Study On The Effect Of Intestinal Flora Changes On The Expression Of PD-1 And CD28 In CD8~+T Cells

Objective:To show that oral administration of live Clostridium butyricum could effectively regulate the colonization of intestinal symbiotic microbiota in preterm mice.And explore the possibility that the changes of intestinal colonization symbiotic microbiota may regulate the development and maturation of the immune system in preterm mice by regulating the expression of the molecular CD28 T-cell co-stimulators and co-inhibition PD-1.Methods:1.Animal model establishment:The BALB/c adult mice aged 6-8weeks were randomly selected and naturally conceived in cages with a 2:1ratio of male and female,some female mice were randomly injected intraperitoneally with mifepristone（RU486）at the 18^thand 19^thdays of pregnancy to induce premature birth,mice with gestational age less than 20days were randomly selected as the preterm birth mouse model;The neonatal mice with a gestational age greater than 20 days were randomly selected as the full-term mouse model.2.Grouping and intervention:20 preterm mice were selected and randomly divided into preterm test group and preterm control group,the full-term mouse model was randomly selected as the full-term control group,with 10 mice in each group.The preterm test group was gavaged with 10ul/g Clostridium butyricum bicombined live powder at 8o’clock in the morning from day one to day seven after birth,and the preterm control group and the full-term control group were gavaged with the same dose of normal saline at the same time,they were fed in IVC environment without any other intervention.3.Specimen retention:10neonatal mice in the three groups were randomly and respectively selected on the postnatal 14 and postnatal 21,they were anesthetized by intraperitoneal injection of 10%chloral hydrate（dose:0.3-0.4ml/100g）,and at least 100ul of peripheral blood was collected by Eyeball extraction for blood,and the blood was anticoagulated with EDTA-K2 anticoagulant tube and placed on ice for later use.P21 mice were killed by cervical dislocation after blood collection,the bodies were soaked in 75%alcohol for 20 minutes,and then the jejunum,ileum and colon parts were intercepted on the super-clean table and placed in a petri dish containing sterile PBS buffer,the intestinal contents were thoroughly rinsed with a 10ml sterile syringe and thoroughly mixed.1-2ml of intestinal contents were absorbed into a 5m L sterile tube to obtain fecal samples,which were sealed with sterile paraffin sealing film,and then quickly transferred to liquid nitrogen and frozen for24h,and placed on-80℃in biological sample storage of cryogenic refrigerator save for later use.4.The expression of microbial richness,diversity and species abundance was detected by high-throughput sequencing（this experiment was completed by the high-throughput sequencing laboratory of Hua Da gene limited company in Wuhan,Hubei province）;Flow cytometry was used to detect the percentages of CD28 and PD-1 and their mean fluorescence intensity on the surface of CD4⁺T cells of newborn mice in the three groups,and the percentages of CD28 and PD-1（PD-1）and their mean fluorescence intensity on the surface of CD8⁺T cells of newborn mice in the three groups.Finally,flow analysis software Flow Jo X was used to analyze the expressions of CD28 and PD-1.Results:1.P21,At the genus level,there was a statistically significant difference among the three groups in 24 genera,which were Firmicutes,Proteobacteria,Bacteroidetes,Verrucomicrobia and Deferribacteres,respectively（P<0.05）.2.P14,the percentage of CD3⁺CD8⁺CD28⁺in peripheral blood of neonatal mice and CD28MFI on the surface of CD3⁺CD8⁺T cells were statistically significant among the three groups（P<0.001）.The percentage of CD3⁺CD8⁺CD28⁺in the preterm control group was higher than that in the preterm test group and the term control group（P<0.05）,The percentage of CD3⁺CD8⁺CD28⁺in the preterm test group was higher than that in the term control group（P<0.05）;CD3⁺CD8⁺T cell surface CD28MFI in preterm test group was higher than that in preterm control group and term control group（P<0.05）,and CD3⁺CD8⁺T cell surface CD28MFI in preterm control group was higher than that in term control group（P<0.05）.P21,the percentages of CD3⁺CD8⁺CD28⁺in peripheral blood of neonatal mice and CD3⁺CD8⁺T cell surface CD28MFI were statistically significant among the three groups（P<0.001）,The percentages of CD3⁺CD8⁺CD28⁺in term control group were higher than those in preterm test group and preterm control group（P<0.05）;CD3⁺CD8⁺T cell surface CD28MFI in term control group was higher than that in preterm test group and preterm control group（P<0.05）,and CD3⁺CD8⁺T cell surface CD28MFI in preterm test group was higher than that in preterm control group（P<0.05）.3.P14,the percentages of CD3⁺CD8⁺PD-1 in peripheral blood and CD3⁺CD8⁺T cell surface PD-1MFI of neonatal mice were statistically significant among the three groups（P<0.001）,the preterm test group was higher than the preterm control group and the term control group（P<0.05）,and the preterm test group was higher than the term control group（P<0.05）.P21,the percentages of CD3⁺CD8⁺PD-1in peripheral blood and CD3⁺CD8⁺T cell surface PD-1MFI of neonatal mice were statistically significant among the three groups（P<0.001）,the percentages of CD3⁺CD8⁺PD-1 in the preterm test group were lower than those in the preterm control group and the term control group（P<0.05）,and the percentages of CD3⁺CD8⁺PD-1 in the term control group were lower than those in the preterm control group（P<0.05）,CD3⁺CD8⁺T cell surface PD-1MFI in term control group and preterm test group was higher than that in preterm control group（P<0.05）.Conclusion:1.Clostridium butyricum bivariate can effectively improve the richness,diversity and species abundance of intestinal microbiota in preterm mice;2.Changes in the richness,diversity and species abundance of intestinal colonization microbiota may play an important role in the development and maturation of the immune system of preterm mice through up-regulation/down-regulation of the expressions of CD28 and PD-1 in CD8⁺T cells.