紫甘薯花色苷对肠道菌群的调节作用

发布时间：2018-10-24 10:18

【摘要】：长期大量使用抗生素将会出现肠道菌群紊乱,临床上抗生素相关性腹泻已经十分普遍。目前已有研究证明花青素具有体外抑菌作用,但是它对抗生素诱导的肠道菌群紊乱是否具有体内调节作用目前还未见报道。本文采取给小鼠灌胃抗生素头孢克肟的方式,制作出肠道菌群紊乱模型小鼠。随后将其分为紫甘薯花色苷组(Anthocyanins from purple sweet potato,APSP)、阴性对照组、阳性对照组和空白对照组,经过灌胃干预十四天后,颈椎脱臼处死,无菌采集回盲部粪便标本。采用溶菌酶法提取粪便标本总DNA,采用传统培养结合聚合酶链式反应-变性梯度凝胶电泳技术(Polymerase Chain Reaction- denaturing gradient gel electrophoresis,PCR-DGGE)观察和分析头孢克柄对小鼠肠道菌群影响。传统培养结果显示高剂量组小鼠粪便中乳酸杆菌数量在所选稀释度由1.48×104 CFU/g下降至30CFU/g以下,而肠球菌数量由30CFU/g以下上升至1.26×10~5CFU/g,大肠杆菌数量由1.38×106 CFU/g上升至1.86×106 CFU/g; PCR-DGGE实验中DGGE图谱的多样性指数分析结果显示,与空白对照组相比,抗生素模型组小鼠肠道菌群的丰富度,香农指数有极显著降低,因此头孢克肟可以不同程度的诱导小鼠肠道菌群紊乱且随剂量的增加肠道菌群紊乱程度加重,模型成功建立且以中剂量为最佳选择。采用PCR-DGGE结合实时荧光定量-聚合酶链式反应(RealTime-PCR,RT-PCR)观察和分析紫甘薯花色苷对肠道菌群紊乱模型小鼠肠道菌群的调节作用。DGGE图谱结果显示,与空白对照组相比,低剂量组、中剂量组、高剂量组小鼠肠道菌群的丰富度,香农指数有极显著差异,与阴性对照组相比,阳性对照组和花色苷干预组的基因组多样性没有显著差异,但可以看出其灰度值明显大于阴性对照组,因此采用RT-PCR实验对基因组的主要基因进行绝对定量,结果显示:在灌胃花色苷后,肠道菌群紊乱小鼠肠道拟杆菌由6.76×107Count/μL增加至1.41×1011Count/μL;乳杆菌由5.89×109 Count/μL 增加至 3.80×1010Count/μL;大肠杆菌由 3.98×109Count/μL 降低至 2.04×108 Count/μL;肠球菌由 7.08×1011Count/μL 降低至 1.05×1010Count/μL。给小鼠灌胃一定剂量的头孢克肟可以成功诱导出肠道菌群紊乱模型。并且紫甘薯花色苷对抗生素头孢克肟诱导的肠道菌群紊乱模型小鼠具有显著的调节作用。
[Abstract]:Long-term use of antibiotics will lead to intestinal microflora disorders, antibiotic-associated diarrhea has been very common in clinical practice. It has been proved that anthocyanin has bacteriostatic effect in vitro, but whether anthocyanin can regulate intestinal flora disorder induced by antibiotics in vivo has not been reported. The intestinal flora disorder model of mice was established by intragastric administration of cefixime. It was then divided into (Anthocyanins from purple sweet potato,APSP group, negative control group, positive control group and blank control group. After 14 days of oral administration, cervical vertebrae dislocations were executed and fecal specimens of ileocecal part were collected. Total DNA, extracted from feces by lysozyme method was used to observe and analyze the effect of ceftilla on intestinal flora of mice by traditional culture combined with polymerase chain reaction-denaturing gradient gel electrophoresis (Polymerase Chain Reaction- denaturing gradient gel electrophoresis,PCR-DGGE). The results of traditional culture showed that the number of Lactobacillus in feces of high dose group decreased from 1.48 脳 10 ~ 4 CFU/g to below 30CFU/g at the selected dilution. The number of Enterococcus increased from below 30CFU/g to 1.26 脳 10 ~ (5) CFU / g, the number of Escherichia coli increased from 1.38 脳 10 ~ (6) CFU/g to 1.86 脳 10 ~ (6) CFU/g; PCR-DGGE. The results showed that compared with the blank control group, the abundance of intestinal flora in the antibiotic model group was higher than that in the control group. The Shannon index was significantly decreased, so cefixime could induce intestinal microflora disorder in mice to varying degrees and increase with the increase of dose. The model was successfully established and the best choice was middle dose. PCR-DGGE combined with real-time fluorescence quantification-polymerase chain reaction (RealTime-PCR,RT-PCR) was used to observe and analyze the regulation of purple sweet potato anthocyanin on intestinal flora in mice with intestinal dysbacteriosis. The results of DGGE map showed that compared with the blank control group, the low dose group was lower dose group. There were significant differences in intestinal flora richness and Shannon index between middle and high dose groups, but there was no significant difference in genomic diversity between positive control group and anthocyanin intervention group. But it can be seen that its gray value is obviously higher than that of the negative control group. Therefore, the RT-PCR experiment was used to quantify the main genes of the genome. The results showed that: after oral administration of anthocyanin, Enterococcus increased from 6.76 脳 107Count/ 渭 L to 1.41 脳 1011Count/ 渭 L, Lactobacillus from 5.89 脳 109 Count/ 渭 L to 3.80 脳 1010Count/ 渭 L, Escherichia coli from 3.98 脳 109Count/ 渭 L to 2.04 脳 108Count/ 渭 L, Enterococcus from 7.08 脳 1011Count/ 渭 L to 1.05 脳 1010Count/ 渭 L. The intestinal flora disorder model was successfully induced by intragastric administration of cefixime in mice. And purple sweet potato anthocyanin has a significant regulatory effect on intestinal flora disorder induced by antibiotic cefixime in mice.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R151

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