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肠道碱性磷酸酶解毒脂多糖并预防炎症在肠道微生物群的响应
来源: | 作者:詹妮弗·M·贝茨1，Janie Akerlund，Erika Mittge，和Karen Guillemin | 发布时间: 2020-06-23 | 11298 次浏览 | 分享到:

微生物群计量和分析
       为了比较myd88-MO和WT同科的微生物群，将这些胚胎放在底部为网眼的聚碳酸酯圆柱体中，并在共享的微生物水生环境中饲养在同一罐中。在8 dpf时，将动物用三卡因甲磺酸盐（MS222，Sigma）安乐死，并在无菌水中漂洗3次。将分别注入10 WT或myd88-MO的幼虫置于100μl无菌水中，匀浆，稀释并在胰蛋白酶大豆琼脂上培养以计数CFU并检查菌落形态。为了估计斑马鱼幼虫肠道中的细菌浓度，据此我们可以推断出LPS的浓度，我们测量了从已知细胞数（斑马鱼微生物群的一个主要成员）维罗纳氏菌生物变种的已知细胞中扩增出的16S rRNA基因的平均数量。大约4份/ CFU。根据我们之前对每条斑马鱼扩增的16S rRNA拷贝的测量结果（Bates等，2006），我们计算出每8 dpf幼虫的细菌载量约为4×105。如果将液体倒入幼虫的肠道，我们估计该器官的体积约为1至4 nl。
统计分析—重复进行AP活性测定，每次治疗至少进行两次试验，并使用两个样本的t检验对数据进行分析，并假设Excel中存在不相等的方差（Microsoft Office）。 存活曲线至少进行两次，每次处理总共合计至少30条鱼。 使用GraphPad Prism（Kaplan-Meier分析，GraphPad软件，http：//www.graphpad.com）对存活曲线的数据作图和分析。 还使用GraphPad Prism（使用一个分组变量进行单向ANOVA的散点图）对中性粒细胞计数进行图形化和分析，并假设Excel中的方差不相等，使用两样本t检验进行中性粒细胞计数分析。
补充材料
有关补充材料，请参见PubMed Central上的Web版本。
致谢
We thank Rose Gaudreau and the staff of the University of Oregon Zebrafish Facility for fish husbandry, Poh Kheng
Loi for histology services, Mimi Shirasu-Hiza for assistance with statistical analysis, Tamara Pozos and Lalita Ramakrishnan for the gift of the myd88-MO, Hilary Clay for sharing unpublished information on the tnfr1-MO, members of the Guillemin lab for insightful discussions, and Sarah Cheesman, Hilary Clay, Tory Herman, Robin Lesley and Lalita Ramakrishnan for critical reading of the manuscript. This research was supported by NIH grants R21 DK067065-01 and R01 DK075549-01 and a Burroughs Wellcome Fund Career Award in the Biomedical Sciences (to K.G.). NIH grant HD22486 provided support for the Oregon Zebrafish Facility.
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