The Research Of IFN-? And IL-4 Gene Methylation Of Mice Offspring Encountered Ovalbumin Exposure In Utero

Part one Effects on the immune function of mice offspring encountered ovalbumin exposure in utero[Objective] To investigate the effects on the immune function of the mice offspring exposed to ovalbumin in pregnancy.[Method] Given female mice ovalbumin(OVA)by intraperitoneal injection in the early pregnancy(EP),the middle pregnancy(MP)and the late pregnancy(LP)to detect the effects on immune function of mice offspring encountered OVA exposure during pregnancy(OEDP).5-day-old mouse spleen lymphocytes cultured with OVA in vitro,The level of IFN-? IL-4 in cultural supernatant was detected by ELISA.The CCK-8 assay was to detect the antigen specific lymphocyte proliferative responses to reflect lymphocyte sensitization.The m RNA expression of IFN-? and IL-4 was reflex by RT-q PCR.The 6-8-weeks-old pups was exposed to OVA for once and OVA-specific Ig E was detected by ELISA to reflect the OVA sensitization of adult offspring.[Results] The IFN-? and IL-4 expression levels and antigen-specific lymphocyte proliferation response of 5-day-old mice offspring of EP and MP did not change significantly,The OVA-specific Ig E that when the offspring of EP and MP were grown to 6-8 weeks after exposure to OVA once also showed no significant increase.The antigen-specific lymphocyte proliferation response of 5-day-old mice offspring of LP was enhanced.The level of IFN-? in 5-day-old mice offspring of LP was decreased,whereas the level of IL-4 was increased.The 6-8-week-old offspring of LP exposed tp OVA once showed the increased OVA-specific Ig E.[Conclusion] 1.The 5-day-old pups that exposed to OVA in LP exposed to OVA again displayed such obvious immune function changes,such as the reduced level of IFN-?,the increases level of IL-4 and antigen-specific lymphocyte reaction,suggesting that offspring were intrauterine sensitization by OVA.It was confirmed again that the sensitive period of OVA intrauterine sensitization was in the late pregnancy.2.The 6-8-week-old pups that exposed to OVA in LP once again exposed to OVA showed increased OVA-specific Ig E,suggesting the sensitization induced by OVA in utero in the LP can be extended to adulthood offspring;3.The intrauterine sensitization may be the initiating factor for offspring of allergic disease,sensitization can be extended to adulthood.Part Two The immunoreaction of adult offspring exposed to OVA during pregnancy that exposed to OVA repeatedly[Objective] To investigate the immunoreaction of adult offspring exposed to OVA during pregnancy that exposed OVA repeatedly.[Method] The 6-8-week-old female pups that exposed to OVA in pregnancy repeatedly exposed to OVA.The symptoms was observed and recorded the scores;The serum OVA-specific Ig E was detected by ELISA.The splenic lymphocytes was cultured with OVA in vitro after 48 h.The IFN-? and IL-4 in supernatant was detected by ELISA.The function of spleen lymphocytes was detected by flow cytometry.[Results] 1.The 6-8-week-old pups that exposed to OVA in different periods of pregnancy was induced different degrees of acute diarrhea after 9 repeatedly OVA exposure.The pups that exposed to OVA in EP and MP had later diarrhea with mild symptoms;those in LP appeared early diarrhea with more severe symptoms.2.All 6-8-week–old OEDP pups showed increased OVA-specific Ig E after OVA repeatedly exposure,especially high in the LP pups.3.The culture supernatant of IFN-? of all 6-8-week-old OEDP pups exhibited no differences after OVA repeatedly exposure,whereas IL-4 were increased,the ration of IFN-? / IL-4 were reduced,especially in LP pups.4.The results of flow cytometry in spleen lymphocyte function of 6-8-week-old OEDP pups that exposed to OVA repeatedly showed increased CD4+ / CD8+ ratio,especially in LP pups.[Conclusion] The 6-8-week-old offspring that encountered repeatedly OVA exposure showed that there were different degrees of diarrhea in different period of OEDP with the changed immunoreaction,including increased OVA-specific Ig E,IFN-? / IL-4 decreased,CD4+ / CD8+ T lymphocytes liter high,suggesting that all pups are OVA allergy with the most obvious changes in LP,proving once again that the period of late pregnancy is an important opportunity for intrauterine sensitization,or "window period."Part Three The DNA methylation of IFN-?& IL-4 in OEDP mice offspring[Objective] To investigate DNA methylation of IFN-?& IL-4 of spleen lymphocyte in OEDP mice offspring that OVA repeatedly exposed at 6-8 weeks old.[Method] ELISA was used to detect serum total Ig E of 6-8-week-old OEPD pups that exposed OVA repeatedly.The DNA from spleen lymphocyte with bisulfite modification was performed Pyrosequencing to detect related loci methylation of 5-day-old pups and pups The relationship between IFN-? and IL-4 gene methylation and those with total Ig E expression,symptom scores was completed by correlation analysis.[Results] All the 6-8-week-old OEPD pups after repeatedly OVA exposure showed increased total serum Ig E with the most obvious high in the l P3 group.The IFN-? and IL-4 gene related sites showed no changes in 5-day-old mice offspring.The IFN-? and IL-4 gene related sites of pups after 6-8 weeks OVA repeatedly exposure had changed.The IFN-? promoter Cp G-205,Cp G-190 site was hypermethylation.The IL-4 promoter Cp G-408,Cp G-393 site was hypomethylation.The relevant changes of DNA methylation in l P3 were significantly.Correlation analysis showed the allergic symptom severity of 6-8-week-old OEDP pups that repeatedly OVA exposure was significantly correlated with the changes of IFN-? and IL-4 gene methylation.There was no correlation between 5-day-old pups and 6-8-week-old pups repeatedly OVA exposure in IFN-? and IL-4 promoter methylation.[Conclusion] 1.The methylation of IFN-? and IL-4 promoter specific sites did not occur in the 5-day-old OEDP pups and has no relationship with the methylation changes of the IFN-? and IL-4 promoter specific site in 6-8 weeks OEDP mice offspring after repeatedly OVA exposure.2 The OEDP pups were exposed to OVA repeatedly at 6-8 weeks old,the IFN-? promoter Cp G-205,Cp G-190 sites showed hypermethylation,IL-4 promoter Cp G-408,Cp G-393 sites exhibited hypomethylation with the significant changes in l P3 offspring as the confirmed participation of epigenetic occur in OVA allergy.The severity of OVA allergy was concerned with the IFN-? and IL-4 promoter specific sites methylation.3 The relationship between IFN-? with IL-4 promoter sites methylation and the expression needs further study.