鼠肺炎沙眼衣原体质粒蛋白pgp5的克隆表达鉴定及免疫原性分析

目的获取鼠肺炎沙眼衣原体(Chlamydia muridarum)质粒蛋白pgp5的基因及纯化的蛋白并鉴定其免疫原性。方法设计引物,PCR扩增目的基因,将其定向插入到原核表达载体pET28a中,然后将重组质粒转化入大肠杆菌E.coli DH5α中,并用PCR扩增及序列测定等方法对重组质粒进行鉴定。再将重组质粒转化入感受肽Rosetta(DE3)并诱导表达,用镍柱纯化pgp5-his融合蛋白。用纯化后的目的蛋白免疫新西兰家兔,酶联免疫吸附试验(ELISA)测定抗体效价,Western blot、细胞免疫荧光方法检测抗体与pgp5蛋白的结合。结果所获得的pgp5基因片段经测序长度为795 bp,检索确认其序列与GeneBank一致。十二烷基硫酸钠一聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹实验均显示获得相对分子质量约29 000的纯化蛋白。ELISA检测多克隆抗体效价达1∶100 000。细胞免疫荧光检测结果显示抗体可与体外培养的鼠肺炎沙眼衣原体特异性结合。结论成功表达pgp5-his融合蛋白,并制备了高效价、高特异性的抗pgp5抗体,为进一步研究奠定了基础。     

细胞凋亡相关基因与银屑病

分子生物学研究证明，细胞凋亡受一系列凋亡相关基因的调控，许多疾病的发生，发展过程中存在着凋亡相关基因的表达异常。在银屑病的研究中发现多种基因以不同的机理和方式介导着其病理生理过程的进行。对细胞凋亡分子机理的了解将有助于揭示该病的发病机理，并可能开创基因治疗前景。     

细胞凋亡相关基因与银屑病

分子生物学研究证明,细胞凋亡受一系列凋亡相关基因的调控,许多疾病的发生、发展过程中存在着凋亡相关基因的表达异常。在银屑病的研究中发现多种基因以不同的机理和方式介导着其病理生理过程的进行。对细胞凋亡分子机理的了解将有助于揭示该病的发病机理,并可能开创基因治疗前景。     

IFN-γ体外诱导沙眼衣原体持续性感染细胞模型的建立

目的：建立γ－干扰素（ IFN-γ）体外诱导沙眼衣原体持续性感染的细胞模型。方法：衣原体感染McCoy细胞,实验组加入0.5 ng／mL IFN-γ诱导沙眼衣原体,对照组无IFN-γ诱导,空白组为正常培养的McCoy细胞,培养72 h后通过光学显微镜、荧光显微镜、电子显微镜分别观察诱导后衣原体的细胞形态。实验组72 h解除IFN-γ诱导,继续培养48 h,观察衣原体细胞形态学变化。结果：实验组在0.5 ng／mL IFN-γ作用72 h后,碘液染色发现衣原体包涵体的数量比对照组减少,包涵体变小;荧光抗体染色显示衣原体包涵体质地疏松,染色不均匀;透射电子显微镜可见包涵体变小和网状体体积增大等特征的典型异形包涵体。去除IFN-γ作用后,包涵体内致密的EB颗粒数量增加。对照组培养72 h后碘液染色和荧光染色可见典型包涵体,透射电子显微镜可见典型的网状体和原体,充满整个包涵体。结论：成功建立了IFN-γ体外沙眼衣原体持续性感染的细胞模型,为进一步进行沙眼衣原体持续性感染形成机制的研究奠定了基础。     

沙眼衣原体pORF5质粒蛋白通过HMGB1抑制细胞凋亡机制初步研究

【摘要】 目的 探讨pORF5质粒蛋白抗凋亡分子机制,为进一步阐明沙眼衣原体致病机制提供实验依据。方法 将HeLa细胞分为两组,一组用凋亡诱导剂碳酰氰基-对-氯苯腙（CCCP）刺激30 min,另一组经pORF5质粒蛋白预处理18 h后,再用CCCP刺激30 min。然后,Western印迹检测细胞凋亡相关蛋白Bcl-2、Bax和胱天蛋白酶3（caspase-3）的表达水平;JC-1荧光探针检测HeLa细胞线粒体膜电位变化;间接免疫荧光观察细胞色素c释放情况。为了分析高迁移率族蛋白1（HMGB1）是否参与pORF5质粒蛋白的抗凋亡作用,分别用HMGB1 shRNA和对照RNA稳定转染HeLa细胞,再用pORF5质粒蛋白与CCCP共刺激两种细胞后,测定Bcl-2、Bax和活化的caspase-3蛋白的表达以及细胞色素c的释放情况。两组间比较采用配对样本t检验。结果 pORF5质粒蛋白能拮抗CCCP 诱导的线粒体膜电位的下降,CCCP单独处理组Hela细胞红/绿荧光强度比率为0.4 ± 0.1,显著低于pORF5与CCCP共处理组（1.7 ± 0.3,t = 6.95,P < 0.01）。与对照组相比,pORF5与CCCP共处理组HeLa细胞Bcl-2蛋白表达水平增加（5.3 ± 0.6）倍（t = 8.62,P < 0.01）,而Bax和活化的caspase-3平均表达水平分别降低79% ± 10%（t = 9.23,P < 0.01）和75% ± 8%（t = 4.26,P < 0.05）。与对照RNA转染组相比,HMGB1 shRNA转染组HeLa细胞线粒体膜电位下降（t = 11.23,P < 0.01）,细胞色素c释放增加,Bcl-2的表达水平降低（t = 7.19,P < 0.05）,而Bax的表达水平增加（t = 13.06,P < 0.01）。结论 沙眼衣原体pORF5质粒蛋白通过HMGB1阻断线粒体凋亡途径发挥抗凋亡作用。     

沙眼衣原体中药体外敏感性试验

为了探讨中药对沙眼衣原体（ＣＴ）的体外敏感性试验方法,我们利用组织细胞培养技术测定了３种中药合剂对４１株ＣＴ临床分离株的体外敏感性。现将实验结果报道如下。一、材料和方法（一）材料:①实验菌株:所有菌株均采自我院性病中心非淋菌性尿道炎（ＮＧＵ）患者泌尿生殖道,经分离、纯化而得。②细胞株:ＭｃＣｏｙ细胞:由美国标准菌库（ＡＴＣＣ）提供,本室保存传代。③培养基的制备:参照文献［１］方法,略加改进。（二）药物和方法:１药物来源:根据ＮＧＵ的３种病征配制３种中药合剂,所有中药均购自于广州市中药材公司。①中药合剂Ⅰ:由…     

衣原体噬菌体及其与宿主细胞结合机制的相关研究

噬菌体(bacteriophage,phage)是感染细菌、真菌、放线菌或螺旋体等微生物的总称。衣原体噬菌体是一种以衣原体为宿主的噬菌体。1982年科学家首次发现了衣原体噬菌体Chp1,此后又相继发现了5种新的衣原体噬菌体,包括φCPG1,Chp2,φCPAR39(φCpn1),Chp3和Chp4。该文主要对目前所发现的6种衣原体噬菌体分别进行了介绍,并简单介绍了它们与宿主细胞的结合机制,为今后对衣原体噬菌体与宿主细胞间相互作用的进一步研究奠定基础。     

角质形成细胞相关基因的研究进展

角质形成细胞是表皮的主要构成细胞，很多皮肤病都有不同程度的角质形成细胞受累，而角质形成细胞受累经常涉及到基凶的变化，目前已知与角质形成细胞相关的基因主要有癌基因、抑癌基因、凋亡调节基因等。     

角质形成细胞相关基因的研究进展

角质形成细胞是表皮的主要构成细胞 ,很多皮肤病都有不同程度的角质形成细胞受累 ,而角质形成细胞受累经常涉及到基因的变化。目前已知与角质形成细胞相关的基因主要有癌基因、抑癌基因、凋亡调节基因等     

Pelvic inflammatory disease in patients infected with Chlamydia trachomatis: in vitro cell mediated immune response to chlamydial antigens.

Blood samples were obtained from 11 women with laparoscopically confirmed acute salpingitis who yielded positive cultures of Chlamydia trachomatis from the cervix. Four patients also had perihepatitis. Lymphocyte transformation assays, using C trachomatis serovars I and L2 as antigens, showed that the patients' lymphocytes responded to antigenic stimulation more strongly than the lymphocytes of age matched controls. Responses to the I and L2 antigens correlated strongly, but greater responses were obtained to the L2 antigen. No correlation was found between the response in the lymphocyte transformation assay and the degree of the inflammatory changes of the fallopian tubes, the presence of perihepatitis, or the titres of humoral antibodies to C trachomatis as measured by microimmunofluorescence. Sequential transformation assays, however, showed that patients with perihepatitis tended to have a more sustained response.     

Taking cell cultures to the patient in an attempt to improve chlamydial isolation.

McCoy cell cultures were inoculated with 121 urethral and cervical specimens taken from patients attending one of two sexually transmitted disease clinics. The mean number of Chlamydia trachomatis inclusions was greater when the cultures were inoculated with the specimens and centrifuged in the clinic than when the specimens were first stored in liquid nitrogen. Furthermore, 18 of the 29 chlamydia-positive specimens produced larger numbers of of inclusions when inoculated immediately. Despite this, the isolation rate from specimens inoculated directly (22%) was about the same as from specimens which had been frozen (21%). Of the 30 occasions on which the cell monolayers were disrupted, 29 followed immediate inoculation. This may possibly have been due to some toxic factor in some of the specimens.     

Epidemiology of genital chlamydial infections in patients with chlamydial conjunctivitis; a retrospective study.

OBJECTIVE: To determine how often chlamydial conjunctivitis is accompanied by a genital chlamydial infection and if there is a correlation between the dominant hand and the eye first infected. METHODS: We retrospectively studied the records of 65 patients with chlamydial conjunctivitis who were referred to the Outpatient Department of Sexually Transmitted Diseases (STD) of the University Hospital Rotterdam by ophthalmologists of the Eye Hospital Rotterdam. The patients have recently been asked by letter if they were left- or right-handed. RESULTS: Twenty of the 37 men (54%) had a positive chlamydial urethral culture. Seventy per cent of these men had no genital symptoms. Eight of the 37 men (22%) had a non-specific urethritis (NSU). Twenty of the 27 women examined (74%) had a positive chlamydial cervical culture. Sixty per cent of these women had no genital symptoms. Eight women with a genital chlamydial infection also had another genital infection. Five women without a genital chlamydial infection had another genital infection. Two women had no genital infection at all. A correlation between the eye infected and left- or right-handedness of the patient could not be found. CONCLUSIONS: A considerable percentage of the patients with a chlamydial conjunctivitis had a concomitant genital chlamydial infection. The majority of them had no genital symptoms. Since patients with chlamydial conjunctivitis and/or their partners possibly have a concomitant genital chlamydial infection, we recommend referral of both patients and sexual partners to an STD clinic for routine examination and systemic treatment when indicated.     

Identification of genes specifically regulated in human melanoma cells

In order to improve the characterization of human malignant melanoma cells and their variant gene expression in vitro, a search for specifically regulated genes was performed. Four melanoma cell lines (M5, MEWO, IGR39, SKMEL13) and newly cultured normal human melanocytes were included in a comparative hybridization (differential screening) of a human melanoma cDNA-library. Six cDNAs were isolated showing a stronger expression (four genes) or a weaker expression (two genes) in melanoma cells than in normal human melanocytes. Quantification of the expression patterns of the two repressed genes in Northern blots revealed general expression in all melanocyte cultures examined, no expression in three cell lines (M5, IGR39, SKMEL13) and weak expression in MEWO. The four induced genes were found to be only weakly expressed in normal human melanocytes, but showed an elevated expression in all of the four melanoma cell lines tested. Thus, using the technique of differential screening, consistent gene regulation at the messenger RNA level was identified, which distinguishes the four melanoma cell lines tested from normal melanocytes. We conclude from the expression patterns that specific gene regulation in melanoma cells in vitro is characterized both by strong repression of some melanocyte genes, as well as by the induction of other genes, but there was no indication of new expression of genes specific for melanoma cells. Because of the uniform induction or repression in different melanoma cell lines, it is conceivable that the cloned genes may be involved in the malignant transformation of melanocytic cells.     

Identification of selectively expressed genes and antigens in CTCL

Abstract:  The knowledge of tumor-associated antigens is required for most types of immunotherapy and can substantially facilitate diagnosis. To identify potential tumor-associated genes expressed in cutaneous T-cell lymphoma (CTCL), we used three complementary strategies: antigens which elicit a humoral immune response in CTCL patients were detected by serological analysis of a recombinant cDNA expression library. cDNAs differentially expressed in CTCL but not peripheral blood monocytes were identified by comparative cDNA hybridization and suppression subtractive hybridization. We identified 43 genes selectively expressed by CTCL cells, that have not yet been described in the context of CTCL development, but most of which had been reported to be associated with cancer. Expression analysis by database mining and subsequently RT-PCR on selected clones confirmed their selective expression in CTCL tissues. Serological tests showed that 15 clones were recognized by sera of CTCL patients but not of healthy donors. Analysis of serological tests for 11 clones using serum antibody detection array (SADA) and 100 sera of controls and CTCL patients each revealed up to 5% reactive sera in the tumor group. The expression pattern of the detected clones and their immunogenicity demonstrates that they might be relevant for the understanding of CTCL and suggests particularly three clones, HD-CL-41 (DRAK2), HD-CL-49 (nudC) and HD-CL-12 (ZNF195) for further analysis with respect to their prognostic and therapeutic value for CTCL.     

Does detection of chlamydial antibodies by microimmunofluorescence help in managing chlamydial lower genital tract infection in women?

A total of 113 women thought to have chlamydial infection of the lower genital tract were studied prospectively to evaluate the effect of antibiotic treatment on antibodies to chlamydiae detected by microimmunofluorescence. Of them, 81 were randomly selected for treatment with a two week course of either triple tetracycline or erythromycin stearate, and 32 who had microimmunofluorescent antibodies to, but did not yield cultures for, chlamydiae were used as controls and left untreated. Results for the treated patients showed that 22 (27%) had at least a fourfold fall in the microimmunofluorescent titre, but there was a similar rise in titre in 14 (17%), and the titre remained unaltered in 45 (56%) patients. In the control group 10 (31%) patients had at least a fourfold fall in titre, but there was a similar rise in titre in seven (22%), and it remained unaltered in 15 (47%) patients. The differences between these percentages in treated and untreated patients were not significant.     

Investigations on in vitro survival and virulence of T. pallidum under aerobiosis.

Motility of pathogenic T. pallidum was maintained in aerobic in vitro cultures for several weeks using a special medium. The latter consisted of McCoy's 5a medium supplemented with glutathione, sodium pyruvate, HEPES buffer, gentamycin (garamycin), and fetal calf serum. The virulence of the organisms was lost in 5 to 6 days. No multiplication of the organisms was observed. Four antibiotics (viomycin, kanamycin, gentamycin (garamycin), and neomycin) were tested for their bactericidal action and possible toxicity to T. pallidum. Gentamycin proved to be superior to the other three antibiotics in being non-toxic to the treponemes and showing a possible stimulatory effect on their motility and longevity. Cultivation of T. pallidum in cultured cells in the presence of the enzymes, superoxide dismutase and catalase, in a special medium showed possibilities for future experimentation under monitored, reduced oxygen pressure with a continuous system to dismutate superoxide radicals.