卡氏棘阿米巴肌动蛋白1的免疫学特性和细胞黏附功能

目的:探讨卡氏棘阿米巴(Ac)肌动蛋白1 (Actin 1)(Ac-Actin 1)的免疫学特性,初步阐明Ac-Actin 1介导Ac虫体黏附宿主细胞并参与Ac虫体入侵的作用。方法:以Ac滋养体cDNA为模板,构建原核表达载体pET 22b (+)-Ac-Actin 1,转化大肠埃希菌感受态细胞BL21 (DE3);1 mmol·L^(-1)异丙基硫代半乳糖苷(IPTG)体外诱导表达重组Ac-Actin 1蛋白(rAc-Actin 1),十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)对rAc-Actin 1进行可溶性分析,通过亲和层析方法纯化带有His标签的rAc-Actin 1。3只新西兰白兔随机分为对照组(1只)和实验组(2只);实验组兔以rAc-Actin 1为免疫原皮下注射400μg,对照组兔注入等量生理盐水,制备抗rAc-Actin 1多克隆兔血清;采用酶联免疫吸附试验(ELISA)法检测2组兔抗rAc-Actin 1多克隆抗体效价并测定IgG亚型,Western blotting法检测抗rAc-Actin 1多克隆兔血清与rAc-Actin 1的免疫反应性,间接免疫荧光实验(IFA)检测Ac-Actin 1在Ac滋养体中的定位。以正常滋养体为对照,抗rAc-Actin 1多克隆兔血清阻断Ac滋养体后与Vero细胞共孵育,显微镜观察Ac-Actin 1对Vero细胞的黏附作用。结果:SDSPAGE和BCA蛋白浓度检测,获得高浓度rAc-Actin 1 (1.7 g·L^(-1))蛋白。ELISA法检测,制备的兔抗rAc-Actin1特异性多克隆抗体效价为1∶6 400, IgG1和IgG2a浓度分别为116.76 g·L^(-1)和1 136.15 mg·L^(-1)。Western blotting法检测,兔抗rAc-Actin 1多克隆抗体可与rAc-Actin 1发生特异性结合,具有良好的免疫反应性。IFA检测,rAc-Actin 1主要定位于Ac滋养体的细胞膜上。显微镜观察,与对照组比较,随着抗体与虫体孵育时间的延长,实验组Ac滋养体对Vero细胞的黏附率明显降低(P<0.01),抗rAc-Actin 1特异性多克隆抗体可有效阻断Ac滋养体与Vero细胞的黏附。结论:rAc-Actin 1具有良好的免疫原性和免疫反应性,参与Ac虫体与宿主细胞的黏附作用。

甲硝唑与护理液对棘阿米巴原虫体外杀伤效果的评估(英文)

目的:分析6种角膜接触镜多功能护理液和加入甲硝唑滴眼液的护理液对自生生活性棘阿米巴原虫的杀伤效果。方法:将6种多功能护理液分别加入96孔板中,每种护理液占用48孔,其中24孔滴入阿米巴悬液,另外24孔先滴入甲硝唑滴眼液后再滴入阿米巴悬液,室温静置8h后在倒置显微镜下观察残存阿米巴的形态变化和数量。将残存的棘阿米巴原虫分别在PYG培养液中培养5d,观察其形态、活性与增殖能力的变化。结果:单纯护理液组1-6号阿米巴检出率分别为0%、80.3%、29.1%、41.7%、62.5%、79.2%,加入甲硝唑滴眼液后护理液1-6号阿米巴检出率分别为0%、0%、4.2%、8.3%、16.7%、16.7%,3-6号护理液加与不加甲硝唑滴眼液杀伤阿米巴的效力差异有统计学意义(3-6组的χ2值分别为3.75、7.11、10.54和18.78,P<0.05)。残存的棘阿米巴原虫经培养后活力与增殖力减弱。结论:部分多功能护理液对棘阿米巴原虫的杀伤效果不佳,添加甲硝唑滴眼液后杀伤效果明显提高。

甲硝唑滴眼液与角膜接触镜多功能护理液体外对棘阿米巴原虫的杀伤效果

目的 了解6种角膜接触镜多功能护理液和加入甲硝唑滴眼液的护理液对自生生活性棘阿米巴原虫的杀伤效果。方法 将6种多功能护理液分别加入96孔培养板中,每种护理液占用48孔,其中24孔滴入棘阿米巴悬液,另外24孔先滴入甲硝唑滴眼液后再滴入棘阿米巴悬液,室温静置8 h后在倒置显微镜下观察棘阿米巴的形态变化和数量。将残存的棘阿米巴原虫分别在PYG培养液中培养5 d,观察其形态、活性与增殖能力的变化。结果 单纯护理液组1～6号棘阿米巴原虫检出率分别为0、8.3%、29.1%、41.7%、62.5%、79.2%,加入甲硝唑滴眼液后护理液1～6号棘阿米巴原虫检出率分别为0、0、4.2%、8.3%、16.7%、16.7%,3～6号护理液加与不加甲硝唑滴眼液杀伤棘阿米巴原虫的效果差异有统计学意义（χ^2=3.75～18.78,P〈0.05）。残存的棘阿米巴原虫经培养后活力与增殖力减弱。结论 部分多功能护理液对棘阿米巴原虫的杀伤效果不佳,添加甲硝唑滴眼液后杀伤效果明显提高。

Helicobacter is preserved in yeast vacuoles! Does Koch's postulates confirm it?

The manuscript titled "Vacuoles of Candida yeast behave as a specialized niche for Helicobacter pylori(H.pylori)" not only has not been prepared in a scientific manner but the methodology used was not adequate, and therefore the conclusion reached was not correct. First of all, "yeast" is a broad terminology covering a great number of genera and species of unicellular micro-organisms. The authors should have defined the organism with its binary scientific name. This measure would allow experiment reproduction by the scientific community. Moreover, the criteria established by Robert Koch to identify a specific microorganism or pathogen was not adopted in the methodology used. Regarding the methodology applied, use of the chicken eggyolk(Ig Y) antibody and PCR of the apparently tainted yeast population to prove H. pylori existence in the yeast vacuoles might be main factors for their wrong conclusions. Bacterial tropism toward yeast extract is a known phenomenon, and yeast extract is one of the main ingredients in culture media. Their internalization through phagocytosis or similar pathways does not seem possible or practical because of the thick and cellulosic yeast wall. While the small size of yeast cells does not support their ability in harboring several H. pylori, other observations such as inefficiency of antifungal therapy as anti-Helicobacter therapy strongly reject the conclusion reached by the above-mentioned article.