Research On TLR2 Expression And Lung Damage In HCMV Intrauterine Infection Neonatal Mice

Objective:To establish neonatal mice lung damage model induced by HCMV intrauterine infection,observe the changes of TLR2 expression and My D88,IL-8 and IFN-?levels,and to explore relationship between lung damage in neonatal mice intrauterine infection of HCMV and TLR2 signaling pathway.Methods:1.Construction and Evaluation of Animal Model:Randomly selected 8-10 weeks old clean grade(SPF)BALB/c mice,divided into three groups: A group,B group and C group.Mice of A group were intraperitoneally injected HCMV AD169 strain suspension 0.5ml(5.0log TCID50)once,mice of B group were injected intraperitoneally with HELF cell suspension 0.5ml,and the C group was not given any injection.Seven days after injection the serum levels of HCMVIgM were detected in three groups of mice respectively.Injected with the virus and serum HCMVIgM positive mice were randomly selected 54(female: male = 2: 1),mice in B group and C group with serum HCMVIgM negative were randomly selected from each of 15(female: male = 2: 1),paired feeding and observed three groups of female mice time to pregnancy,fetal death rate of newborn mice.Half of newborn mice on the first day(within 24 hours after birth,P1)anesthesia hypothermia,extract lung tissue and serum samples.Hemi lung tissue for HCMV-IgM(gold-labeled immunosorbent assay)used to detect and pathology(HE staining),the other hemi lung tissue stored at-80 ?.2.TLR2 expression and related molecules relations: Used the first step of the neonatal mice lung tissue specimens as the research object.Group A newborn mice lung tissue that HCMV IgM were positive both in serum and lung tissue,were divided into A1(lung damage)and A2 group(no lung damage)according to the pathological results.Randomly selected group B and group C newborn mice lung tissue samples that serum HCMVIgM and lung HCMV-IgM negative and pathology confirmed no lung damage,divided into the B1 group(blank control group)and the C1 group(normal control group).Half newborn mice of A,B and C group in seventh days after birth(P2)processed like the first day.Every group(Day 1,Day 7)were detected TLR2-m RNA expression(RTq PCR)and My D88,IL-8,IFN-? levels(ELISA)in neonatal mice lung.Results:1.Seven days after injection the serum positive rate of HCMVIgM antibodies in mice of A group(85%)was significantly higher than mice of B group(7.6%)and C group(0%),the difference has statistical significance(P <0.05).After paired feeding the time of pregnancy(day)were respectively(8.7 + 1.5,6.5 + 1.1,6.4 + 0.8),average each newborn rats number(only)respectively(+ 6.5 1.3,8.0 + 1.2,7.7 + 1.1),neonatal mice serum HCMV IgM positive rate(%)respectively(53.3,4.2,3.1),neonatal mouse lung tissues of HCMV IgM positive rate(%)respectively(27.7,0,0)in three mice of female groups,the differences were statistically significant(P<0.05).The lung tissue pathological biopsy showed obvious inflammatory changes in experimental group A,not found in the other two groups.2.The lung tissue TLR2-m RNA ?Ct value on the first day were respectively 9.8�1.2,12.0�2.2,12.7�1.6,12.7�2.3,in A1 group,A2 group and B1 group and C1 group,the difference was statistically significant(P < 0.05).There was no significant difference between ?Ct values on the 7th day(P > 0.05).On the first day the levels of My D88,IL-8,IFN-? in A1 group were respectively 5.8�0.9 ng/ml,39.8�2.8pg/ml,20.3�1.8ng/L,significantly higher than the other three groups,and the difference was statistically significant(P < 0.05).The level of My D88 and IL-8 in A1 group decreased significantly in seventh day compared with the first day,and the difference was statistically significant(P < 0.05).Compared with the two control group,the differences of the levels of My D88,IL-8 and IFN-? in A2 group were not statistically significant(P > 0.05).Conclusion:Intraperitoneal injection of HCMVAD169 strain can successfully infect mice,and cause fetal rats HCMV intrauterine infection,part of the newborn mice appear lung inflammation,show that lung damage mouse model caused by HCMV intrauterine infection be successfully constructed.Lung damage in the model may be related to TLR2 high expression in lung tissue.High expression of TLR2 involve in lung damage may primarily through My D88-dependent pathway induce inflammatory factors release.