大麻哈鱼胚胎耳石微结构及其群体环境标记

采用人工调控环境方法对黑龙江、绥芬河大麻哈鱼发眼期胚胎群体耳石日轮进行周期性持续标记。实验分４组: 1组为对照组, 2－3组为变温标记组, 4组为“暴气”-变温标记组, 实验用发眼卵1.2万粒。待胚胎发育至耳石日轮结构形成后实施标记。实验胚胎耳石随着标记期间环境周期性变化及其持续的时间, 形成相应变化节律的日轮标记区。获得各实验组设定环境的日轮标记图谱。人工环境标记的耳石日轮图谱, 暗带色度加深, 明带亮度增大, 并可形成生长轮距不同的标记轮, 与对照组耳石微结构有明显区别, 标记率达到100%。初步建立鱼类耳石标记及其识别技术, 适用于大麻哈鱼等鲑鳟鱼类群体标记。作为安全有效、成本低廉的群体标记技术方法, 鱼类耳石日轮标记在鱼类资源评估和增殖放流效果评价中将会得到广泛应用。       

Cdk4/cyclin D1通路异常参与C型Niemann-Pic病小鼠神经元变性

以Balb／cnihnpc-1小鼠模型为对象,使用免疫组织化学和免疫印迹技术来研究cdk4／cyclin D1／Rb2通路的激活与神经元神经丝蛋白的过度磷酸化及神经元变性的相互关系,探讨C型Niemann-Pick病（NPC）病理性球状神经轴突形成的机制。实验表明,cdk4／cyclin D1在该小鼠模型脑白质球状神经轴突中异常聚集,其下游因子Rb2／p130呈现过度磷酸化并分布在上述病理性结构中。它们的分布随鼠龄增加而逐渐从脑干扩大到其他脑白质区域。球状神经轴突的主要成分之一为过度磷酸化神经丝蛋白,它与cdk4的分布在时空中具有高度的一致性。提示cdk4／cyclin D1／Rb2通路激活参与NPC神经元球状神经轴突形成,并与神经丝蛋白的异常磷酸化关系密切。该通路可作为干预神经元变性的一个新的靶点来挽救病损的神经元。     

Ap、Km和Cm抗性细菌在土壤中的差异分布及成因

从不同区域土壤中分离细菌：对其进行氨苄青碡素、卡那霉素和氯霉素的抗性测试,以及抗药性质粒分析。结果表明,对照区和生活区土壤细菌抗氨苄青霉素和卡那霉素菌株比例未见显著性差异,未检出抗氯霉素细菌。保护区中抗氨苄青霉素、卡那霉素菌株含质粒比例均为18.2％,生活区中抗氨苄青霉素和卡那霉素菌株含质粒比例分别为36.4％和27．3％。随机抽提质粒转化无抗忡菌,表明部分抗菌素抗性基因是由质粒携带。实验结果认为本地土壤中抗菌素抗性菌分布差异尚未有显著性表现。     

研究生病理学实验带教的探索与实践

医学研究生教育的目的是培养高素质创新型医学人才,创新离不开科研,病理学是多学科科研究工作的基础。建立科研平台提高科研能力是研究生教育十分关注的问题。如何培养研究生病理学实验能力,使学到知识技术在今后的科研活动中得以运用,本文就近几年我们在研究生病理学实验带教实践过程中的一些方法作一介绍。     

多肽抗体检测原发性肝癌血清标志物ELISA方法的建立及应用

血清多肽是癌症诊断信息的重要来源,建立、优化了检测多肽标志物的直接ELISA法,并应用于肝癌血清中的多肽标志物的检测。制备及纯化针对多肽标志物Pep5的单克隆抗体并进行辣根过氧化物酶标记,用其建立检测相应抗原的直接ELISA法。方法线性范围为1.5-20 ng/mL,检测限为1.24 ng/mL;标准品批内及批间CV分别小于3.66%及4.89%,血清样本批内及批间CV分别小于11.69%及18.18%;线性范围内(9、12和15 ng/mL)的回收率分别为98.98%,99.61%和101.58%。应用该方法共检测160例正常血清、104例肝硬化及156例肝癌患者血清,正常组与肝硬化组及肝癌组间差异显著(P<0.001),Pep5诊断肝癌的敏感性和特异性分别为80.8%和96.2%。同时检测94例HCC血清中的AFP和Pep5,AFP检出率为63.8%,Pep5检出率为90.4%,AFP联合Pep5检测时,能将HCC的检出率提高至94.7%。     

细菌抗生素和重金属协同选择抗性机制研究进展

随着各类抗生素和新型复合金属材料的不断开发和使用,环境中抗生素和重金属离子协同污染的机率不断提高,对环境选择最为敏感的细菌通过自身的进化和发展形成了二者协同选择的抗性机制,如协同抗性、交叉抗性、协同调控和生物膜诱导机制。     

SDS-PAGE法测定His-tag融合蛋白分子量产生偏差的原因

Ｈｉｓｔａｇ／ＮｉＮＴＡ系统是新发展起来的一个亲和纯化重组蛋白的有用工具,现常用于基因编码产物的特性研究中。ＳＤＳＰＡＧＥ是实验室测定蛋白质分子量通常采用的方法,而许多实验室用此方法检测Ｈｉｓｔａｇ融合蛋白时却常发现测得的分子量偏大,产生偏差的原因尚未阐明。为弄清这一问题,本实验室在研究一个Ｈｉｓｔａｇ融合蛋白Ｐ７３Ｈｉｓ时,首先用ＳＤＳＰＡＧＥ法测得其分子量确实比理论计算值大,然后对其进行Ｃ末端氨基酸顺序测定、电喷雾质谱分析,结果证实其实际分子量与理论值一致。酶切去除包括Ｈｉｓｔａｇ在内的部分肽段使ＳＤＳＰＡＧＥ法测量蛋白分子量的偏差大大降低,证实Ｈｉｓｔａｇ确实是造成偏差的原因之一。推测由于Ｈｉｓｔａｇ中的碱性氨基酸的作用造成蛋白在ＳＤＳＰＡＧＥ中迁移变慢,而导致偏差。这一现象值得引起有关研究者的注意。     

Store-dependent and -independent modes regulating Ca2+ release-activated Ca2+ channel activity of human Orai1 and Orai3

We evaluated currents induced by expression of human homologs of Orai together with STIM1 in human embryonic kidney cells. When co-expressed with STIM1, Orai1 induced a large inwardly rectifying Ca(2+)-selective current with Ca(2+)-induced slow inactivation. A point mutation of Orai1 (E106D) altered the ion selectivity of the induced Ca(2+) release-activated Ca(2+) (CRAC)-like current while retaining an inwardly rectifying I-V characteristic. Expression of the C-terminal portion of STIM1 with Orai1 was sufficient to generate CRAC current without store depletion. 2-APB activated a large relatively nonselective current in STIM1 and Orai3 co-expressing cells. 2-APB also induced Ca(2+) influx in Orai3-expressing cells without store depletion or co-expression of STIM1. The Orai3 current induced by 2-APB exhibited outward rectification and an inward component representing a mixed calcium and monovalent current. A pore mutant of Orai3 inhibited store-operated Ca(2+) entry and did not carry significant current in response to either store depletion or addition of 2-APB. Analysis of a series of Orai1-3 chimeras revealed the structural determinant responsible for 2-APB-induced current within the sequence from the second to third transmembrane segment of Orai3. The Orai3 current induced by 2-APB may reflect a store-independent mode of CRAC channel activation that opens a relatively nonselective cation pore.     

Meningitic Escherichia coli K1 penetration and neutrophil transmigration across the blood-brain barrier are modulated by alpha7 nicotinic receptor

Alpha7 nicotinic acetylcholine receptor (nAChR), an essential regulator of inflammation, is abundantly expressed in hippocampal neurons, which are vulnerable to bacterial meningitis. However, it is unknown whether α7 nAChR contributes to the regulation of these events. In this report, an aggravating role of α7 nAChR in host defense against meningitic E. coli infection was demonstrated by using α7-deficient (α7(-/-)) mouse brain microvascular endothelial cells (BMEC) and animal model systems. As shown in our in vitro and in vivo studies, E. coli K1 invasion and polymorphonuclear neutrophil (PMN) transmigration across the blood-brain barrier (BBB) were significantly reduced in α7(-/-) BMEC and α7(-/-) mice. Stimulation by nicotine was abolished in the α7(-/-) cells and animals. The same blocking effect was achieved by methyllycaconitine (α7 antagonist). The tight junction molecules occludin and ZO-1 were significantly reduced in the brain cortex of wildtype mice infected with E. coli and treated with nicotine, compared to α7(-/-) cells and animals. Decreased neuronal injury in the hippocampal dentate gyrus was observed in α7(-/-) mice with meningitis. Proinflammatory cytokines (IL-1β, IL-6, TNFα, MCP-1, MIP-1alpha, and RANTES) and adhesion molecules (CD44 and ICAM-1) were significantly reduced in the cerebrospinal fluids of the α7(-/-) mice with E. coli meningitis. Furthermore, α7 nAChR is the major calcium channel for nicotine- and E. coli K1-increased intracellular calcium concentrations of mouse BMEC. Taken together, our data suggest that α7 nAChR plays a detrimental role in the host defense against meningitic infection by modulation of pathogen invasion, PMN recruitment, calcium signaling and neuronal inflammation.     

Combined effect of sodium dodecyl sulfate and enzyme on waste activated sludge hydrolysis and acidification

The combined effect of sodium dodecyl sulfate (SDS) and enzyme system on hydrolysis and acidification of waste activated sludge (WAS) was investigated. The results showed that the combined system was more effective in the promotion of sludge hydrolysis than sole SDS and sole enzyme, and the SDS + mixed-enzymes (ME) system had better hydrolysis performance than SDS + single enzyme system. Compared with SDS + protease and SDS + amylase systems, the soluble protein concentration in SDS + ME system increased respectively by 20.0% and 44.4%, and the soluble carbohydrate concentration increased by 78.3% and 37.0%, respectively. During the WAS acidification stage, the SDS, ME and SDS + ME system could make the maximum short-chain fatty acids (SCFAs) concentration increased by 1.82 (6th day), 2.04 (5th day), 2.32 (7th day) times, respectively. The composition analysis of SCFAs produced in SDS + ME system indicated that acetic acid was the most prevalent product and propionic acid was the second one.