PES1慢病毒表达载体的构建及其对乳腺癌ZR75-30细胞生长的影响

目的:利用慢病毒载体表达PES1基因,研究其对乳腺癌ZR75-30细胞生长的影响.方法:以乳腺文库为模板,PCR扩增PES1基因,克隆到pCDH载体,构建成pCDH-PES1,将其与包装载体共转293T细胞,包装成Lenti-PES1慢病毒载体并测定病毒滴度,感染乳腺癌ZR75-30细胞,Western印迹鉴定病毒载体...     

定量比格犬血浆中去甲文拉法辛LC-MS/MS方法的建立及比格犬血浆中富马酸去甲文拉法辛药代动力学研究

目的:建立一种快速灵敏的液质联用方法定量比格犬血浆中去甲文拉法辛,并进行比格犬体内富马酸去甲文拉法辛药代动力学研究,同时与琥珀酸去甲文拉法辛比格犬体内药代参数进行对比。方法:样品处理采用叔丁基甲醚进行液液萃取,液相分离采用Agilent Eclipse Plus C18色谱柱(2.1 mm×100 mm,3.5μm),以乙腈-乙酸铵缓冲液(1 mmol/L)(体积比80∶20)进行等度洗脱。采用单次给药和多次给药试验进行比格犬体内药代动力学研究。结果:此方法具有良好的线性、精密度、准确度,定量下限(0.5 ng/mL)高于文献报道。液液萃取的样品提取方法简便,液质联用分析时间相对较短(3 min)。结论:此方法可成功地应用于比格犬血浆中去甲文拉法辛定量分析。比格犬体内富马酸去甲文拉法辛的药代参数(单次给药和多次给药试验)与对照品琥珀酸去甲文拉法辛没有明显的统计学差异。     

蛋白质组学关键技术研究进展

蛋白质组学是后基因组时代的一门新兴科学,是对生物体在蛋白质水平上定量、动态、整体性的研究。简要综述了高通量蛋白质分离和鉴定技术,如双向电泳、生物质谱、蛋白质芯片、酵母双杂交、同位素亲和标签及生物信息学的原理、方法、应用及存在的问题与局限,并对蛋白质组学研究的发展前景进行了展望。     

Identification and functional analysis of an ovarian form of the egg activation factor phospholipase C zeta (PLCζ) in pufferfish

Recent studies suggest that egg activation in mammals is triggered by a sperm-specific phospholipase C, PLCzeta. In other vertebrate species such as medaka fish, chickens, and quail, PLCzeta is also expressed as a testis-specific mRNA. Functional studies suggest that PLCzeta plays a similar role as a trigger of egg activation in these species. Here, we report the identification of PLCzeta orthologues in pufferfish species Takifugu rubripes (Fugu) and Tetraodon nigroviridis (Tetraodon). Unexpectedly in these species PLCzeta is expressed not in the testis, but in ovary and brain. Injection of pufferfish PLCzeta copy ribonucleic acid (cRNA) into mouse eggs failed to trigger calcium oscillations, unlike medaka PLCzeta cRNA. Our findings provide the first evidence that PLCzeta may be expressed in the egg, rather than the sperm, in some vertebrate species, and that its mechanism of action and physiologic role at fertilization may differ in different vertebrate species.     

Analytical ultracentrifugation combined with X-ray and neutron scattering: Experiment and modelling

Analytical ultracentrifugation and solution scattering provide different multi-parameter structural and compositional information on proteins. The joint application of the two methods supplements high resolution structural studies by crystallography and NMR. We summarise the procedures required to obtain equivalent ultracentrifugation and X-ray and neutron scattering data. The constrained modelling of ultracentrifugation and scattering data is important to confirm the experimental data analysis and yields families of best-fit molecular models for comparison with crystallography and NMR structures. This modelling of ultracentrifugation and scattering data is described in terms of starting models, their conformational randomisation in trial-and-error fits, and the identification of the final best-fit models. Seven applications of these methods are described to illustrate the current state-of-the-art. These include the determination of antibody solution structures (the human IgG4 subclass, and oligomeric forms of human IgA and its secretory component), the solution structures of the complement proteins of innate immunity (Factor H and C3/C3u) and their interactions with macromolecular ligands (C-reactive protein), and anionic polysaccharides (heparin). Complementary features of joint ultracentrifugation and scattering experiments facilitate an improved understanding of crystal structures (illustrated for C3/C3u, C-reactive protein and heparin). If a large protein or its complex cannot be crystallised, the joint ultracentrifugation-scattering approach provides a means to obtain an overall macromolecular structure.     

肺炎支原体液体培养法检测的实验评价

目的比较液体培养法和固体培养法平行检测肺炎支原体结果的一致性;评价液体培养法检测肺炎支原体的可靠性。方法采用液体培养基和固体琼脂培养基平行检测1 648份临床标本的肺炎支原体,比较同一份标本在2种培养基上的检测结果。结果液体培养法阳性296例,阳性率为18%;固体培养法阳性244例,阳性率为14.8%;液体培养法阳性而固体培养法阴性57例;固体培养阳性而液体培养法为阴性5例。2种方法的阳性检出率比较差异有统计学意义(P<0.05)。结论肺炎支原体快速液体培养法与固体培养有较好的一致性,具有方便、简单、准确且可以用于早期检测等优点,适合临床大批量标本筛查。需结合患者临床症状等排除真菌和耐药菌造成的假阳性。     

新疆伊犁地区马匹亨德拉病毒脑内感染情况调查

目的检测新疆伊犁地区天然牧场放养马群脑组织中亨德拉病毒（Hendra Virus,HeV）的核酸片段,调查该地区马群中枢神经系统HeV感染流行状况。方法针对HeV高度保守区核蛋白（N）基因设计特异性引物和探针,采用一步法实时荧光定量RT-PCR检测中枢神经系统感染样本中低浓度HeV RNA的方法,检测新疆伊犁草原地区天然放养且未接种HeV疫苗的183例马匹脑组织。结果最低特异性检出浓度可低至2.6×102copies/μL,与其他单股负链RNA病毒如同属的尼帕病毒（NiV）无交叉反应,对183例马脑组织进行一步法实时荧光定量RT-PCR检测,未检出阳性样本。结论初步流行病学研究尚未发现我国新疆伊犁地区天然牧场放养马匹中存在HeV感染的证据,提示该地区短时间内爆发亨德拉病毒感染的可能性小。     

氨溴索与碳酸氢钠序贯灌洗佐治支气管扩张感染疗效分析

目的探讨氨溴索与碳酸氢钠灌洗治疗支气管扩张反复感染的疗效及意义。方法将支气管扩张反复感染住院患者,分为灌洗组(常规治疗方法 +支气管局部注药灌洗34例)、对照组(常规治疗方法 38例),治疗2周后进行临床评估,随访半年。结果灌洗组总有效率与对照组相比差异有统计学意义(P<0.05),半年急性加重次数与对照组相比差异有统计学意义(P<0.01)。结论在常规治疗基础上局部氨溴索、碳酸氢钠序贯灌洗能提高支气管扩张感染疗效、减少半年急性加重次数、改善生活质量,通过改善微环境来维持菌群平衡值得借鉴、推广。     

1800例女性阴道分泌物检测分析

目的分析北京丰台地区女性阴道感染情况。方法随机抽取2010年1月至2010年8月期间在北京市丰台区铁营医院就诊女性1800例,采用湿片镜检方法对就诊者的阴道分泌物进行检测。结果通过对1800患者的阴道分泌物进行检测发现,检出细菌性阴道病(BV)患者、外阴阴道念珠菌病(VVC)患者、滴虫性阴道炎(TV)患者共计887例(占总数的49.28%),其中单一性感染718例(占发病人数的80.95%),混合感染169例(占发病人数的19.05%)。结论细菌性阴道病的发病率较高,阴道混合感染日趋严重。及时正确的检测出阴道感染的病原菌、合理用药,是阴道感染提高治愈率、降低复发率的有效手段。     

牛源隐孢子虫分离株的分子鉴定

目的对安徽4个牛源隐孢子虫分离株进行鉴定,旨在阐明感染牛隐孢子虫主要虫种。方法采用PCR和Nested-PCR方法分别对4个分离株18S rRNA、HSP70基因进行扩增和测序,所测定的序列经生物信息学分析它们同源性和构建种系发育进化树,以确定各分离株与其他隐孢子虫虫种之间的亲缘关系。结果 （1）它们的18S rRNA基因和HSP70基因与安氏隐孢子虫（C.andersoni）AY954887相似性可达88.3%和98.3%;（2）在遗传进化树方面,它们与AY954892（河南株）亲缘关系接近。结论此次分离的4个牛源隐孢子虫分离株均为安氏隐孢子虫（Cryptosporidium andersoni）。