Study On The Structural Characteristics Of Intestinal Flora Of Yang - Deficiency

Objectives:Previous studies have shown that yang-deficiency exist imbalance intestinal flora and disorder of energy metabolism, lipid metabolism and immune function. However, there is no complete and accurate understanding about what have happened to the intestinal micro environment of yang-deficiency. Through the research of the microbial molecular ecology of feces samples of yang-deficiency and the normal constitution we obtained the index characteristics of flora diversity. Besides,we obtained the markers of intestinal flora that can reflect characteristics of yang-deficiency and further provide objective basis for TCM constitution with the application of multivariate statistical analysis. Based on the intestinal flora, this research is of great significance to carry out individualized diagnosis, treatment and prevention. To get the markers of the intestinal flora of yang-deficiency is another key scientific problem of this project. The balanced structure of intestinal flora will play an important role in metabolism of its host. To reveal the characteristics of Yang-deficiency from the microbial level, this project adopted the microbial molecular ecology technology, and the multivariate statistical method, with the latter screening and identifying the markers.Methods:This study has two groups,30 cases of yang-deficiency and 30 cases of the single type normal constitution in Beijing area. Regular physical examination of all the cases was required to be normal, and they should have no clear diagnosis. Besides, they were also required not to use antibiotics, gastrointestinal medicine, and microecological regulating agents at least one month before sampling. We ensured that the gender, age and other baseline data of the two groups were comparable. After that, we went to collect fresh dejection and immediately put it into the box of sterility and, then preprocess.Afterwards, we extracted and purified the total DNA of the fecal bacteria. According to the V3-V4 area of 16 s rRNA, we designed specific primers for amplification and purification of PCR.100 ng a sample,20 samples a set, in this way, we mixed the purified sample and installed the library, thus obtaining an abundant library. We used Miseq sequencer to sequence microbial diversity and, we adopt sequencing model of 2*300 according to the length of the amplified fragment (V3-V4). Then we evaluated the sequencing results, including sequence quality, quantity and the basic information of the sample such as cover depth, and OTU clustering. The results are statistically computed, for the diversity index of each sample. We have a comprehensive understanding of the evolutionary relationships and different relative abundance of all the microbes in a single sample with reference to silva databases. With the application of multivariate statistical methods, such as PCoA, we found the potential trend of classification between the two groups and the featured flora of yang-deficiency.Results:We received 2281881 high quality sequences. The results of PCoA, metastat index of diversity and other statistical analysis are as follows:1, compared with the normal constitution, abundance of intestinal flora of yang-deficiency presents a more uneven characteristics; 2, from the angle of community structure, the difference between the structure of intestinal flora of yang-deficiency and the normal constitution is in the proportion of some groups of bacteria; 3, the bacteria of significant differences between the two groups obtained through statistical methods are as follows:yang-deficiency has more Brevundimonas, Leucon-ostoc, Turicibacter, Defluviitaleaceae_incertae_sedis, Rothia, Butyricimonas, while the normal constitution has more Megasphaera, Ruminococcus, Lachnospira.Conclusions:This study shows that the difference between the intestinal flora of yang-deficiency and the normal constitution is not that great and mainly in the structure of intestinal flora.Through statistical analysis, we found some significant different bacteria between the two groups and some bacterias only different in proportion. The discovery of the characteristic flora in the Yang deficiency, has some certain significance to the study of the interaction between Yang-deficiency host and its intestinal flora in the metabolism and immune area.