细胞内外膜跨膜电位频率响应模型及滤波器特性的研究

提出球形单细胞内外膜跨膜电位频率响应模型和仿真计算方法。通过对频率响应的仿真分析，发现内外膜跨膜电位分别表现出一阶带通和低通滤波器特性，并在内膜频率响应中心频率到上限截止频率的范围内，内膜跨膜电位大于外膜同时能保持在较高水平，这将有利于诱导细胞内电处理效应。计算结果与动物实验吻合得很好，为纳秒脉冲电场治疗肿瘤的窗口参数合理选择提供了理论依据。     

纳秒级陡脉冲电场诱导癌细胞凋亡的实验及作用机理研究

观察纳秒级陡脉冲电场诱导人浆液囊腺性卵巢癌SKOV3癌细胞凋亡效应,及其对细胞内钙离子浓度即[Ca2＋]i的影响并探讨其作用机理。采用场强为10kV/cm、脉宽为100ns、重复频率为1Hz的纳秒级陡脉冲电场作用于SKOV3癌细胞悬液5min。4h后采用Annexin V-FITC/PI双标记并结合流式细胞仪分析SKOV3癌细胞的凋亡率,采用扫描和透射电子显微镜观察癌细胞凋亡的超微结构变化;以Fluo-3/AM为细胞内钙离子的荧光指示剂,用激光扫描共聚焦显微镜检测纳秒级陡脉冲电场对SKOV3癌细胞[Ca2＋]i的影响,及[Ca2＋]i变化时的Ca2＋来源。结果显示,流式细胞术检测到癌细胞的早期凋亡率为（22.21±2.71）%,明显高于对照组的（3.04±0.44）%（P〈0.01）,扫描和透射电子显微镜均观察到典型的凋亡细胞形态,证实纳秒级陡脉冲电场能够有效诱导SKOV3癌细胞发生凋亡;同时纳秒级陡脉冲电场可明显升高细胞[Ca2＋]i（P〈0.01）,而与细胞外钙离子浓度无关（P〉0.05）。由于纳秒级陡脉冲电场的等值频率很高,容易穿透细胞膜,其诱导凋亡的机制之一可能是通过激活细胞内钙库（内质网、线粒体）,引起细胞内钙离子升高,从而介导凋亡信号通路。     

不同方法处理牛心包的体外力学和抗钙化性能评价

比较传统GA法（A组）、三氯化铝与乙醇联合处理法（B组）、多聚环氧化物处理法（C组）和光氧化法（D组）四种不同方法处理牛心包后力学与抗钙化性能。对新鲜牛心包随机分为四组,用四种方法处理后,测定组织的极限抗拉强度、断裂伸长率、热皱缩温度,比较其力学性能;建立大鼠皮下植入模型,包埋2月后取出标本测定组织钙含量,并做HE染色及von Kossa染色。极限抗拉强度：B组〉A组=C组〉D组;断裂伸长率：C组=D组〉B组〉A组;热皱缩温度：A组=B组〉C组〉D组;钙含量测定：A组〉〉C组〉B组〉D组（P〈0.05）;HE染色和von Kossa染色结果符合以上测定值。三氯化铝与乙醇联合处理组的组织交联程度与力学强度最佳,光氧化处理组与多聚环氧化物（简称PC）处理组的组织柔韧性更好。在抗钙化性能方面,光氧化处理组最好,三氯化铝与乙醇联合处理组与多聚环氧化物处理组次之,GA处理组最差。     

人乳头状瘤病毒11型E7抗原HLA-A*0201限制性细胞毒性T淋巴细胞表位的功能鉴定

目的 筛选和鉴定人乳头状瘤病毒11型E7抗原(HPVllE7)HLA-A*0201限制性细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)表位.方法 预测HPVllE7抗原HLA-A*0201限制性CTL表位并合成相对应的表位多肽和四聚体(tetramer),即HPVllE7 7-15(TLKDIVLDL)、15-23(LQPPDPVGL)、47-55(PLTQHYQIL)、81-89(DLLLGTLNI)和82-90(LLLGTLNIV).从健康HLA-A*0201成人外周血单一核细胞诱导树突状细胞(DC)并负载上述表位多肽,流式细胞技术检测DC成熟分化标记及ELISA法检测DC分泌的IL-12;成熟DC负载各组多肽后观察DC激活T淋巴细胞的效应,ELISA法检测T细胞分泌的IFN-γ;四聚体检测抗原特异性CD8+ T细胞及乳酸脱氢酶(LDH)释放法评价DC诱导的CTL对靶细胞的特异性体外杀伤效应.结果 预测的5条HPVllE7表位多肽均能诱导DC的成熟分化;E7 7-15、82-90和15-23多肽负载的DC能激活T淋巴细胞分泌高水平IFN-γ;E7 7-15多肽负载的DC能刺激特异性tetramer+CD8+细胞增殖且其诱导的CTL对HPVllE7/293细胞产生高效率的特异性杀伤作用(P<0.05).结论 筛选并鉴定出1条HPVllE7HLA-A*0201限制性CTL表位E7 7-15(TLKDIVLDL),负载该表位肽的DC体外可诱导高效、特异性的CTL效应,抗原性较强,有可能作为HPV感染治疗用肽疫苗的候选表位.     

阈值校正方式对PET脑显像统计参数图分析显示结果的影响

目的 探讨各种阈值校正方法对统计参数图(SPM)软件统计比较结果显示的影响.方法 利用Hoffman标准脑模型制作缺损模型PET成像与正常模型PET成像,进行统计参数图的统计比较.并选取脑梗死患者与健康检查者进行统计参数图的统计比较.结果 校正与非校正产生的结果有差异,其中族错误率(FWE)校正(P=5×10-2)得到的统计分析结果激活区最少及最小,其次是错误发现率(FDR)校正(P=5×10-2),非校正方式(P=1×10-3)得到的激活区最多及最大.但FDR校正效果不稳定,有时不如不校正.结论 统计参数图软件中FWE校正方法可明显降低假阳性,得到的结果可信度稳定地高于非校正方法.     

Expression and characterization of the human YWK-II gene, encoding a sperm membrane protein related to the alzheimer betaA4-amyloid precursorprotein

The YWK-II cDNA, RSD-2, encoding a sperm membrane protein was isolated from a rat testis cDNA expression library. Using the RSD-2 insert in combination with rapid amplification of cDNA ends (RACE), the corresponding human gene was isolated from a human testis cDNA expression library. The human testis cDNA, HSD-2, is 3654 bp in length and contains an open reading frame of 763 codons. Hydropathicity analysis showed that the deduced polypeptide is a single strand transmembrane protein. The deduced polypeptide has partial homology with the amyloid precursor protein (APP) and high homology with the amyloid precursor homologue, APLP2/APPH. The YWK-II gene was mapped and assigned to human chromosome locus: 11q24-25. Northern blotting of various human tissue RNAs using the HSD-2 cDNA as a probe showed that the gene is transcribed ubiquitously. The cytoplasmic domain of HSD-2 was expressed in Escherichia coli. In-vitro studies showed that the recombinant polypeptide bound to a GTP-binding protein (G(o)) and was phosphorylated by protein kinase C and cdc2 kinase. In mammalian F11 cells, the recombinant polypeptide was found to be coupled to G(o). Thus, the YWK-II component has the characteristics of a G(o)-coupled receptor and may be involved in G(o)-mediated signal transduction pathway. Protein kinase C and cdc2 kinase may regulate this pathway in spermatozoa by phosphorylating the cytoplasmic domain of the YWK-II component.     

Clinical experiences of bacteremia caused by metallo-beta-lactamase-producing gram-negative organisms.

The emergence of acquired metallo-beta-lactamase (MBL) in gram-negative bacilli is regarded as a therapeutic challenge since such enzymes are capable of hydrolyzing all beta-lactams in vitro except the monobactams. The clinical characteristics and outcome of 8 episodes of gram-negative bacteremia caused by MBL-producing isolates from January 1997 through December 2000 (Klebsiella pneumoniae, 6 isolates; Pseudomonas stutzeri, 4; Pseudomonas aeruginosa, 1; and Pseudomonas putida, 1) were analyzed. The median age of the patients was 61 years (range, 2-95 years). Most patients (n = 6, 75%) had more than 1 comorbid illness or condition and 6 patients acquired bacteremia in the intensive care unit. The median time from admission to the first positive culture was 34.5 days (range, 1-99 days). Pneumonia was the most common site of infection. Five patients (62.5%) received a carbapenem to treat bacteremia. The median time to defervescence was 6 days (range, 2-12 days). No bacteriologic failure was noted during or after antimicrobial therapy. The overall mortality rate from bacteremia caused by gram-negative, MBL-producing organisms was nil at 14 or 28 days.     

单细胞凝胶电泳在遗传学损伤检测中的应用

单细胞凝胶电泳或彗星试验是在暴露人群中评价遗传学损伤的新式方法。这种分析可以快速的探测单一细胞中不同形式的DNA损伤，因此适于在暴露人群分析应用。本文将对彗星试验在临床研究及监测职业、环境、合理饮食、生活方式等对DNA损伤的危险性，以及在DNA损伤与修复研究中的应用等方面进行综述。     

恙虫病东方体Sta56抗原的原核表达、纯化及其在间接ELISA中的应用

目的　构建含恙虫病东方体sta5 6基因的重组表达质粒 ,在E .coli中表达Sta5 6重组抗原 ,纯化后用于间接ELISA检测恙虫病。方法　从含有恙虫病东方体Karp株sta5 6基因的重组质粒TOPO sta5 6扩增出截短的sta5 6 ,定向插入pET30a载体 ,转化大肠杆菌BL2 1(DE3) ,IPTG诱导表达 ,用SDS PAGE及Westernblot进行分析 ;采用电洗脱方法纯化重组抗原并应用于间接ELISA检测恙虫病。结果　以截短的sta5 6基因片段构建了重组表达质粒pETOt95 7,重组的Sta5 6抗原可在E .coli中以融合蛋白的形式有效表达 ,SDS PAGE显示了一条相对分子质量 (Mr)为 4 0 .3× 10 3 的蛋白表达带 ,West ernblot证实该融合蛋白能被恙虫病患者阳性血清所识别。电洗脱纯化的重组抗原应用于间接法ELISA检测恙虫病 ,与间接免疫荧光法IFAT比较 ,其敏感性和特异性分别为 91.7%和 76 .5 %。结论　在大肠杆菌中表达的Sta5 6重组抗原具有免疫反应性 ,纯化后可用作免疫诊断试剂。     

尖锐湿疣患者外周血Thl/Th2及Tc1/Tc2亚群的检测及意义

Objective To investigate the role of T-helper (Th) and cytotoxic T (Tc) lymphocyte polarization in the pathogenesis of condyloma acuminatum (CA) and its correlation with recurrence. Methods Three-colour immunofluorescent flow cytometry was used to detect the proportion of CD3+ CD8-/IFN-γ+ (Th1),CD3+CD8-/IL-4+ (Th2), CD3+ CD8+/IFN-γ+ (Tel) and CD3+ CD8+/IL-4+ (Tc2) cells in the peripheral blood of CA patients and health controls. Results Compared to health controls, CA patients showed a decreased number of Thl (P < 0.01) and Tcl cells (P < 0.05), as well as a decreased Th1/Th2 and Tc1/Tc2 ratio (P <0.05). Furthermore, in 15 recurrent CA patients the ratio of Th1/Th2 was remarkably decreased (P < 0.01),while the ratio of Tc1/Tc2 had no significant change in comparison with health controls. Conclusion The decrease of Th1 and Tc1 subsets results in relative Th2 and Tc2 predominance, and this tendency is more significant in recurrent CA patients. The Th1 to Th2 and Te1 to Tc2 shifts in CA patients could be responsible for the fact that human papilloma virus (HPV) is hard to be eliminated.