2型猪链球菌烯醇化酶的原核表达及其对体外血脑屏障模型通透性影响的检测

通过原核表达的方式获得能在多种病原茵表面表达的烯醇化酶（Enolase,Eno）,探索Eno在脑膜炎致病过程中的作用。根据GenBank上公布的基因序列设计Eno特异性引物,通过PCR技术扩增2型猪链球菌（Streptococcussuis serotype2,SS2）的Eno基因序列,克隆到pMD18-T载体,进-步亚克隆到原核表达载体pET28a（＋）,构建重组表达质粒pET28a—Eno,转化入大肠杆菌BL21CodonPlus（DE3）感受态中诱导表达,经镍离子螯合柱纯化后,检测其对体外血脑屏障模型通透性的影响。结果显示,经1mmol／LIPTG诱导5h为最佳诱导条件,目的蛋白约54000,与预期蛋白大小相符合;纯化的蛋白Westernblotting分析表明,其具有较好的免疫原性;Eno可致体外血脑屏障模型通透性增加。结果表明,Eno作为SS2潜在的毒力因子在SS2引起脑膜炎的致病过程中起到重要的作用。     

Localization of Inhibin in Some Rat Tissues and Exploration of Its Transport Mechanism

The aims of the study were to determine the distribution of inhibin and its α subunit in some rat tissues by an immunohistochemical method of streptavidin-biotin complex （SABC） and to assess the transport mechanism of inhibin by investigating the localization of inhibin and α subunits in the central nervous system （mainly in hypothalamus and pituitary） of ovariectomized rats. Investigations on the extragonadal tissues of ovariectomized rats showed positive expression of inhibin and its α subunit in heart, kidney, spleen, pancreatic gland cells, but no positive reaction sites were seen in lung, liver, submaxillary gland, and adrenal gland. After injection with inhibin α subunit fragment or inhibin extract, positive reaction sites were observed in hypothalamus and pituitary of ovariectomized rats by SABC. Inhibin and its subunit was present in a wide variety of nonreproductive organs and tissues, and its expression was tissue specific, which indicated that inhibin might play a role in regulating tissue function through autocrine/paracrine mechanisms. Inhibin dimer and α subunit could be transported through the BBB by the method of “separation and reconstruction”.     

三七总皂苷对大鼠脊髓损伤后GS的表达及运动功能恢复的影响

目的探讨三七总皂苷(total panax notoginseng saponins,TPNS)对大鼠脊髓半横断损伤后运动功能恢复的作用以及对谷氨酰胺合成酶(glutaminesynthetase,GS)表达的影响。方法将大鼠随机分为正常组、溶媒对照组和TPNS组,实验组大鼠建立脊髓半横断模型,TPNS组损伤处为脊髓右侧T10段。损伤后15 min,腹腔注射剂量为20 mg/kg的三七总皂苷,每天给药1次,溶媒对照组注射等量生理盐水。术后1、3、7、14、21、28 d进行行为学指标检测;采用免疫生物化学方法检测脊髓损伤远侧端GS表达的变化。结果行为学结果表明,该浓度的三七总皂苷能促进大鼠脊髓损伤后运动功能的恢复,其中损伤后7 d和14 d的BBB评分表明,三七总皂苷组大鼠运动功能恢复程度明显高于溶媒对照组。免疫组化结果表明脊髓半横断损伤后,在损伤脊髓远侧端GS的表达损伤侧强于对侧,损伤侧GS的表达趋势为1、3 d逐渐增强,7 d达高峰,在14 d时GS的表达逐渐下降,至28 d仍略高于正常组。三七总皂苷组和溶媒对照组相比,GS的表达在相同时间点优于对照组,尤其是3 d和7 d。结论三七总皂苷促进大鼠脊髓损伤后运动功能的恢复,这可能与其促进GS表达,从而改善脊髓再生的微环境有关。