發(fā)布時間：2018-10-24 10:18

【摘要】：長期大量使用抗生素將會出現(xiàn)腸道菌群紊亂,臨床上抗生素相關(guān)性腹瀉已經(jīng)十分普遍。目前已有研究證明花青素具有體外抑菌作用,但是它對抗生素誘導(dǎo)的腸道菌群紊亂是否具有體內(nèi)調(diào)節(jié)作用目前還未見報(bào)道。本文采取給小鼠灌胃抗生素頭孢克肟的方式,制作出腸道菌群紊亂模型小鼠。隨后將其分為紫甘薯花色苷組(Anthocyanins from purple sweet potato,APSP)、陰性對照組、陽性對照組和空白對照組,經(jīng)過灌胃干預(yù)十四天后,頸椎脫臼處死,無菌采集回盲部糞便標(biāo)本。采用溶菌酶法提取糞便標(biāo)本總DNA,采用傳統(tǒng)培養(yǎng)結(jié)合聚合酶鏈?zhǔn)椒磻?yīng)-變性梯度凝膠電泳技術(shù)(Polymerase Chain Reaction- denaturing gradient gel electrophoresis,PCR-DGGE)觀察和分析頭孢克柄對小鼠腸道菌群影響。傳統(tǒng)培養(yǎng)結(jié)果顯示高劑量組小鼠糞便中乳酸桿菌數(shù)量在所選稀釋度由1.48×104 CFU/g下降至30CFU/g以下,而腸球菌數(shù)量由30CFU/g以下上升至1.26×10~5CFU/g,大腸桿菌數(shù)量由1.38×106 CFU/g上升至1.86×106 CFU/g; PCR-DGGE實(shí)驗(yàn)中DGGE圖譜的多樣性指數(shù)分析結(jié)果顯示,與空白對照組相比,抗生素模型組小鼠腸道菌群的豐富度,香農(nóng)指數(shù)有極顯著降低,因此頭孢克肟可以不同程度的誘導(dǎo)小鼠腸道菌群紊亂且隨劑量的增加腸道菌群紊亂程度加重,模型成功建立且以中劑量為最佳選擇。采用PCR-DGGE結(jié)合實(shí)時熒光定量-聚合酶鏈?zhǔn)椒磻?yīng)(RealTime-PCR,RT-PCR)觀察和分析紫甘薯花色苷對腸道菌群紊亂模型小鼠腸道菌群的調(diào)節(jié)作用。DGGE圖譜結(jié)果顯示,與空白對照組相比,低劑量組、中劑量組、高劑量組小鼠腸道菌群的豐富度,香農(nóng)指數(shù)有極顯著差異,與陰性對照組相比,陽性對照組和花色苷干預(yù)組的基因組多樣性沒有顯著差異,但可以看出其灰度值明顯大于陰性對照組,因此采用RT-PCR實(shí)驗(yàn)對基因組的主要基因進(jìn)行絕對定量,結(jié)果顯示:在灌胃花色苷后,腸道菌群紊亂小鼠腸道擬桿菌由6.76×107Count/μL增加至1.41×1011Count/μL;乳桿菌由5.89×109 Count/μL 增加至 3.80×1010Count/μL;大腸桿菌由 3.98×109Count/μL 降低至 2.04×108 Count/μL;腸球菌由 7.08×1011Count/μL 降低至 1.05×1010Count/μL。給小鼠灌胃一定劑量的頭孢克肟可以成功誘導(dǎo)出腸道菌群紊亂模型。并且紫甘薯花色苷對抗生素頭孢克肟誘導(dǎo)的腸道菌群紊亂模型小鼠具有顯著的調(diào)節(jié)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.
【學(xué)位授予單位】：天津科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R151

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