| Objective:Glioma is a primary malignant tumor of central nervous system.Although it has been treated by surgery,radiotherapy and chemotherapy,its survival time is still short and its clinical prognosis is poor.Intestinal flora can affect the occurrence and development of central nervous system diseases through immune,metabolic and endocrine pathways.Immunotherapy has become a new treatment for solid tumors,but it has little effect in glioma.Therefore,this study aims to explore the characteristics of intestinal flora in patients with different levels of glioma,further detect and analyze the differences of T lymphocyte subsets in patients with different levels of glioma,and then further analyze the relationship between intestinal flora and cellular immunity in patients with glioma,in order to provide more basis for the prevention and treatment of glioma in the future.Methods:According to the inclusion and exclusion criteria,28 patients with gliomas treated in the Affiliated Hospital of Qingdao University from November 2019 to February 2022 were selected and divided into low grade glioma(LGG)and high grade glioma(HGG),including 14cases of low grade glioma and 14 cases of high grade glioma,and 12 healthy people in the same period as the control,aged between 18～65 years.Collect feces,extract and detect the DNA of feces samples by 16S r DNA amplicon sequencing method,and conduct computer sequencing based on Illumina novaseq sequencing platform.After the sequencing results are obtained,conduct splicing,quality control,OTU clustering and species annotation,and then analyze the differences of intestinal flora through dilution curve,species composition,alpha and beta diversity;Collect fasting morning venous blood,detect the number of CD3~+T,CD4~+T,CD8~+T cells and CD4~+/CD8~+ratio by flow cytometry,and analyze the difference of cellular immunity;The relationship between genus level flora difference and cellular immunity in high grade glioma was evaluated by spearson correlation coefficient.Results:16S r DNA amplicon sequencing of intestinal flora:By drawing the dilution curve,it was found that under the same sequencing depth,the number of species observed in the high grade glioma group was significantly higher than that in the control group and low grade glioma group,while the number of species observed in the low-grade glioma group was lower than that in the control group;Through the analysis of species composition at different grades,there are some differences in species composition between low grade glioma,high grade glioma and control.The species composition of high grade glioma is significantly higher than that of control,and the proportion of main flora is reduced;Species difference analysis:at the phylum level,there were differences in cyanobacteria between low grade glioma and control(P<0.05),and unidentified between high grade glioma and control.There was significant difference in bacteria(P<0.01),verrucomicrobiota and unidentified in low grade glioma and high grade glioma.There were significant differences in bacteria(P<0.05)and acidobacteriota(P<0.01);At the department level,there were differences in Enterobacteriaceae between high grade glioma and control,low grade glioma and control(P<0.05);At the genus level,there were differences in Escherichia Shigella and Ruminococcus between the low grade glioma and the control(P<0.05),Escherichia Shigella and Klebsiella between the high grade glioma and the control(P<0.05),sutterella,anaerostipes and parasutterella between the low grade glioma and the high grade glioma(P<0.05),and there were significant differences in(Clostridium)-innocum group,coprobacillus and blautla(P<0.01);Through alpha diversity analysis,there were significant differences in chao1 index and shannon index between high grade glioma and control(P<0.05),and there were significant differences in chao1 index between high grade glioma and low grade glioma(P<0.05),but there was no significant difference in different indexes between low grade glioma and control(P>0.05);Through beta diversity analysis,the high grade glioma,low grade glioma and the control are far from each other in weighted Uni Frac and unweighted Uni Frac,and there are significant differences in flora.Cellular immunity with the increase of glioma grade,CD4~+T and CD4~+/CD8~+ratio decreased gradually,while CD3~+T and CD8~+T increased gradually.One way ANOVA found that there were significant differences in CD8~+T cell level and CD4~+/CD8~+ratio among the three groups(P<0.05).Further statistical analysis found that the main difference was the significant difference between the high grade glioma and the control(P<0.05).Spearson correlation coefficient was used to evaluate the correlation between bacterial species and CD8~+T cell level and CD4~+/CD8~+ratio in the high grade glioma.It was found that bacterial Sutterella and Dialister were negatively correlated with CD8~+T cells(P<0.05),and CD4~+/CD8~+ratio was positively correlated with Dialister(P<0.05).There was no correlation between other bacteria and CD8~+T and CD4~+/CD8~+ratio.Conclusion:In the high grade glioma,the species and total number of intestinal flora increased,the main dominant flora decreased and the diversity of flora increased;The intestinal flora of patients with low grade glioma increased,the species increased,the total number of species decreased,the main dominant flora decreased,and the diversity of flora didn’t change significantly.In the high grade glioma,there was an imbalance in cellular immunity,CD8~+T cells increased and CD4~+/CD8~+ratio decreased;There was no significant difference in cellular immunity between patients with low grade glioma.There is a correlation between intestinal flora and cellular immunity in high grade glioma.In the future,it may improve cellular immune function by regulating intestinal flora,so as to provide a new method for the prevention and treatment of glioma. |
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