鲑鱼鱼白DNA延缓小鼠衰老的实验研究

目的 探讨鲑鱼鱼白DNA(salmon milt DNA，SMD)对小鼠胸腺增龄性萎缩的作用及作用机制。方法 l0月龄雌性：BALB／c小鼠按体重随机分成缓冲液对照组(control group，C组)、低剂量组(1ow dosage group，L组)和高剂量组(high dosage group，H组)，每组26只。在标准饲料基础上分别每日灌胃0．1mo|／L的柠檬酸钠缓冲液和166，67mg／(kg&;#183;d)，333，33mg／(kg&;#183;d)的SMD.5周后，无菌取胸腺，测量胸腺脏器指数；每个胸腺单独蜡块包埋，切片用Image—pro Plus专业图像分析系统(4．0版)进行细胞计数和皮质厚度测量并经SAS(6．12版)统计软件分析数据。应用基因芯片技术在c组和H组胸腺组织中筛选表达差异的基因片段、RT．PCR鉴定部分片段。结果 SMD对小鼠的体重、胸腺重量和胸腺指数均无影响(各指标，&;lt;3．0，P&;gt;0．05)；高剂量组小鼠的胸腺皮、髓质细胞数量与对照组相比均显著增多[皮质D(H．C)=9．46，P&;lt;0．0l；髓质t(H.C)=2．53，P&;lt;0．05]；低剂量组的胸腺皮、髓质细胞数量与对照组相比差异均不显著[皮质D(L,C)=3．65，P&;gt;0．05；髓质t(L.C)=0．8，P&;gt;0．05]；高、低剂量组的胸腺皮质平均厚度均显著高于对照组[t(H,C)=4．0l，P&;lt;0．0l；t(L.C)=2．80，P&;lt;0．05]；经基因芯片技术初筛出112条差异表达的基因片段；Genebank登录号为Aw209102．U23789，X80232的3条上调表达的基因片段，经RT-PCR鉴定，在高剂量组确实存在上调表达。结论 SMD有通过促进细胞增殖相关基因的表达并同时促进发育、分化相关基因表达而逆转胸腺增龄性萎缩的可能。     

有氧运动延缓衰老线粒体DNA突变的机制

目的:线粒体DNA突变在衰老过程中起核心作用。总结有氧运动在延缓机体衰老过程中对线粒体DNA突变的影响,探讨有氧运动延缓衰老的机制。资料来源:应用计算机检索Medline数据库1996-01/2006-01期间关于线粒体DNA突变与有氧运动延缓衰老的文章,检索词为"aerobics exercise,aging,mtDNA mutation",限定文章语言种类为English。同时计算机检索中国期刊全文数据库、万方数据库1994-01/2006-12期间的有氧运动、衰老和线粒体DNA突变相关的文章,检索词"衰老,线粒体DNA突变,有氧运动",并限定文章语言种类为中文。资料选择:对资料进行初审,纳入标准:①有氧运动延缓衰老关系的理论研究。②线粒体DNA突变与衰老关系的基础与临床研究。③有氧运动对线粒体DNA突变的影响研究。资料提炼:共收集到36篇线粒体DNA突变与有氧运动延缓衰老相关的文献,均为全文,32篇符合纳入标准,排除4篇重复性研究。资料综合:①有氧运动可以通过减少线粒体DNA突变,从而达到延缓机体的衰老。有氧运动可能通过影响自由基及抗氧化系统、细胞凋亡、线粒体的结构和功能,对衰老进程产生重要影响。②线粒体DNA突变随增龄而积累,达到一定阈值后,可导致细胞能量供应的严重障碍,从而造成组织器官生理功能的减退。③长期有氧运动可通过刺激心肌、骨骼肌线粒体的生成及蛋白质的合成而延缓线粒体形态结构的改变,有利于维持线粒体功能以满足机体对其能源的需求。结论:中、低强度有氧运动可以提高机体有氧工作能力,增进线粒体氧化磷酸化的功能,对延缓衰老具有一定的积极作用。有氧运动可以减少心肌、骨骼肌等线粒体DNA突变,提示有氧运动在延缓衰老机制中,减少线粒体DNA突变是可能机制之一。     

衰老小鼠线粒体DNA缺失突变的研究

目的测定不同年龄组及D-半乳糖诱导的衰老小鼠线粒体DNA(mtDNA)缺失突变,探讨mtDNA缺失突变与衰老的关系。方法用聚合酶链反应技术和琼脂糖凝胶电泳检测mtDNA缺失片段,用密度扫描技术对扩增片段进行相对定量。缺失片段构建质粒后,直接测序进行鉴定。结果全部小鼠的肝、脑与海马组织内均存在4239bp的mtDNA缺失片段。老年鼠肝、脑与海马组织内mtDNA缺失的比例较青幼年鼠明显增加(P<均0.01)。衰老模型鼠不同组织mtDNA缺失的相对百分含量均明显高于实验对照组(P均<0.01),肝组织中mtDNA缺失的比例较大脑皮层和海马组织更高。结论4239bp的mtDNA缺失片段普遍存在于小鼠中,与增龄和衰老相关,可作为衰老的一种分子生物标志。     

马齿苋粉延缓衰老的实验研究

目的:研究马齿苋粉延缓衰老的作用,为马齿苋的应用提供理论依据。方法:将大鼠分成4组,每组10只:对照组(喂普通饲料)、马齿苋大、中、小剂量组(喂普通饲料并分别加入5.00,2.50和1.25g马齿苋粉)连喂4周。于实验第29天大鼠采血测血清丙二醛和超氧化物歧化酶(superoxidedismutase,SOD)。果蝇生存实验,将果蝇分成4组,对照组只用普通玉米培养基,实验组按大、中、小剂量将马齿苋粉分别按20%,10%,5%加入培养基中,计算果蝇半数死亡时间、平均寿命、最高寿命。结果:马齿苋组大鼠血清丙二醛降低,大、中、小剂量3组分别为:(3.84±0.43),(4.06±0.32)和(4.58±0.45)μmol/L,与对照组比较有显著差异(t=10.22,13.59,13.43,P<0.01)。血清SOD活性高于对照组,大、中、小剂量3组分别为:(3.46±0.24),(3.25±0.20),(3.00±0.15)kat/L,与对照组比较,差异有显著性意义(t=6.03,4.50,1.96;P<0.05～0.01)。马齿苋粉延长了果蝇的半数死亡时间、平均寿命和最高寿命。结论:马齿苋粉有抗氧化作用,能够延缓机体衰老,是有开发价值的保健品。     

马齿苋粉延缓衰老的实验研究

目的：研究马齿苋粉延缓衰老的作用，为马齿苋的应用提供理论依据。方法：将大鼠分成4组，每组10只：对照组(喂普通饲料)、马齿苋大、中、小剂量组(喂普通饲料并分别加入5．00，2．50和1．25g马齿苋粉)连喂4周。于实验第29天大鼠采血测血清丙二醛和超氧化物歧化酶(superoxide dismutase，SOD)。果蝇生存实验，将果蝇分成4组，对照组只用普通玉米培养基，实验组按大、中、小剂量将马齿苋粉分别按20％，10％，5％加入培养基中，计算果蝇半数死亡时间、平均寿命、最高寿命。结果：马齿苋组大鼠血清丙二醛降低，大、中、小剂量3组分别为：(3．84&;#177;0．43)，(4．06&;#177;0．32)和(4．58&;#177;0．45)μmol／L，与对照组比较有显著差异(t=10．22，13．59，13．43，P&;lt;0．01)。血清SOD活性高于对照组，大、中、小剂量3组分别为：(3．46&;#177;0．24)，(3．25&;#177;0．20)，(3．00&;#177;0．15)kat／L，与对照组比较，差异有显著性意义(t=6．03，4．50，1．96；P&;lt;0．05—0．01)。马齿苋粉延长了果蝇的半数死亡时间、平均寿命和最高寿命。结论：马齿苋粉有抗氧化作用，能够延缓机体衰老，是有开发价值的保健品。     

珍珠粉延缓衰老作用的实验研究

珍珠是我国传统名贵药材 ,长期以来都被用作延缓衰老、美容养颜 ,为观察珍珠粉的抗衰老作用 ,作者自2002年4～8月进行了实验研究 ,现将结果报告如下。1抗氧化作用试验1.1样品珍珠粉(××有限公司提供 ) ,为白色粉末。1.2剂量设计设低、中、高3组剂量 ,分别为0.5、1.5、3g/kg ,分别相当于人体剂量的5、10、30倍。并设高龄(12～18个月龄)动物对照组、低龄(3～6个月龄)动物对照组 (以蒸馏水灌胃 )。1.3样品处理取样品0.5、1.5、3g,分别加蒸馏水至10ML,使成均匀混悬液 ,即为低、中、高3组剂量的供试液。1.4实验动物SD大白鼠 ,20个月龄 ,雄性 ,…     

黄芪延缓果蝇衰老作用的实验研究

目的：探讨黄芪对果蝇的延缓衰老作用及其机理。方法：将未交配的果蝇随机分成5组，给予含维生素E和不同浓度黄芪的培养基喂养，用生存试验检测平均寿命、最高寿命；羟胺法测定SOD活性；TBA法测定MDA含量。结果：黄芪能显著延长果蝇的平均寿命和最高寿命，能使果蝇体内的SOD活性明显升高。使果蝇体内的MDA含量降低。结论：黄芪具有抗氧化、延缓衰老的作用。     

雌激素对衰老小鼠海马神经元受损DNA的修复作用

目的观察模拟绝经及D-半乳糖模型小鼠空间认知的改变,评价雌激素保护海马神经元功能的分子机制。方法成年雌性C57BL/6小鼠双侧卵巢切除(OVX)并皮下注射D-半乳糖100 mg/kg造模。雌激素替代治疗(ERT)组腹腔注射17β-雌二醇(E2)50 μg/kg。造模与ERT共8周。Morris水迷宫测试空间学习记忆功能,试剂盒检测雌激素与氧化应激酶。免疫组化染色8-氧鸟嘌呤脱氧核苷 (8-oxo-dG),Western blotting检测脑海马MTH1表达。结果模型组血中E2水平仅为假手术组的1/5(P<0-01),而ERT组明显升高(P<0-01);模型组寻台时间明显延长(P<0-01),ERT组缩短(P<0-05);模型组SOD、GSH-Px明显降低而MDA明显增高(P<0-05),ERT组接近正常。8-oxo-dG作为DNA氧化损伤标志物在模型组海马明显增多,而DNA修复蛋白MTH1表达明显减少(P<0-05),而两者在ERT组恢复正常(P<0-05)。结论雌激素可通过对海马神经元受损DNA的修复及抗氧化作用改善衰老模型的空间认知功能。     

回春胶囊延缓衰老的临床观察和实验研究

我院自1985—1988年应用回春胶囊进行延缓衰老的研究,现将临床观察资料分析报告以下: 一、临床资料采用抽签式随机分组方法选择病例.各组病人均由门诊及住院患者中选择,符合中医衰老辨证标准.共观察212例病人,其中合并冠心病的104例,高脂血症的100例,其它无严重心、脑、肺、肝、肾等疾病的老年期及老年前期患者106例.以上病例均符合全国统一诊断标准.     

表达谱芯片与DNA甲基化芯片综合分析探索鼻咽癌发生、发展的分子靶标

目的综合分析表达谱芯片与DNA甲基化芯片,探索鼻咽癌发生、发展的分子靶标与潜在治疗靶点。方法在GEO公共数据库下载编号为GSE64634的表达谱芯片数据以及编号为GSE52068的DNA甲基化芯片数据;利用R语言的相关工具包对表达谱芯片进行差异表达分析,对DNA甲基化芯片进行差异甲基化位点分析;利用DAVID数据库对筛选出的差异表达基因进行基因功能分析和信号通路分析;利用实时荧光定量PCR和甲基化特异性PCR进一步验证生物信息学分析的结果。结果表达谱芯片分析获得筛选出2 327个差异表达基因,其中1 777个基因表达下调,550个基因表达上调;DNA甲基化芯片分析获得2 321个差异甲基化位点,其中2 228个低甲基化位点,93个高甲基化位点;对表达谱芯片和DNA甲基化芯片进行综合分析,找到4个表达水平升高且甲基化修饰水平降低的基因,并利用定量PCR、表达谱芯片和DNA甲基化芯片成功验证该结果。结论综合分析表达谱芯片和DNA甲基化芯片,筛选出4个与鼻咽癌发生、发展相关的分子靶标和潜在的治疗靶点。     

Optimized PCR amplification of influenza A virus RNA using Tth DNA polymerase,incorporating uracil N glycosylase (UNG) in a single tube reaction

An optimized reaction condition for amplification of influenza A virus RNA, by thermus thermophilus (Tth) DNA polymerase-based PCR, incorporating uracil N glycosylase (UNG) and dUTP in the reaction has been determined. DUTP could not be substituted for all dTTP sites when UNG was present in the reaction. The relative concentration of dUTP and dTTP has been optimized for allowing amplification of the target RNA. It has been verified that the amplified product DNA had sufficient dUTP and was digestable by UNG. Using the optimized reaction condition, influenza A virus-specific DNA fragment could be amplified and detected in 15 of 15 culture positive (for influenza A virus) nasopharyngeal specimens. J. Clin. Lab. Anal. 11:323–327, 1997. © 1997 Wiley-Liss, Inc.     

肺炎克雷伯菌介导碳青霉烯类耐药的基因型检测

目的对临床分离的碳青霉烯类抗生素耐药的肺炎克雷伯菌进行耐药基因型分析。方法采用琼脂稀释法检测菌株最低抑菌浓度(MIC),用聚合酶链反应(PCR)、DNA测序、脉冲场琼脂糖凝胶电泳(PFGE)、等电点聚焦电泳(IEF)和质粒分子杂交等技术对实验菌株进行基因组DNA同源性、介导耐药的基因型、耐药酶pI和耐药基因携带状态等研究。结果12株肺炎克雷伯菌的亚胺培南和美罗培南M IC值分别为16~64μg/m l和8~256μg/m l;对其他被测的β-内酰胺类和喹诺酮类药物广泛耐药,氨基糖苷类耐药型不定。12株菌均扩增出编码碳青霉烯类抗生素耐药的blaKPC-2和编码β-内酰胺酶的blaSHV基因;部分菌株扩增出blaTEM或/和blaCTX-M基因。IEF和分子杂交揭示KPC-2的等电点为6.8,编码基因被携带在一个约56 kb大小的质粒上;PFGE结果显示12株细菌可分为2个克隆型。结论产KPC-2型碳青霉烯酶是本院肺炎克雷伯菌耐碳青霉烯类药物的主要原因,垂直传播以及通过质粒传递耐药基因可能是其在本医院流行的主要方式。     

Biodegradable poly(ethylenimine) for plasmid DNA delivery

Poly(ethylenimine) (PEI) has been known as an efficient gene carrier with the highest cationic charge potential. High transfection efficiency of PEI, along with its cytotoxicity, strongly depends on the molecular weight. Synthesis of cationic copolymers derived from the low molecular weight of PEI and hydrophilic poly(ethylene glycol) (PEG), which are water soluble and degradable under physiological conditions, was investigated for plasmid delivery. Hydrophilic PEG is expected to reduce the toxicity of the copolymer, improve the poor solubility of the PEI and DNA complexes, and help to introduce degradable bonds by reaction with the primary amines in the PEI. Considering the dependence of transfection efficiency and cytotoxicity on the molecular weight of the PEI, high transfection efficiency is expected from an increased molecular weight of the copolymer and low cytotoxicity from the introduction of PEG and the degradation of the copolymer into low molecular weight PEIs. Reaction conditions were carefully controlled to produce water soluble copolymers. Results from a gel retardation assay and zetapotentiometer indicated that complete neutralization of the complexes was achieved at the charge ratios of copolymer/pSV-β-gal plasmid from 0.8 to 1.0 with the mean particle size of the polyplexes ranging from 129.8±0.9 to 151.8±3.4 nm. In vitro transfection efficiency of the synthesized copolymer increased up to three times higher than that of starting low molecular weight PEI, while the cell viability was maintained over 80%.     

Evaluation of an optimized system for random amplified polymorphic DNA (RAPD)-analysis for genotypic mapping of Candida albicans strains

A simple, rapid, and cost-effective protocol has been developed for a PCR-based molecular typing method for Candida albicans, which includes the use of a commercially available medium (Chelex® 100 Resin) for DNA extraction and a single set of two arbitrarily chosen oligonucleotide (10 nt length) primers for random amplified DNA(RAPD)-analysis. The optimized parameters for the amplification components and conditions for the selected primer combination have been determined to avoid artifactual variation (absence/presence of bands) in RAPD banding patterns in repeated assays. The optimized RAPD-assay consistently generated DNA-patterns of 33 genetically unrelated C. albicans isolates that contained ten polymorphic markers in the non-artifactual banding patterns. The intralaboratory reproducibility of RAPD patterns was efficient and consistent provided the optimized amplification conditions were rigidly controlled. Interlaboratory reproducibility was tempered by slight variations in time of cyclers of different thermocyclers. In comparison, the RAPD assay was almost equal to restriction enzyme analysis (REA) (Eco RI digested chromosomal DNA) in discrimination, and the RAPD assay was able to group isolates of C. albicans that were untypable by REA. The protocol outlined for an optimized RAPD-assay of C. albicans has the potential to be widely useful epidemiological screening tool that can be easily applied in the clinical laboratory. © 1996 Wiley-Liss Inc.     

Application of multiple DNA fingerprinting techniques to study the genetic relationships among three members of the subgenus Trypanozoon (Protozoa: Trypanosomatidae)

Three different DNA fingerprinting techniques, the mobile genetic element (MGE)-PCR, simple sequence repeat (SSR)-PCR and random amplified polymorphic DNA (RAPD)-PCR, were used to define a large set of genetic markers to study genetic similarity within and among Trypanosoma brucei, Trypanosoma equiperdum and Trypanosoma evansi strains (n=18) from China, Africa and South America and to investigate their genetic relationships. Using the three fingerprinting techniques, >890 bands (ranging in size from 0.2 to 2kb) were defined for all 18 strains of Trypanosoma. Within each of the strains, 39-59 bands were defined. The similarity coefficients between strains ranged from approximately 41 to 94%, with a mean of 65%. There was more genetic similarity among strains within T. evansi (mean of approximately 79%) compared with T. equiperdum ( approximately 65%) and T. brucei ( approximately 59%). The similarity coefficient data were used to construct the dendrogram, which revealed that (irrespective of species) the majority of strains from China and South America grouped together to the exclusion of those from Africa. The exceptions were a T. brucei strain from Africa and a T. equiperdum strain of unknown origin. Hence, employing data sets generated using the three different fingerprinting methods, it was not possible to unequivocally distinguish among T. brucei, T. evansi and T. equiperdum, although there was a tendency for T. evansi strains to group together to the exclusion of T. brucei. The findings provide support for the hypothesis that T. evansi originated from a mutated form of T. equiperdum and stimulate further investigations of the genetic make-up and evolution of members of the subgenus Trypanozoon.     

A new RT-PCR assay specific for influenza A(H2), multiplexed with an assay specific for HPAI A(H5N1)

Worldwide efforts are ongoing to improve influenza pandemic preparedness, including from the perspective of the clinical virology laboratory. In particular, much work has been devoted to the development of diagnostic assays targeted at the Highly Pathogenic Avian Influenza (HPAI) A(H5N1); much less efforts have been devoted to the A(H2) subtype. Yet, A(H2) subtype has a proven capacity to cause pandemics and is among the subtypes prioritized for surveillance and control. Although the human A(H2N2) virus that caused the pandemic of 1957 no longer circulates, many related avian A(H2) viruses circulate in avian population and could conceivably adapt to infect humans and cause a new pandemic.In this study the design and development of an RT-PCR assay specific for A(H2) subtype is presented. It is shown that the assay is highly sensitive and specific, able to detect human and avian A(H2) viruses, and can be incorporated into a multiplex assay with another previously described assay for HPAI A(H5N1).     

献血者HBV DNA存在的确认与S蛋白变异特征分析

目的对HBsAg阴性和阳性献血者血样HBV DNA存在的确认并分析隐匿性乙型肝炎病毒S区变异特征。方法使用EIA/NAT方法筛查深圳地区19 397份无偿献血者血样,把109例乙肝不合格样品分成3类(HBs Ag+/NAT+、HBs Ag+/NAT-、HBs Ag-/NAT+),通过跟踪检测,确认为OBI毒株10例、HBV窗口期感染期3例和5例缺失追踪的HBs Ag-/HBV DNA+样品,采用荧光定量聚合酶链反应(QPCR)测定HBV病毒载量,应用NestedPCR技术扩增S基因片段并测定序列,与B/C基因型HBs Ag+/HBV DNA+阳性野毒株序列比对。结果深圳市无偿献血者经乙肝表面抗原胶体金快速试纸筛查后的HBs Ag阳性检出率为0.34%(66/19 397);隐匿性乙型肝炎病毒感染(OBI)的流行率范围为1∶1 939-1∶1 293,HBV窗口期感染流行率范围为1∶6 465-1∶2 424;10例OBI样品其病毒载量介于不能定量至112.0 IU/m L(中位数98.5 IU/m L)。10例OBI样本在S蛋白区(nt215-710)出现随机变异,OBI样品S区氨基酸置换率显著高于野毒株(P0.000 1),有4、2、3个OBI样品分别在CTL表位21-29、86-96、172-180出现L21S(2)、K/R24E(1)、I25M(1)、L88P(2)、S172F/L(2)、V178T(1)变异;OBI非CTL表位免疫区的氨基酸置换率亦显著高于野毒株(P0.05);其中1个OBI样品在nt636发生缺失变异。结论深圳献血者OBI流行率有增高趋势,OBI发生机制与乙型肝炎病毒的S蛋白区变异,特别是免疫活性区的变异密切相关。     

Electrical activity of the diaphragm (EAdi) as a monitoring parameter in difficult weaning from respirator: a pilot study

Introduction

A reliable prediction of successful weaning from respiratory support may be crucial for the overall outcome of the critically ill patient. The electrical activity of the diaphragm (EAdi) allows one to monitor the patients’ respiratory drive and their ability to meet the increased respiratory demand. In this pilot study, we compared the EAdi with conventional parameters of weaning failure, such as the ratio of respiratory rate to tidal volume.

Methods

We studied 18 mechanically ventilated patients considered difficult to wean. For a spontaneous breathing trial (SBT), the patients were disconnected from the ventilator and given oxygen through a T-piece. The SBT was evaluated by using standard criteria.

Results

Twelve patients completed the SBT successfully, and six failed. The EAdi was significantly different in the two groups. We found an early increase in EAdi in the failing patients that was more pronounced than in any of the patients who successfully passed the SBT. Changes in EAdi predicted an SBT failure earlier than did conventional parameters.

Conclusions

EAdi monitoring adds valuable information during weaning from the ventilator and may help to identify patients who are not ready for discontinuation of respiratory support.     

A noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for salmon calcitonin: Determination of the bioavailability of subcutaneous salmon calcitonin and its correlation with the hypocalcemic activity in rats

A noncompetitive enzyme immunoassay method (hetero-two-site enzyme immunoassay) for salmon calcitonin (SCT) and its usability for the pharmacokinetic study are described. The method in brief proceeds as follows: centrifugal filtration through a polysaccharide membrane to remove plasma proteins, biotinylation, trapping onto an anti-SCT IgG-coated polystyrene ball, acid elution, coupling with affinity-purified anti-SCT Fab¹-peroxidase conjugate, final trapping onto streptavidin-coated polystyrene balls, and measurement of peroxidase activity bound to the balls by fluorometry. The practical detection limit of SCT was 0.1 pg (30 amol)/assay and 2 pg/ml as the assay sample's concentration, which was at least fivefold lower than those previously reported by competitive radioimmunoassays. The application of this method has enabled us to 1) directly estimate the bioavailability of SCT dosed subcutaneously at the therapeutic levels (1.2 and 4.7 μg/kg) for its antiosteoporotic effect as compared to an intravenous dose (1.2 μg/kg) and 2) search for the relationship between blood level and the hypocalcemic activity of SCT. The pharmacokinetic parameters of subcutaneous SCT (1.2 and 4.7 μg/kg) thus estimated were as follows: the area under the blood concentration-time curve (AUC) = 89 and 550 pg hr/ml, and mean residence time (MRT) = 44 and 65 minutes, respectively, when the AUC for an intravenous SCT (1.2 μg/kg) = 160 pg hr/ml and the MRT = 10 minutes. © 1996 Wiley-Liss, Inc.