A患者中就已经发生骨量减少㊂1.3维生素D缺乏与肌少症维生素D缺乏与自身免疫性疾病的发病机制有关,低维生素D水平被认为是R A发病的危险因素,可抑制单核细胞向树突状细胞分化,减少了抗原提呈细胞向T细胞分化,且作用于B细胞,从而使免疫球蛋白的产生减少[31]㊂约有半数R A患者存在25-羟基维生素D缺乏(<20 n g/m l),中老年人低25-羟基维生素D水平与低骨量的相关性已被证明,25-羟基维生素D除了参与骨骼和钙代谢,还在免疫调节中也发挥重要作用㊂有研究报道,R A患者血清25-羟基维生素D水平明显降低,且与R A易感性有关,与疾病活动呈负相关[32]㊂肌少症是一种以肌肉质量㊁力量和功能低下为特征的全身性疾病,增加患者身体残疾㊁生活质量低下和死亡的风险㊂缺乏物理锻炼被认为是肌少症的首要危险因素,除此之外,激素和细胞因子失衡也会导致肌肉质量和力量的损失,极端肌肉损失发生由与年龄相关的激素浓度下降及促炎细胞因子(T N F-α㊁I L-6)所介导[33]㊂L i a n等[34]招募了549名来自中国的R A患者和158名对照受试者,并对其进行骨密度和骨骼肌质量测量,结果显示肌少症在R A患者中的发生率为61.7%,患有肌少症R A患者的年龄㊁病程㊁血沉㊁C反应蛋白水平㊁28个关节疾病活动度评分和健康评估问卷-残疾指数显著升高㊂并且C h u 等[35]研究显示,肌少症和维生素D缺乏对R A患者椎体骨质疏松性骨折具有协同作用,可能与维生素D 不足损害肌肉功能有关㊂2R A相关治疗药物对O P的影响2.1激素糖皮质激素(g l u c o c o r t i c o i d s,G C)在R A的治疗中运用广泛,长期大剂量G C对骨骼产生的负面影响是众所周知的,然而短期小剂量G C治疗的作用仍存在争议㊂全身性应用G C对骨形成和骨吸收有很大的抑制作用,特别是长期大剂量给药可促进破骨细胞形成,抑制成骨细胞活性,且促进肾脏对钙的排泄,抑制肠道对钙的吸收,减少性激素分泌,导致肌肉萎缩和步态障碍,进而增加骨质流失和骨折风险,即使在G C停药后,骨折风险仍可持续1年[36]㊂实际上,小剂量G C(<10m g/d)可以减少R A患者的全身炎症反应,对骨的保护作用能够抵消其对骨的不良影响,处于疾病早期和活动期的R A 患者使用小剂量G C不会出现显著的骨密度改变,尤其是泼尼松剂量<5m g/d[37-38]㊂然而,临床上G C 的不规范使用并不少见,受文化水平及诊疗条件等影响,大多R A患者将G C当做灵丹妙药而长期大剂量口服,糖皮质激素性骨质疏松患者大量出现,骨质疏松性骨折也越来越多㊂一项基于中国人的队列研究显示,R A使用过G C组O P的发生率为41.6%,明显高于未使用G C组(29.4%),并与使用疗程有关,而与G C每日剂量无关,且骨量丢失多发生在G C治疗的第一年,在最初治疗的3~6个月,糖皮质激素性骨质疏松的患病率没有明显增加,维持在30.0%左右,但在治疗6~12个月迅速上升,12个月后随着G C疗程的延长而向上波动[39]㊂为了最大限度地降低骨折风险,临床实践指南建议,对于预计在3~6个月内每天接受相当于5~7.5m g泼尼松治疗的患者,应给予抗O P药物治疗[40]㊂2.2传统合成改善病情抗风湿药(d i s e a s e-㊃182㊃‘临床荟萃“2023年3月20日第38卷第3期 C l i n i c a l F o c u s,M a r c h20,2023,V o l38,N o.3Copyright©博看网. All Rights Reserved.m o d i f y i n g a n t i r h e u m a t i cd r u g s,D MA R D s)临床上普遍使用G C联合传统合成D MA R D s治疗R A,目前常用的传统合成D MA R D s包括甲氨蝶呤(m e t h o t r e x a t e,MT X)㊁羟氯喹(h y d r o x y c h l o r o q u i n e,H C Q)㊁来氟米特㊁柳氮磺吡啶和新型小分子抗风湿药艾拉莫德,这些药物不仅在治疗R A中具有突出作用,对骨密度也具有显著影响㊂低剂量MT X(ɤ25m g/周)是欧洲风湿病防治联合会(E U L A R)指南推荐治疗R A的一线用药, MT X通过上调血清O P G,降低R A N K L的表达,竞争性抑制R A N K和R A N K L的结合以及调节R A N K L/R A N K/O P G通路来抑制骨破坏,与G C联合使用可预防骨侵蚀和关节破坏[41]㊂然而,近年来越来越多长期使用MT X的患者出现了所谓的 MT X骨病 ,发生多发性跟骨/胫骨远端和近端以及股骨远端干骺端不全性骨折,可能与MT X抑制原始骨髓基质细胞增殖,损害成骨细胞增殖及活性有关[42]㊂H C Q可能影响成骨细胞和破骨细胞形成,体外研究表明,H C Q降低了成熟破骨细胞的细胞内p H值,抑制破骨细胞的形成,导致骨吸收减少[43]㊂来氟米特抑制基质金属蛋白酶(MM P-1㊁MM P-9)的表达,从而抑制关节骨的破坏,是唯一一个与腰椎骨密度显著增加相关的传统合成D MA R D s[44-45]㊂柳氮磺吡啶可能会增加氧化应激,通过减少R A N K L 的表达㊁增加O P G的表达来抑制破骨细胞的形成[46]㊂艾拉莫德抑制环氧合酶-2而起到抗炎作用,抑制骨吸收,促进骨形成[47]㊂临床上MT X单一疗法失败的R A患者常联合传统合成D MA R D s进行治疗,C a r b o n e等[48]研究发现,单独或联合使用MT X㊁H C Q㊁柳氮磺吡啶对骨折风险没有显著影响㊂2.3生物制剂随着生物制剂的问世,R A的治疗有了重大突破,以T N F-α拮抗剂(英夫利昔单抗㊁阿达木单抗㊁依那西普)为代表的早期生物制剂作为治疗R A的第四代药物,具有高选择性药理作用及低毒副作用的特点,较传统合成D MA R D s起效更快㊁作用更强㊂T N F-α拮抗剂已经被证实可减轻R A患者关节骨侵蚀㊁全身骨量减少及滑膜炎,英夫利昔单抗不仅可以缓解关节炎症,还可以减少全身骨损失和抑制肌腱炎症[49]㊂阿达木单抗减轻脊柱和髋关节骨质流失,特别是在开始治疗的第一年,而手部和髋部的骨密度分别在治疗1年和4年后持续下降[50]㊂I L-6抑制剂托珠单抗能够有效控制R A炎症反应,延缓关节损伤,并且托珠单抗联合MT X消除了疾病活动和关节损伤间的相关性[51]㊂P o u t o g l i d o u等[52]通过构建C I A模型发现,托珠单抗可使肌腱组织中T N F-α和I L-23显著降低,并且明显改善小梁微结构,改善肌腱形态㊂靶向小分子药物J A K抑制剂托法替尼抑制J A K/S T A T信号传导进而减弱I L-6功能,促进成骨分化,降低破骨细胞的分化和活性,并且不损害软骨分化[53]㊂新型生物制剂阿巴西普是治疗R A的第五代药物,直接抑制T细胞活化,对股骨颈骨密度的增加有显著影响[54]㊂3结论综上,R A和O P的发病在信号转导通路及炎症因子间相互作用㊁相互联系,并且与维生素D水平及物理锻炼息息相关,R A治疗药物对O P的影响也不容小觑㊂进一步探究这些因素与R A合并O P的关系可提供更有效的治疗方式,减少R A患者骨折的发生风险,改善R A患者生活质量㊂参考文献:[1] T a n a k aY.M a n a g i n g o s t e o p o r o s i s a n d j o i n t d a m a g e i n p a t i e n t sw i t hr h e u m a t o i d a r t h r i t i s:A n o v e r v i e w[J].J C l i n M e d,2021,10(6):1241.[2] M i a oC G,Y a n g Y Y,H eX,e t a l.W n t s i g n a l i n gp a t h w a y i nr h e u m a t o i da r t h r i t i s,w i t hs p e c i a le m p h a s i so nt h ed i f f e r e n tr o l e s i ns y n o v i a l i n f l a mm a t i o na n db o n er e m o d e l i n g[J].C e l lS i g n a l,2013,25(10):2069-2078.[3] C i c iD,C o r r a d o A,R o t o n d o C,e ta l.W n ts i g n a l i n g a n db i o l o g ic a l t h e r a p y i n r h e u m a t o id a r t h r i t i s a n d s p o n d y l o a r t h r i t i s[J].I n t JM o l S c i,2019,20(22):5552.[4] Z o u M L,C h e nZ H,T e n g Y Y,e ta l.T h eS m a dd e p e n d e n tT G F-βa n d B M Ps i g n a l i n gp a t h w a y i n b o n er e m o d e l i n g a n dt h e r a p i e s[J].F r o n tM o l B i o s c i,2021,8:593310. 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