The Effects Of Strain And Age On The Maturation,Function And Apoptosis Of In Vitro Cultured BMDC

Background:Dendritic cells(DCs)are the dominant class of antigen-presenting cells(APC)and the initiation of innate immune responses dependent on DCs.DCs sense infection or tissue damage and pathogens.Based on the the diversity of DC subtypes in maturation level and location in tissues and organs,DCs share specific functions.Therefore the diversity of DC subsets influence the types and intense of immune response.It is proved that gene-environment interactions determine the type or intense of individual's immunological response.Changes in the gene sequence can alter the immune responsiveness of an individual to infections either negatively or positively.For example,BALB/c mice bias to Th2 response comparing with C57BL/6 mice which tend to develop to Th1 response.The same species might display different response to the same antigen or infection.Our preceding research shows that neonate mice bias to Th2 immune response to RSV infection comparing to adult mice.It has been investigated that DCs affect the type of immune types and intense by the phenotype of molecule and secreting cytokines.Therefore the type and intense of immune response of different strains and ages of mice might be tightly related with the quantity and quality of DCs.In vivo,DCs are located in all the organs and tissues except for brain,but the quantity of DC is rare.It resulted that DC directly isolated from organs and tissues is not abundant to conduct an integrated experiment,especially for neonate mice.Yet the BMDC induced in vitro derived from bone marrow solved the problem and made it possible to investigate the phenotype and function of DC in neonate mice.It may also be a new immune therapy strategy of deficiency of DC quantity or function in a variety of ages especially for infants.Objective:Our investigation established the cell culture system of dendritic cell derived from bone marrow of different strains and ages of mice in vitro.Through comparative analysis of the yield,purity,surface marker,cytokines expression level as well as the capacity of BMDC to induce T cells proliferation,the study demonstrates the influence of strain,age and the absence of TLR4 on DC,providing the evidencethat these factors might affect the immune response eventually.Method:1.Design experiment grouping.Dependent on ages,C57BL/6 or BALB/c mice were divided into 3groups which include neonate,3weeks and adult.The adult mice of wide type C57BL/6 and TLR4knock-out were studied,too.2.Reform the method of cell-culture system of BMDC in vitro.First,we isolate BMSC from Tibia and femur of mice.Cells treated by Red Blood Cell Lysis Buffer were cultured in RPMI-1640 supplied with IL-4(10ng/ml)and GM-CSF(10ng/ml)for 7 days to induce the differentiation of progenitor cells.In the 7th day,the cells were stimulated with LPS(500ng/ml)for 24 hours and collected nonadherent cells.Cells were centrifuged for further detection.3.The measurant of expression level of surface molecule(MHCII,CD80,CD86)were detected by FCM,which reflects the maturation of BMDC to a certain extent.4.ELISA(enzyme linked immunosorbent assay)were performed to measure the production of TNF-?,IL-6 and IL-12 from supernatant of DC culture5.The capacity of BMDC to induce T cells proliferation: Co-cultured BMDCs with na?ve CD4+ from allogeneic mice to test the capacity of BMDC to induce T cells proliferation.6.The apoptosis of BMDC before and after LPS stimulation were performed through FCM,too.Result:1.The cell culture system of BMDC was established and reform.And the method has been applied to the neonate mice.Our date shows the yield of C57BL/6 is higher than BALB/c(p<0.05)in any age grade,but the yield of different age stages demonstrates no significant difference.The purity of all the groups shows no significant difference(p>0.05).2.The maturation of BMDC of C57BL/6 is higher than BALB/c whether before or after LPS stimulation(p<0.01)in all the age grades by the measurement of percentage of CD11c+MHCII+cells.It has been proved that the maturation DC of neonate before LPS treated In C57BL/6 mice isconsiderably subnormal than 3w or adult(p<0.01)..LPS stimulation promotes the maturation of DC in all the groups(p<0.01).In BALB/c mice,before LPS treated,the maturation of BMDC in neonate is lower than adult or 3w(p<0.01).LPS stimulation improved the maturation in adult and 3w(p<0.01),however there is no evident difference in neonate(p>0.05).The intensity of CD80 and CD86 is almost similar to the expression of MHC-II mentioned above.3.All the groups secret rare cytokines(IL-6,IL-12,TNF-?)without LPS stimulation.LPS stimulation improved the production of TNF-? in both C57BL/6 and BALB/c mice(p<0.01)in some extent,however the TNF-? secreted by C57BL/6 is much higher than BALB/c(p<0.01).In the same strain,the TNF-? secreted by neonate is significantly lower than other groups(p<0.01).The secretion of IL-6 is almost similar to TNF-?,except for the higher expression level of IL-6 in neonate mice before LPS stimulation.4.The co-culture of BMDC and naive CD4T+ cells show that the proliferation induced by mice of 3w and 8w demonstrate significant difference compared to neonate mice before LPS treated(p<0.01)in the strain of C57BL/6.After the cells treated by LPS,the capacity of DC derived from mice of 3w or 8w to induce proliferation is much stronger than the neonate mice(p<0.01).Similar to the C57BL/6 mice,the intensity induced by neonate is lower than the other groups in BALB/c mice.After LPS treated,the proliferation initiated by neonate is lower than other groups(p<0.01)and adult shows significant difference comparing to 3w mice(p<0.01).5.In the same age grades,both C57BL/6 and BALB/c mice demonstrate significant difference whether in the presence of LPS stimulation(p<0.01).LPS stimulation promotes apoptosis in all the groups especially for BALB/c(p<0.01).Before LPS treated,the apoptosis of the neonate of BALB/c mice reaches up to 40%,which is the highest in all the groups.After stimulation,the apoptosis of the neonate of BALB/c mice is up to 70% which is the highest among all the groups.6.The purity and yield of TLR4 KO mice shows no evident difference with the WT(p>0.05).Before LPS treated,the BMDC derived from TLR4 KO shows no significant difference with the WT in the maturation molecule expression,cytokines secretion as well as the capacity to induce proliferation and apoptosis(p>0.05).LPS stimulation demonstrated almost no effect on TLR4 KO mice(p>0.05).However the detection index of TLR4 KO mice mentioned above show significant difference with WT after LPS stimulation(p<0.05).Conclusion:1.The yield of BMDC shows no relationship with age,but the yield of C57BL/6 is higher than BALB/c.The age and strain almost don't affect the purity of DC.2.The strain and age impact the maturation of DC and the function of DC in immune response to LPS.C57BL/6 seems to be more mature than BALB/c.In the same strain,the maturation of neonate mice is the lowest.3.In the same age,the capacity of DC from C57BL/6 to secret cytokine is stronger than BALB/c.In the same strain,the expression level of TNF-? and IL-12 secreted by neonate derived BMDC is much dimmer than other ages.However,the neonate BALB/c secreted IL-6 and the expression of IL-6doesn't change after LPS.4.Except for neonate of BALB/c,other groups improve proliferation of T cells after LPS treated.In the strain of C57BL/6,the capacity of inducing proliferation by neonate is lower than other ages.However there are no considerate changes among 3w and adult.In BALB/c mice,the capacity of inducing proliferation by neonate of is weaker than other ages and LPS stimulation almost has no effect on it.5.BALB /c mice are sensitive to LPS than C57BL/6 mice in apoptosis.The apoptosis of neonate of BALB/c is highest whether before or after LPS treatment.6.Sharing the same genetic background,the expression of TLR4 has no effect on the differentiation of BMDC.However the expression level of TLR4 affects the phenotype,function and apoptosis of BMDC after LPS stimulation.