不同年龄段川金丝猴肠道菌群结构差异分析
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摘要
本试验旨在分析幼年、成年、老年川金丝猴肠道菌群结构以及随年龄变化的趋势。将提取的幼年、成年、老年川金丝猴粪样总DNA进行聚合酶链反应-变性梯度凝胶电泳(PCRDGGE),对结果进行UPGMA聚类分析、主成分分析、生物多样性比较。用荧光定量PCR技术检测肠道菌群中真细菌、乳酸杆菌属(Lactobacillus spp.)、肠杆菌科(Enterobacteriaceae)、双歧杆菌属(Bifidobacterium spp.)的数量。结果显示:无论是UPGMA聚类分析,还是主成分分析以及生物多样性比较均表明,不同年龄段川金丝猴肠道菌群多样性存在明显差异;指纹图谱中成年与幼年川金丝猴的优势条带较多,老年川金丝猴的优势条带则较少。各年龄段川金丝猴肠道菌群中真细菌的数量都为同一数量级(1011);幼年川金丝猴肠道菌群中Enterobacteriaceae的数量显著少于成年和老年川金丝猴(P<0.05);成年川金丝猴肠道菌群中Lactobacillus spp.的数量极显著多于幼年和老年川金丝猴(P<0.01);老年川金丝猴肠道菌群中Bifidobacterium spp.的数量极显著少于成年和幼年(P<0.01)。由此得出,川金丝猴肠道菌群随年龄增长,多样性呈幼年到成年增加、成年到老年降低的趋势,老年时期优势菌群显著减少;此外,川金丝猴肠道各菌群的数量在不同年龄段有较大差异。
This experiment aimed at analyzing the structure of gut microbiome in young,adult and old Rhinopithecus roxellana,and analyzing the change trend of the gut microbiome with the age increasing.Performed the polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE) with the total DNA extracted from the faeces of young,adult and old Rhinopithecus roxellana,and the results were analyzed by UPGM A cluster and principal component,and the biological diversity of Rhinopithecus roxellana in different ages was compared.The number of eubacteria,Lactobacillus spp.,Enterobacteriaceae,Bifidobacterium spp.in gut microbiome was detected by fluorescence quantitative PCR technology.The results showed as follows: not only the UPGM A cluster analysis,but also the principal component analysis and the biological diversity comparison indicated that the diversity of gut microbiome of Rhinopithecus roxellana in different ages existed obvious differences.The advantages of the band of the young and adult Rhinopithecus roxellana were more than the old Rhinopithecus roxellana in the fingerprint.The number of eubacteria in the gut microbiome of Rhinopithecus roxellana in different ages was all in the level 1011.The number of Enterobacteriaceae in the gut microbiome of the young Rhinopithecus roxellana was significantly less than the adult's and the old's(P < 0.05).The number of Lactobacillus spp.in the gut microbiome of the adult Rhinopithecus roxellana was significantly more than the young 's and the old's(P < 0.01).The number of Bifidobacterium spp.in the gut microbiome of the old Rhinopithecus roxellana was significantly less than the adult's and the young 's(P < 0.01).In conclusion,along with the age increasing,the diversity of the gut microbiome of Rhinopithecus roxellana shows an increasing trend from childhood to adulthood and shows a reducing trend from adulthood to senectitude.And,the number of advantage bacterium group is reduced in the senile period.M oreover,the number of gut microbiome of Rhinopithecus roxellana has great differences in different ages.
This experiment aimed at analyzing the structure of gut microbiome in young,adult and old Rhinopithecus roxellana,and analyzing the change trend of the gut microbiome with the age increasing.Performed the polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE) with the total DNA extracted from the faeces of young,adult and old Rhinopithecus roxellana,and the results were analyzed by UPGM A cluster and principal component,and the biological diversity of Rhinopithecus roxellana in different ages was compared.The number of eubacteria,Lactobacillus spp.,Enterobacteriaceae,Bifidobacterium spp.in gut microbiome was detected by fluorescence quantitative PCR technology.The results showed as follows: not only the UPGM A cluster analysis,but also the principal component analysis and the biological diversity comparison indicated that the diversity of gut microbiome of Rhinopithecus roxellana in different ages existed obvious differences.The advantages of the band of the young and adult Rhinopithecus roxellana were more than the old Rhinopithecus roxellana in the fingerprint.The number of eubacteria in the gut microbiome of Rhinopithecus roxellana in different ages was all in the level 1011.The number of Enterobacteriaceae in the gut microbiome of the young Rhinopithecus roxellana was significantly less than the adult's and the old's(P < 0.05).The number of Lactobacillus spp.in the gut microbiome of the adult Rhinopithecus roxellana was significantly more than the young 's and the old's(P < 0.01).The number of Bifidobacterium spp.in the gut microbiome of the old Rhinopithecus roxellana was significantly less than the adult's and the young 's(P < 0.01).In conclusion,along with the age increasing,the diversity of the gut microbiome of Rhinopithecus roxellana shows an increasing trend from childhood to adulthood and shows a reducing trend from adulthood to senectitude.And,the number of advantage bacterium group is reduced in the senile period.M oreover,the number of gut microbiome of Rhinopithecus roxellana has great differences in different ages.
引文
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[25]BARTOSH S,FITE A,MACFARLANE G T,et al.Characterization of bacterial com m unities in feces from healthy elderly volunteers and hospitalized elderly patients by using real-tim e PCR and effects of antibiotic treatm ent on the fecal m icrobiota[J].Applied and Environm ental M icrobiology,2004,70(6):3575-3581.
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[28]MARIAT D,FIRMESSE O,LEVENEZ F,et al.The Firm icutes/Bacteroidetes ratio of the hum an m icrobiota changes with age[J].BM C M icrobiology,2009,9(1):123.
[29]BIAGI E,NYLUND L,CANDELA M,et al.Through ageing,and beyond:gut m icrobiota and inflam m atory status in seniors and centenarians[J].PLo S One,2010,5(5):e10667.
[30]STSEPETOVA J,SEPP E,KOLK H,et al.Diversity and m etabolic im pact of intestinal Lactobacillus species in healthy adults and the elderly[J].British Journal of Nutrition,2011,105(08):1235-1244.
[2]FREDRIK BACKHED,RUTH E L,JUSTIN L S,et al.Host-bacterial m utualism in the hum an intestine[J].Science,2005,307:1915-1920.
[3]O’HARA A M,SHANAHAN F.The gut flora as a forgotten organ[J].EM BO reports,2006,7(7):688-693.
[4]YATSUNENKO T,REY F E,MANARY M J,et al.Hum an gut m icrobiom e viewed across age and geography[J].Nature,2012,486(7402):222-227.
[5]ECKBURG P B,BIK E M,BERNSTEIN C N,et al.Diversity of the hum an intestinal m icrobial flora[J].Science,2005,308(5728):1635-1638.
[6]MUYZER G,DE WAAL E C,UITTERLINDEN A G.Profiling of com plex m icrobial populations by denaturing gradient gel electrophoresis analysis of polym erase chain reaction-am plified genes coding for 16S rRNA[J].Applied and Environm ental M icrobiology,1993,59(3):695-700.
[7]毛华明,马松成,陈静,等.大额牛瘤胃细菌16S rRNA基因序列分析[J].中国农业科学,2008,41(11):3769-3775.
[8]FREY J C,ROTHMAN J M,PELL A N,et al.Fecal bacterial diversity in a wild gorilla[J].Applied and Environm ental M icrobiology,2006,72(5):3788-3792.
[9]XU B,HUANG Z X,WANG X Y,et al.Phylogenetic analysis of the fecal flora of the wild pygm y loris[J].American Journal of Primatology,2010,72(8):699-706.
[10]梁冰,戚汉君.笼养川金丝猴不同年龄阶段的发育特征[J].动物学报,2001,47(4):381-387.
[11]WALTER J,HERTEL C,TANNOCK G W,et al.Detection of Lactobacillus,Pediococcus,Leuconostoc,and Weissella species in hum an feces by using groupspecific PCR prim ers and denaturing gradient gel electrophoresis[J].Applied and Environm ental M icrobiology,2001,67(6):2578-2585.
[12]倪学勤,曾东,周小秋.采用PCR-DGGE技术分析蛋鸡肠道细菌种群结构及多样性[J].畜牧兽医学报,2008,39(7):955-961.
[13]RINTTILA T,KASSINEN A,MALINEN E,et al.Developm ent of an extensive set of 16S r DNA-targeted prim ers for quantification of pathogenic and indigenous bacteria in faecal sam ples by real-tim e PCR[J].Journal of Applied M icrobiology,2004,97(6):1166-1177.
[14]HEILIG H G H J,ZOETENDAL E G,VAUGHAN E E,et al.M olecular diversity of Lactobacillus spp.and other lactic acid bacteria in the hum an intestine as determ ined by specific am plification of 16S ribosom al DNA[J].Applied and Environm ental M icrobiology,2002,68(1):114-123.
[15]WU C,YANG F,GAO R,et al.Study of fecal bacterial diversity in Yunnan snub-nosed m onkey(Rhinopithecus bieti)using phylogenetic analysis of cloned 16S rRNA gene sequences[J].African Journal of Biotechnology,2013,9(38):6278-6289.
[16]PALMER C,BIK E M,DIGIULIO D B,et al.Developm ent of the hum an infant intestinal m icrobiota[J].PLo S Biology,2007,5(7):e177.
[17]HUURRE A,KALLIOMAKI M,RAUTAVA S,et al.M ode of delivery-effects on gut m icrobiota and hum oral im m unity[J].Neonatology,2007,93(4):236-240.
[18]KOENIG J E,SPOR A,SCALFONE N,et al.Succession of m icrobial consortia in the developing infant gut m icrobiom e[J].Proceedings of the National Academ y of Sciences,2011,108(Suppl.1):4578-4585.
[19]贺锐,张翀,赵翠生,等.不同月龄婴儿肠道微生态改变的临床研究[J].国际检验医学杂志,2014(24):153-155.
[20]MITSUOKA T.Significance of dietary modulation of intestinal flora and intestinal environm ent[J].Bioscience and M icroflora,2000,19(1):15-25.
[21]SAUNIER K,DORE J.Gastrointestinal tract and the elderly:functional foods,gut m icroflora and heal thy ageing[J].Digestive and Liver Disease,2002,34:19-24.
[22]HOLT P R.Gastrointestinal disease in the elderly[J].Current Opinion in Clinical Nutrition and M etabolic Care,2003,6:41-48.
[23]PANDA A,ARIONA A,SAPEY E,et al.Human innate im m unosenescence:causes and consequences for im m unity in old age[J].Trends in Im m unology,2009,30(7):325-333.
[24]MUELLER S,SAUNIER K,HANISCH C,et al.Differences in fecal m icrobiota in different European study populations in relation to age,gender and country:a cross-sectional study[J].Applied and Environm ental M icrobiology,2006,72(2):1027-1033.
[25]BARTOSH S,FITE A,MACFARLANE G T,et al.Characterization of bacterial com m unities in feces from healthy elderly volunteers and hospitalized elderly patients by using real-tim e PCR and effects of antibiotic treatm ent on the fecal m icrobiota[J].Applied and Environm ental M icrobiology,2004,70(6):3575-3581.
[26]HOPKINS M J,SHARP R,MACFARLANE G T.Age and disease related changes in intestinal bacterial populations assessed by cell culture,16S rRNA abundance,and com m unity cellular fatty acid profiles[J].Gut,2001,48(2):198-205.
[27]TIIHONEN K,TYNKKYNEN S,OUWEHAND A,et al.The effect of ageing with and without non-steroidal anti-inflam m atory drugs on gastrointestinal m icrobiology and im m unology[J].British Journal of Nutrition,2008,100(1):130-137.
[28]MARIAT D,FIRMESSE O,LEVENEZ F,et al.The Firm icutes/Bacteroidetes ratio of the hum an m icrobiota changes with age[J].BM C M icrobiology,2009,9(1):123.
[29]BIAGI E,NYLUND L,CANDELA M,et al.Through ageing,and beyond:gut m icrobiota and inflam m atory status in seniors and centenarians[J].PLo S One,2010,5(5):e10667.
[30]STSEPETOVA J,SEPP E,KOLK H,et al.Diversity and m etabolic im pact of intestinal Lactobacillus species in healthy adults and the elderly[J].British Journal of Nutrition,2011,105(08):1235-1244.