| Objective:Hepatocellular carcinoma,HCC,is a kind of high-mortality and short-survival histologic subtype of primary liver cancer.Transcatheter arterial chemoembolization,TACE,is the acknowledged treatment plan of first choice for non-removable liver cancer at home and abroad[23-24].In recent years,people’s cognition of intestinal flora function has gradually been strengthened and more and more evidence shows that the imbalance of intestinal microecology affects the emergence and development of liver cancer possibly through various ways like immune,metabolism and nutrition and it is closely related with etiology of liver cancer[21-22].However,it is not clear now whether intestinal flora changes after patients with hepatocellular carcinoma receive the treatment of TACE,how the change of this kind of microbiology flora will impact TACE curative effect and whether it has positive effect of the long-term survival of patients with hepatocellular carcinoma.Through using 16S rDNA sequencing technology,this experiment observes the change of intestinal flora in HCC patients after TACE treatment and provide theoretical and experimental basis for the prevention and cure of the imbalance of intestinal flora during the process of HCC patients’TACE treatment.Method:Select 30 cases of patients with primary liver cancer in first visit in The First Hospital attached to Hainan Medical University from January 2019 to 31st,December 2019.All patients in group receive TACE treatment and separately collect fresh feces once in one day before receiving TACE treatment and within 1 week after it,and fresh sample is stored in cell freezing tube and transferred into-80℃refrigerator.Use dry ice to deliver them to Guangzhou Ruibo Micro Technology CO.,LTD,which is responsible for the sequencing and analysis of 16s rDNA in the feces sample of HCC patients,the statistical analysis of flora diversity of sample of HCC patients and macro analysis of intestinal flora difference before and after TACE.Result:1.In the 60 cases of samples from 30 HCC patients,there are 54 cases of samples,in which the density reaches to the experimental requirement and 7 cases of samples,disqualified.There are 26 patients whose samples are qualified before and after TACE.The effective data are obtained after processing the data with sequencing and collected as OTUs.The OUT amount of all qualified sample is between50000-100000,showing that the species of sample are relatively abundant.2.After separately calculate Ace index,Chao1 index and Shannon index,it finds that index difference is not big before and after TACE,showing intestinal flora diversity has no distinct difference（P>0.05）but it can find that flora diversity decreases obviously.According to Chao1 index,Shannon index,different sequencing depth index of sample and Observed species,it separately establishes the rarefaction curves.Chao l curve shows that the sample curve tends to be gently in the levels of20000reads-50000reads,proving that the sequencing amount of sample can basically reflect the specie abundant degree of sample.Shannon diversified curve proves that all samples curves enter plateau in the level of 50000reads,showing that the continuous detection with existing sequencing depth can basically include all species in samples.1.To further analyze the difference of microbe species diversity in samples before and after TACE,we construct PCo A analysis to analyze the major composition difference in first three types.It finds out that there is difference among samples before and TACE but there is still similarity,which needs a further discrimination.Conduct cluster to the species composition information according to samples and the distance between species,investigate the aggregation degree and abundance difference of sample species.It finds that there is similarity in intestinal flora composition to some degree with difference still before and after TACE.2.According to the sequencing result of feces samples of HCC patients,draw bar chart with grades of“phylum”,“genus”.It detects 13 kinds of bacterium phylum and143 kinds of bacterium genus.In the level of phylum,the composition structure of each sample’s microbe abundance is majorly made up of four kinds of predominant bacteria:50.1%Bacteroidetes,29.7%Firmicutes,12.4%Proteobacteria and 1.6%Actinobacteria,which make up 93.8%of all species.3.According to the species represented by the obtained OTU from the sample sequencing of HCC patients,the bar chart is made separately about the statistics before and after TACE in the grades of“phylum”,“genus”to have a direct look the species ratio difference of different grades before and after TACE.It finds that 12kinds of bacterium phylum before TACE and 10 kinds of bacterium phylum after TACE are detected.For HCC patients after TACE,the average abundance of Proteobacteria increases（16.44%-23.66%）,and the average abundance of Bacteroidetes,Firmicutes and Actinobacteria decreases（49.75%-49.36%、29.7%-20.49%、0.50%-0.25%）.In the level of genus,there are 120 kinds of bacteria detected before TACE and 103 kinds of bacteria detected after TACE.After TACE,except for Sutterella,the bacteria genera with the decrease of abundance belong to the intestinal probiotics like Bacteroidetes and Firmicutes,and the abundance of pathogenic bacteria and conditioned pathogen like Enterobacteriaceae,Parabacteroides and Klebsiella obviously increase after TACE.Conclusion:1.For patients with hepatocellular carcinoma before and after TACE treatment,the flora diversity of intestinal flora decreases.2.For patients with hepatocellular carcinoma after TACE treatment,the relative abundance of Actinobacteria,Firmicutes and Bacteroidetes decreases and the relative abundance of Proteobacteria increases.3.In samples after TACE treatment,the probiotics in intestinal flora like Bacteroidetes,Lachnospiraceae and Faecalibacterium decrease and conditioned pathogen like Enterobacteriaceae,Parabacteroides,Klebsiella and Fusobacterium increase. |
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