| Objective:To explore the effect of live clostridium butyricum and bifidobacterium on BTLA,HVEM expression on T cell and the percent of lymphocyte subsets in peripheral blood,spleen,and small intestine Peryer pathes at different time points in preterm mice,and to provide basis for the regulation of immune imbalance in preterm infants by probiotics.Methods:To establish a sterile,preterm animal model by intraperitoneal injection of mifepristone(RU486).On the 18th day of pregnancy,pregnant mice were given intraperitoneal injection of mifepristone(0.15mg/kg,dissolved in castor oil in advance)and used for two days.Live Preterm infants born within 20 days of pregnancy were randomly divided into preterm experimental group(n=40)and the preterm control group(n=40),the non-intervented full-term mice were divided into term control group(n=40).The premature experimental group was administered via gavage with live clostridium butyricum and bifidobacterium(commercial name:Changlekang?,Clostridium butyricum live bacteria concentration 1.0*10~7 CFU/mL,Bifidobacterium concentration 1.0*10~6 CFU/m L,gavage volume was 100 ul/10 g),the preterm control group and the term control group were given gavage with the same amount of normal saline at the same time,three groups of mice were fed back after the above treatment.Every group of newborn mice were randomly selected 10 mice from postnatal day 1(P1),P7,P14,and P21 and were anesthetized with ether.The blood was taken by eyeball at least 500ul of peripheral blood,EDTA-K2 anticoagulant,placed on ice for use.Sterile spleen and small intestine Peryer pathes(PP)were used(P14 and P21 mice were selected only for PP).One-third spleen and PP were randomly harvested,and pathological HE staining was used to observe the activation of lymphocytes.Another two thirds was prepared mononuclear cells by mechanical milling.Peripheral blood,spleen and small intestine PP mononuclear cell suspension were all used to prepare lymphocyte suspension via red blood cell lysate.Flow cytometry was used to detect the expression of BTLA and HVEM on T cells,and the percentage of B,T and NK cells.Results:1.In P1,P7,P14,and P21,the weight of the mice in the preterm control group was lower than that in the term control group(P<0.05).The weight of mice in the preterm experimental group of P1 and P7 was lower than that in the term control group(P<0.05)and there was no difference between these two groups of P14 and P21(P>0.05).There was no difference between preterm experimental group and preterm control group in P1(P>0.05).Preterm experimental group in P7,P14 and P21 were all higher than those of preterm control group(P<0.05).2.The weight of spleen in P1,P7,P14 and P21 of preterm control group mice was lower than those in the term control group(P<0.05).The preterm experimental group in P1 and P7 was lower than those of the term control group(P<0.05),and there was no significant difference between groups in P14 and P21(P>0.05).Preterm experimental group in P21 was heavier than preterm control group(P<0.05).There was no difference in the spleen index between the three groups in P1,P7,P14 and P21(P>0.05).The physiological structure of the small intestine PP and spleen of newborn mice in P1,P7,P14 and P21 gradually became complete,and the proportion of lymphocyte activation gradually increased in three groups.3.The levels of CD4~+T,CD8~+T,and NK cells in the spleen and peripheral blood of newborn mice in P1 were higher in the term control group than in the preterm control group and the preterm experimental group(P<0.001).There was no difference between the preterm experimental group and preterm control group(P>0.05).There was no difference among the three groups in CD4~+/CD8~+and B cell levels(P>0.05).4.About CD4~+T cell levels of the spleen and peripheral blood in P7,small intestine PP in P14 and P21,spleen in P21,preterm experimental group was higher than the preterm control group(P<0.05).About CD4~+T cell levels of the spleen in P7,small intestine PP,spleen and peripheral blood in P14 and P21,preterm experimental group was lower than the term control group(P<0.05).The level of CD4~+T cell in peripheral blood of the preterm experimental group in P7 was higher than that of the term control group and the preterm control group(P<0.05).The level of CD8~+T cell in spleen and peripheral blood in P7,small intestine PP and spleen in P14 and P21,peripheral blood in P14,preterm experimental group was higher than the preterm control group(P<0.05).The levels of CD8~+T cell in spleen in P7 and P14,small intestine PP and peripheral blood in P14 and P21,preterm experimental group were lower than the term control group(P<0.05).The levels of CD8~+T cell in peripheral blood in P7 and spleen in P21,the preterm experimental group were higher than the term control group(P<0.05).The CD4~+/CD8~+ratio of spleen in P7 and P21,small intestine PP and peripheral blood in P14 of preterm control group was higher than the term control group and preterm experimental group(P<0.05).The CD4~+/CD8~+ratio of peripheral blood in P14 of term control group was higher than that in the preterm experimental group(P<0.05),but the CD4~+/CD8~+ratio of spleen in P14 of term control group was lower than that the preterm experimental group(P<0.05).There was no difference among three groups of small intestine PP and peripheral blood in P21(P>0.05).5.The levels of BTLA on the CD4~+T cell surface of peripheral blood in P7 and spleen in P21,preterm experimental group were lower than the term control group and the preterm control group(P<0.05).The BTLA levels of CD4~+T cells of spleen in P7 and P14,small intestine PP and peripheral blood in P14,and peripheral blood in P21 were higher in the preterm experimental group than in the term control group and the preterm control group(P<0.05).The level of BTLA on CD4~+T cells in P21 small intestine PP was higher in the preterm experimental group than that in the preterm control group(P<0.05).The levels of HVEM on CD4~+T cells of spleen in P7,P14 and P21,and peripheral blood in P14 and P21,and small intestine PP in P21 in the preterm experimental group were higher than those in the preterm control group(P<0.05),but lower than those in the term control group(P<0.05).The levels of HVEM on CD4~+T cells of peripheral blood in P7 and small intestine PP in P14 in the preterm experimental group were higher than those in the term control group and the preterm control group(P<0.05).The levels of BTLA on CD8~+T cells of peripheral blood in P7,P14 and P21,and spleen in P7 and P14 in the preterm experimental group were higher than that in the preterm control group(P<0.05),but lower than that in the term control group(P<0.05).The level of BTLA on CD8~+T cells of small intestine PP in P14 in the preterm experimental group was higher than those in the term control group and the preterm control group(P<0.05).The levels of BTLA on CD8~+T cells of small intestine PP and spleen in P21 in the preterm experimental group were lower than those in the term control group and the preterm control group(P<0.05).The levels of HVEM on CD8~+T cells of spleen and peripheral blood in P7,P14 and P21 in the preterm experimental group were higher than those in the term control group and the preterm control group(P<0.05).The levels of HVEM on CD8~+T cells of small intestine PP in P14 and P21 in the preterm experimental group were higher than those in the preterm control group(P<0.05),but lower than those in the term control group(P<0.05).6.The levels of B and NK cells in the small intestine PP,spleen and peripheral blood increased gradually after birth.The level of B cells in the preterm experimental group was higher than those in the term control group and preterm control group(P<0.05).The level of NK cells in P7 spleen was lower in the preterm experimental group than that in the term control group(P<0.05).The levels of NK cells in the P14 and P21 small intestine PP and peripheral blood in the preterm experimental group were higher than those in the term control group and the preterm control group(P<0.05).There was no difference among three groups in the levels of NK cells in the P14 and P21 spleen and P7 peripheral blood(P>0.05).Conclusions:1.Preterm mice have low innate and adaptive immunity.Live clostridium butyricum and bifidobacterium may promote the development and maturation of peripheral immune organs and immune cells in premature mice.2.Live clostridium butyricum and bifidobacterium may mediate the decrease or rise of BTLA/HVEM expression on the surface of CD4~+T and CD8~+T cells,prevent T cell over-activation or incompetence,and modulate T cells immune balance in preterm mice effectively.3.Live clostridium butyricum and bifidobacterium may improve the adaptive immunity and innate immunity by promoting the maturation of B-cells and NK cells in premature mice,and the effect is related to the length of intervention time. |
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