重组腺病毒HIV-1 vpr基因的构建和表达

目的 构建携带HIV-1 vpr基因的重组腺病毒,使CD4 T淋巴细胞C8166内源性的高表达Vpr蛋白.方法 利用AdEasy-1系统, 通过将含有目的 基因片段的穿梭载体pAdTrack-CMV-vpr和骨架质粒pAdEasy-1在BJ5183细菌内同源重组的方法构建重组腺病毒质粒Ad-vpr, 用脂质体法将重组质粒转染至HEK293A细胞包装, 获得重组腺病毒Ad-vpr, 荧光显微镜观察Ad-vpr感染C8166细胞GFP的表达 , Western blotting鉴定Vpr在C8166细胞内的特异性表达,流式细胞术检测Ad-vpr感染 C8166细胞的效率.结果 成功构建携带HIV-1 vpr基因的重组腺病毒 ,Western blotting结果表明重组腺病毒Ad-vpr感染的C8166细胞内源性的高表达Vpr 蛋白,流式细胞术检测结果表明Ad-vpr感染C8166细胞效率高(44.07±3.62)%.结论 成功构建出携带HIV-1 vpr基因的重组腺病毒,使C8166细胞内源性的高表达Vpr蛋白.     

Stromal cell-derived factor-1/CXCR4 signaling modifies the capillary-like organization of human embryonic stem cell-derived endothelium in vitro.

The molecular mechanisms that regulate human blood vessel formation during early development are largely unknown. Here we used human ESCs (hESCs) as an in vitro model to explore early human vasculogenesis. We demonstrated that stromal cell-derived factor-1 (SDF-1) and CXCR4 were expressed concurrently with hESC-derived embryonic endothelial differentiation. Human ESC-derived embryonic endothelial cells underwent dose-dependent chemotaxis to SDF-1, which enhanced vascular network formation in Matrigel. Blocking of CXCR4 signaling abolished capillary-like structures induced by SDF-1. Inhibition of the SDF-1/CXCR4 signaling pathway by AMD3100, a CXCR4 antagonist, disrupted the endothelial sprouting outgrowth from human embryoid bodies, suggesting that the SDF-1/CXCR4 axis plays a critical role in regulating initial vessel formation, and may function as a morphogen during human embryonic vascular development.     

C. elegans ORFeome Version 3.1: Increasing the Coverage of ORFeome Resources With Improved Gene Predictions

The first version of the Caenorhabditis elegans ORFeome cloning project, based on release WS9 of Wormbase (August 1999), provided experimental verifications for ~55% of predicted protein-encoding open reading frames (ORFs). The remaining 45% of predicted ORFs could not be cloned, possibly as a result of mispredicted gene boundaries. Since the release of WS9, gene predictions have improved continuously. To test the accuracy of evolving predictions, we attempted to PCR-amplify from a highly representative worm cDNA library and Gateway-clone ~4200 ORFs missed earlier and for which new predictions are available in WS100 (May 2003). In this set we successfully cloned 63% of ORFs with supporting experimental data (“touched” ORFs), and 42% of ORFs with no supporting experimental evidence (“untouched” ORFs). Approximately 2000 full-length ORFs were cloned in-frame, 13% of which were corrected in their exon/intron structure relative to WS100 predictions. In total, ~12,500 C. elegans ORFs are now available as Gateway Entry clones for various reverse proteomics (ORFeome v3.1). This work illustrates why the cloning of a complete C. elegans ORFeome, and likely the ORFeomes of other multicellular organisms, needs to be an iterative process that requires multiple rounds of experimental validation together with gradually improving gene predictions.     

The significance of 'anti-HBc only' in the clinical virology laboratory.

BACKGROUND: Isolated detection of hepatitis B core antibody (anti-HBc) in the absence of surface antigen (HBsAg) or antibody (anti-HBs) has been reported, particularly among individuals infected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV). The significance of this phenomenon is unknown and it is unclear whether all individuals with such serological pattern need further molecular investigations. OBJECTIVES: To determine the prevalence of 'anti-HBc only' in samples referred to a clinical virology laboratory and to evaluate its significance and possible mechanisms. STUDY DESIGN: Samples identified as anti-HBc positive (389/4359, 8.9%) during an 11-month period were investigated for HBsAg, anti-HBs, anti-HCV and anti-HIV. 'Anti-HBc only' samples were tested for HBV DNA using a nested qualitative PCR. Viral loads were measured in samples with detectable HBV DNA and the DNA sequences were analysed. RESULTS: Of 379 samples with detectable anti-HBc, 155 (40.9%) were 'anti-HBc only'. HBV DNA was detected in 6/151 (4%), all of which had a viral load <400 copies per ml. Anti-HIV was found in 50/151 (33.1%) and anti-HCV in 14/151 (9.3%). Of these, only one of the HIV infected patients had detectable HBV DNA. Phylogenetic analysis of the HBV surface gene from three patients showed a variety of genotypes (A, E and G). One sequence had a mutation in codon 144, which has previously been reported to give false negative HBsAg results. CONCLUSIONS: 'Anti-HBc only' is a common phenomenon in the clinical virology laboratory but only a small proportion of samples had detectable HBV DNA. The presence of HBsAg mutants with possible false negative HBsAg test result is of concern. Samples with 'anti-HBc only' could be used to monitor the emergence of these mutants.     

mic2/CD99在经典型霍奇金淋巴瘤H/RS细胞中的表达及与Eber-1/LMP-1相关性的研究

目的： 检测mic2基因与CD99蛋白和Eber-1基因与潜伏膜蛋白-1（LMP1）在经典型霍奇金淋巴瘤（cHL）H/RS细胞中的表达，探讨mic2/CD99表达与Eber-1/LMP-1的相关性。 方法： 采用分子原位杂交和免疫组化技术并结合组织芯片技术检测59例石蜡包埋淋巴瘤组织标本［43例cHL,16例非霍奇金淋巴瘤（NHL）］mic2/CD99和Eber-1/LMP-1的表达，比较分析mic2/CD99在两组中的表达及与Eber-1/LMP-1的关系。 结果： cHL组CD99蛋白表达阳性率为2.3％，mic2基因表达为55.8％，LMP1表达为58.1％，Eber-1表达为53.5％；NHL组CD99蛋白与mic2基因表达高于cHL组（P<0.05）；LMP1和Eber-1的表达低于cHL组（P<0.05）；mic2基因的表达高于CD99蛋白的表达（P<0.05）；Eber-1与LMP1的表达无统计学意义（P>0.05）。CD99蛋白与LMP1的表达呈负相关（P<0.05），各项指标的表达与性别无关。CD99蛋白与LMP1的表达与年龄呈负相关（P<0.05），mic2与Eber-1的表达与年龄无关（P>0.05）。 结论： CD99蛋白在cHL H/RS细胞中低表达，并与LMP1的表达呈负相关。     

差异筛选肝癌凋亡细胞cDNA文库的简便方法

目的差异筛选人肝癌凋亡细胞ｃＤＮＡ文库。方法消减杂交和点杂交相结合的噬菌斑原位杂交法筛选ｃＤＮＡ文库，首先采用（－）ｃＤＮＡ探针杂交，挑取（－）的噬菌斑克隆，再用（－）和（＋）两种ｃＤＮＡ探针与初筛出的噬菌斑克隆杂交的差异筛选方法。结果得到４个充分孤立的噬菌斑克隆，其插入片段长度为１．５ｋｂ左右。结论该方法简便、快速，是差异筛选ｃＤＮＡ文库的一种较为可行的简便方法。     

桑色素对小鼠T淋巴细胞体外活化、增殖和细胞周期的影响

目的:研究桑色素(morin)对小鼠T淋巴细胞活化、增殖和细胞周期的影响。方法:以刀豆蛋白A(ConA)刺激培养的淋巴结来源的小鼠淋巴细胞,再以不同终浓度的morin与T细胞共培养,利用流式细胞术(FCM),检测早期T细胞活化的标志CD69分子的表达,以羧基荧光素双醋酸盐琥珀酰脂(CFDA-SE)染色检测T细胞的增殖;以碘化丙锭(PI)染色分析T细胞的细胞周期。结果:小鼠T细胞培养6 h后,未经ConA刺激的对照组中CD69 T的细胞比率为(2.97±0.12)%,经ConA刺激的CD69 T细胞的比率明显增高,达到(72.52±0.66)%,与对照组相比差别明显(P<0.01)。终浓度为25、50、100μmol/L的morin均下调CD69 T细胞的比率,其中,100μmol/L的morin抑制作用最强,为(48.95±0.81)%,与对照组比较具有统计学意义(P<0.01)。CFDA-SE染色分析显示,ConA组培养48 h和72 h的T细胞的增殖指数(PI)分别为(1.58±0.04)和(1.95±0.02),各浓度的morin对ConA刺激的T细胞增殖,具有明显地抑制作用,以100μmol/L的morin抑制作用最明显。培养48 h的ConA组T细胞的PI为(1.02±0.02)、培养72 h的ConA组T细胞的PI为(1.03±0.01),与相应时间的对照组比较,均有统计学意义(P<0.01)。PI染色后流式细胞术分析的结果表明,ConA组处于S期的T细胞的比率为(27.05±0.39)%,显著高于对照组的比率(5.10±0.07)%。morin组中S期的细胞比率较高。结论:Morin可显著抑制ConA刺激的T细胞活化及增殖;其对增殖的抑制作用主要表现为S期的细胞的阻滞。     

LIS、HIS无缝连接及检验样本条码化应用体会——以海河医院为例

通过HIS系统升级增加医生工作站、LIS系统建设、LIS与HIS无缝连接、条码技术在LIS中的应用,实现病人基本信息及检验结果信息全医院联网.实践证明,该方案切实可行,可大大优化和规范医院工作流程,降低出错率,医患共同受益.     

体外厌氧条件下载铜蒙脱石杀菌效果的研究

载铜蒙脱石的杀菌作用通过脑心浸液肉汤 (BHI)二倍系列稀释法来判定其最小抑菌浓度 (MIC)。释放入肉汤和生理盐水中Cu2 的量通过原子吸收光谱仪测定。杀菌动力学研究采用改良的振荡瓶法。结果表明 :厌氧条件下 ,载铜蒙脱石具有很强的杀菌性能。载铜蒙脱石对放线共生放线杆菌的最小抑菌浓度为 2 .5 6mg/ml;对血液链球菌为 5 .12mg/ml。测得肉汤二倍稀释的载铜蒙脱石释放出的Cu2 量在 2 .39～ 38.6 5 ppm之间 ,在生理盐水中释放的Cu2 量为 1.85～ 15 .82ppm。本试验结果表明 ,蒙脱石无抗菌性能。     

肝再生过程中肝和脑垂体纤维粘连蛋白表达的变化

目的；探讨大鼠肝大部分切除再生过程中肝和垂体内纤维粘连蛋白变化变化，方法：用免疫组织化学法观察鼠肝大部切除术０．５天，１天，１．５天，２天，３天，７天时，肝内和垂体远侧部滤泡状细胞细胞纤维粘连蛋白的变化，并用图像分析仪进行定量测定，结果：肝大部切除术后１．５天，肝内纤维粘连蛋白阳性染色增强，基平均光密度高于对照组（Ｐ〈０．０５）；术后２～３天，镜下可见纤维粘连蛋白染色呈强阳性，尤其在肝组织增生部位