VEGF真核表达载体的构建及在内皮与心肌细胞内的表达

目的：探讨外源性人VEGF165基因在内皮细胞与心肌细胞内表达的可行性。方法：构建了(vaseular endothelium growth factor,VEGF)与增强型绿色荧光蛋白(enhanced green fluorescent protein，EGFP)基因其表达载体pIRES2—FGFP—VEGF165，以脂质体法转染内皮细胞和心肌细胞，采用荧光显做镜检测内皮细胞与心肌细胞中EGFP的表达，同时利用免疫组织化学方法检测VEGF的表达。结果：成功地构建了真核表达载体pIRES2—EGFP—hVEGF165，采用脂质体法转染内皮细胞与心肌细胞后，经荧光显微镜观察，可见细胞内有EGFP的表达，同时经免疫组化证实有VEGF的表达。结论：采用脂质体法可以成功地将外源性VEGF165基因转染到内皮细胞与心肌细胞中，并进行表达。本研究为今后利用VEGF基因治疗心肌缺血等疾病提供了实验基础。     

P2x7 deficiency suppresses development of experimental autoimmune encephalomyelitis

Background  

The purinergic receptor P2x7 is expressed on myeloid cells as well as on CNS glial cells, and P2x7 activation has been shown to increase both glial and T-cell activation. These properties suggest a role in the development of autoimmune disease including multiple sclerosis.     

V3 sequence analysis and biological characterization of HIV-1 isolates from asymptomatic and early symptomatic Tanzanian individuals

The aim of this study was to determine HIV-1 V3 sequences, in vitro biological characteristics and co-receptor usage of virus isolates from Tanzania. Virus was isolated from 14 of 17 samples investigated. Four of the isolates induced syncytia in MT-2 cells and used the CXCR4 co-receptor, while the remaining 10 isolates used the CCR5 co-receptor characteristic of non-MT-2 tropic viruses. One of the four MT-2 tropic isolates also used the CCR5 and CCR3 co-receptors. Proviral DNA was detected in all 14 isolates and PCR products were subjected to DNA sequencing. Unambiguous V3 amino acid sequences were obtained from 11 amplificates. Phylogenetic analysis indicated that these sequences were divergent and clustered in HIV-1 subtypes A, C or D. Sequences from the viruses that induced syncytia in MT-2 cells presented characteristic V3 phenotype-associated amino acids. Results of co-receptor analysis are in concordance with the isolate phenotype as determined by replication and induction of syncytia in MT-2 cells. The considerable diversity illustrated by a limited number of isolates from Tanzania is in accordance with reports from other regions of Africa.     

The effect of dopamine D2, D5 receptor and transporter (SLC6A3) polymorphisms on the cue-elicited heroin craving in Chinese.

Heroin dependence is resulted from the interaction between multiple genetic and environmental factors. Subjective craving is considered to be a central phenomenon, which contributes to the continuation of drug use in active abuser and the occurrence of relapse in detoxified abusers. Dopamine pathway has been implicated in the cue-elicited craving for a variety of addictive substances. The objective of this study was to test the hypothesis that heroin addicts carrying specific variants in dopamine-related genes would have higher levels of craving following exposure to a heroin-related cue. Craving induced by a series of exposure to heroin-related cue was assessed in a cohort of Chinese heroin abuser (n = 420) recruited from natural abstinence center at Shanghai. Significantly stronger cue-elicited heroin craving was found in individuals carrying D2 dopamine receptor gene (DRD2) TaqI RFLP A1 allele than the non-carriers (P < 0.001). Furthermore, we did not observed significant association of cue-elicited craving with the nine-repeat allelic variants in dopamine transporter gene (DAT) SLC6A3 and with the dinucleotide repeat polymorphism (DRP) 148bp allele in D5 dopamine receptor gene (DRD5). The results of our study suggest that human dopamine pathway be involved in cue-induced heroin craving, and indicate a potential genetic risk factor for persistent heroin behavior and relapse.     

Diversity of Hepatitis G Virus within a Single Infected Individual

The extent of population diversity among GB virus C (GBV-C)/hepatitis G virus (HGV) within a persistently infected individual (Iw) was investigated by sequence analysis of multiple clones generated from polymerase chain reaction (PCR)-amplified products of cDNA analogous to fragments of 5 non-coding region (5NC), envelope region 1/2 (E1/E2) and non-structural region 3 (NS3) of viral genome. Although nucleotide substitutions were more common in coding regions than in the 5NC region, there was no region corresponding to the hypervariable region of hepatitis C virus in the E1/E2 region. Transition substitution exceeded transversion by 7 to 12-fold, and 79.4% of substitutions were synonymous. This bias against substitutions producing amino acid replacements and the use of Pfu DNA polymerase with an error rate 10 times lower than the observed frequency of substitution, suggests that most substitutions were not artefactual. This data suggests that individual genomes of HGV within an infected individual may differ from each other at 0.23–0.84% nucleotide position and at 0.42–0.61% amino acid position.     

白血病骨髓瘤单抗对白血病细胞株细胞内环核苷酸的影响

本文应用放射免疫测定法,检测了单克隆抗体(λ-44-5-15,λ26-4-15,SMb,OKT10和抗B)作用于相应白血病瘤细胞后细胞内cAMP、cGMP的含量变化。结果发现:抗原、抗体比例在80∶1时经McAb作用16～24h后,瘤细胞内cAMP含量明显高于对照组,cGMP含量明显低于对照组(P<0002-0001)。从而提示:McAb可通过对细胞膜作用,调控细胞内cAMP和cGMP的水平。本文还对McAb作用浓度及作用时间进行了探讨。     

趋化因子受体CCR5反义RNA在真核细胞中的表达

目的 构建趋化因子受体CCR5反义RNA真核表达载体并获取重组假病毒颗粒以用于抗HIV-1研究，方法 用RT-PCR法从健康人外周血单个核细胞（PBMCs)中获得趋化因子受体CCR5翻译起始区的基因片段，并以正、反两个方面定向插入到真核表达载体pLXSN上，重组载体用脂质体转染剂（lipofectAMINE)转染PA317包装细胞，抗-G418克隆的细胞上清经逆转录后用荧光定量PCR（FQ-PCR）测定假病毒滴度，进一步感染NIH/3T3细胞。结果 CCR5正、反义RNA的真核表达载体。经PA317细胞包装形成的假病毒颗粒已成功地感染NIH/3T3细胞，目的基因在该细胞中得到整合与表达。结论 从PBMCs中获得的趋化因子受体CCR5基因片段通过逆转录病毒载体可转移至真核细胞中并得到表达，为进一步研究CCR5反义RNA的抗HIV-1作用奠定了基础。     

STRAIN OF HEPATITIS A VIRUS CAUSING CYTOPATHIC EFFECTS ISOLATED IN A549 CELL LINE

A strain (named BJ-1) of hepatitis A virus (HAV) causing cytopathic effects (CPE) has been isolated in A549 cell cultures from a fecal specimen of an infant with hepatitis A. Hepatitis A antigen (HAAg) w3s detectable in about 50To of the cells by immunofluo- rescence (IF) assay. The viral antigenicity was spe- cifically identified by monoclonal anti-HAV and chim- panzee reference antiserum to HAV (NIH). HAV particles in crystal arraDgement were observed in cell cytoplasm by electron microscopy in ultrathin sec tions. The particles appeared both "empty" and "full". It was observed that many 27-30 nm virtts particles were aggregated by convalescent human he- patitis A sera and chimpanzee HAV antisera observ- ed by immune electron microscopic (IEM) ex- amination. BJ-1 HAAg could be detected on day 34 after inoculation at the initial passage. The virus titers increased and the virus replication cycle short- ened with serial passages. The highest TCID 50 was 10-5/0.1 ml and the shortest replication cycle 2-3 days. IF kit consisting of BJ-1 HAV infected A549 coverslip culture and sheep fluorescence antibody to human IgM were made to detect anti-HAV IgM. Comparing the BJ-t IF with the Abbott EIA HAVAB- M kit. the coincidence rate was 98.4% (246/250).     

降低重型胸部损伤患者肠内营养喂养不耐受发生率

目的降低重型胸部损伤患者肠内营养喂养不耐受发生率。方法成立品管圈小组,针对腹胀、反流或呕吐、腹泻三大改善重点展开原因分析,从多样化培训、6S管理、风险评估等方面进行针对性改进。结果重型胸部损伤患者肠内营养喂养不耐受发生率由63.01%降低至20.55%。结论肠内营养喂养不耐受是重症患者营养实施过程中的常见问题,通过品管圈活动,建立了科学的评估体系和规范的干预措施,改善了重型胸部损伤患者肠内营养喂养不耐受情况,确保了患者安全。