Acylation of the lipooligosaccharide of Haemophilus influenzae and colonization: an htrB mutation diminishes the colonization of human airway epithelial cells

Haemophilus influenzae is a commensal and opportunistic pathogen of the human airways. A number of surface molecules contribute to colonization of the airways by H. influenzae, such as adhesins, including structures found in the lipooligosaccharide (LOS). A human bronchiolar xenograft model was employed to investigate the host-bacterial interactions involved in the colonization of the airway by H. influenzae. Differential display was used to identify H. influenzae mRNA that reflect genes which were preferentially expressed in the xenograft compared to growth. Eleven mRNA fragments had consistent increased expression when the bacteria grew in xenografts. On sequencing these fragments, eight open reading frames were identified. Three of these had no match in the NCBI or the TIGR database, while an additional three were homologous to genes involved in heme or iron acquisition and utilization: two of the mRNAs encoded proteins homologous to enzymes involved in LOS biosynthesis: a heptosyl transferase (rfaF) involved in the synthesis of the LOS core and a ketodeoxyoctonate phosphate-dependent acyltransferase (htrB) that performs one of the late acylation reactions in lipid A synthesis. Inoculation of human bronchiolar xenografts revealed a significant reduction in colonization capacity by htrB mutants. In vitro, htrB mutants elicited lesser degrees of cytoskeletal rearrangement and less stimulation of host cell signaling with 16HBE14o(-) cells and decreased intracellular survival. These results implicate acylation of H. influenzae lipid A as playing a key role in the organisms' colonization of the normal airway.     

类风湿性关节炎RF阳性与阴性患者外周血CD4+细胞基因表达的差异

目的:研究RF因子阳性及阴性RA患者之间是否存在基因组学的差异,探询差异存在的基因表达基础.方法:应用基因表达谱方法来检测上述两型患者CD4 淋巴细胞基因表达情况.结果:RF因子阳性与阴性患者之间有55条基因表达差异有统计学意义.结论:RF因子阳性与阴性RA患者之间存在差异表达基因,这些差异基因多与免疫应答相关.     

小鼠β_2m反义核酸重组荧光蛋白表达载体的构建及表达研究

本研究应用分子生物学技术 ,构建小鼠 β2 m反义核酸重组荧光蛋白表达载体pEGFP β2 mAN ,经过酶切、PCR鉴定 ,以及序列分析证实克隆的正确性。然后用脂质体转染NIH3T3细胞 ,经过RT PCR及Westernblot检测 ,观察重组质粒对细胞β2 mmRNA和蛋白表达的影响 ,同时在荧光显微镜下直接观察细胞转染的结果。结果证明pEGFP β2 mAN已成功导入NIH3T3细胞中 ,且有效表达 ,在荧光显微镜下可见梭形绿色荧光细胞 ;与同步转染的小鼠 β2 m正、反义核酸表达载体pcDNA3 β2 mSN、pcDNA3 β2 mAN的转染结果一起进行分析 ,转染pcDNA3 β2 mSN细胞的 β2 mmRNA和蛋白表达水平升高 ,而转染pcD NA3 β2 mAN和pEGFP β2 mAN的细胞 β2 mmRNA和蛋白表达水平降低。小鼠 β2 m反义核酸重组荧光蛋白表达载体pEGFP β2 mAN的转染结果显示 :它不但可以降低NIH3T3细胞 β2 m基因的表达 ,而且为观察脂质体转染效果提供了直观、即时的方法     

Muscle-specific expression of the smyd1 gene is controlled by its 5.3-kb promoter and 5'-flanking sequence in zebrafish embryos.

Zebrafish SmyD1 is a SET and MYND domain-containing protein that plays an important role in myofiber maturation and muscle contraction. SmyD1 is required for myofibril organization and sarcomere assembly during myofiber maturation. Whole-mount in situ hybridization revealed that smyd1 mRNAs are specifically expressed in skeletal and cardiac muscles in zebrafish embryos. However, it is unknown if smyd1 is expressed in other striated muscles, such as cranial and fin muscles, and moreover, the regulatory elements required for its muscle-specific expression. We report here the analyses of smyd1 expression using smyd1-gfp transgenic zebrafish. smyd1-gfp transgenic zebrafish were generated using the 5.3-kb smyd1 promoter and its 5'-flanking sequence. GFP expression was found in the skeletal and cardiac muscles of smyd1-gfp transgenic embryos. GFP expression appeared stronger in slow muscles than fast muscles in transgenic zebrafish larvae. In addition, GFP expression was also detected in cranial and fin muscles of smyd1-gfp transgenic zebrafish larvae. In situ hybridization confirmed smyd1 mRNA expression in these tissues, suggesting that the expression of the smyd1-gfp transgene recapitulated that of the endogenous smyd1 gene. Deletion analysis revealed that the 0.5-kb sequence in the proximal promoter of smyd1 was essential for its muscle specificity. Together, these data indicate that smyd1 is specifically expressed in most, if not all, striated muscles, and the muscle specificity is controlled by the 5.3-kb promoter and flanking sequences.     

杭州地区8516例孕中期妇女产前筛查回顾性分析

目的分析杭州地区孕中期妇女运用甲胎蛋白(AFP)和游离绒毛膜促性腺激素β亚基(Free-βhCG)进行胎儿先天缺陷产前筛查的结果;并据此修正两筛查指标的孕周中位数值。方法对杭州地区孕14~20w的妇女进行产前AFP和Free-βhCG的检测。胎儿先天畸形由染色体核型分析、B超检查确诊,或经引产证实。应用非线性加权回归求得本地区两筛查指标的中位数,并计算中位倍数值进行分布拟合。比较标化前后检测效果。结果8516例受检孕妇,共筛查出唐氏综合征3例、爱德华综合征2例,神经管畸形7例,其他胎儿异常17例;杭州地区孕中期妇女AFP及Free-β-hcG中位数比欧洲妇女分别高18%和14%;用修正后的中位数重新分析,孕中期假阳性率降低。结论血清AFP、Free-βhcG联合检测,可作为杭州地区孕中期妇女产前筛查优选项目。而根据地域人群的差异,对Mu ltiCalc软件内嵌中位数进行修正后更适于杭州地区孕中期妇女的产前筛查。     

免疫耐受机制研究进展

免疫耐受是机体免疫系统在接触某种抗原后所产生的对该抗原特异性免疫无应答状态,是免疫应答的一种特殊形式,免疫应答的复杂性决定了免疫耐受诱导的复杂性和困难性.随着免疫学的发展,人们对免疫耐受产生机制有了较多的认识.本文对免疫耐受与细胞凋亡、调节性T细胞及树突状细胞的研究进展进行综述.     

纳曲酮对移植排异反应的抑制作用

以小鼠心肌组织异位移植和混合淋巴细胞反应为整体和离体模型，观察了阿片受体阻断剂纳曲酮对移植排异反应的影响。结果显示：给动物从术前开始腹腔注射纳曲酮共１０天（每日二次，每次５ｍｇ／ｋｇ）可明显延长移植心肌组织的存活时间；加入纳曲酮（１０－４～１０－８ｍｏｌ／Ｌ）对混合淋巴细胞反应有抑制作用并呈量效关系。同时还观察到，给正常小鼠腹腔注射纳曲酮３天以上，可引起动物脾细胞由ＣｏｎＡ诱导的淋巴细胞转化反应受抑制。以上结果说明纳曲酮可抑制移植排异反应，此作用有可能是通过阻断内源性阿片肽所致。     

转染survivin反义mRNA对Jurkat淋巴瘤细胞生长和化疗敏感性的影响

目的 研究转染survivin反义mRNA对Jurkat淋巴瘤细胞生长的影响以及转染后淋巴瘤细胞对化疗药物的敏感性。方法 构建survivin反义mRNA真核表达质粒pcDNA3．1-反义（As）survivin;利用脂质体转染法将其转入高表达survivin mRNA T淋巴母细胞淋巴瘤Jurkat细胞系,用逆转录聚合酶链反应（RT—PCR）、免疫组织化学SP法、Western印迹法检测细胞中survivin表达;用细胞计数、流式细胞术（FCM）检测其细胞生长曲线、细胞凋亡指数,并进行光镜、电镜形态学观察;并对转染pcDNA3．1-Assurvivin前后Jurkat细胞分别加入4-羟基-环磷酰胺（CTX）、甲氨蝶呤（MTX）72h后,常规MTT检测细胞存活率。结果 RT—PCR检测转染pcDNA3．1-Assurvivin后48h、5和6周Jurkat细胞survivin mRNA表达,发现survivin mRNA表达皆低于对照组;转染后survivin蛋白表达也明显降低。转染pcDNA3．1-Assurvivin后Jurkat细胞生长倍增时间（52h）明显延长;用FCM检测细胞凋亡发现,转染pcDNA3．1-Assurvivin后Jurkat细胞凋亡指数[20．2％（48h）]明显高于对照组（转染空质粒和未转染组,2．1％和1．3％）;5和6周为6．2％和6．8％,明显高于未转染细胞（1．3％和1．0％）。光镜、电镜观察见转染细胞出现较多凋亡细胞及一些变性肿胀细胞;MTT检测结果显示Jurkat细胞转染前后,经化疗药物4-羟基-环磷酰胺和甲氨蝶呤作用,转染细胞的抑制率明显大于未转染组,差异有统计学意义（P〈0．05）。结论 survivin基因对Jurkat细胞系的生长起着重要的作用,抑制survivin基因表达在T淋巴母细胞淋巴瘤治疗中可能有重要的意义,该基因似可能作为治疗的靶点。