| Objective:①To establish an animal model of mice infected with Toxoplasma gondii, different dose of Toxoplasma gondii infected mice by intraperitoneal(i.p.) on the second trimester and select the appropriate dose.②Use of animal model infected with Toxoplasma gondii, investigate the concentration of placental lactogen,acetylcholine, epidermal growth factor and observed effect on material transport of placenta.③Use of animal mdel infected with Toxoplasma gondii,investigate the concentration of IGF-II and expression of 11β-hydroxysteroid dehydrogenase-2 of placenta to fetal development.④Use of animal mdel infected with Toxoplasma gondii, investigate the concentration of corticotropin-releasing hormone (CRH) of serume from maternal and expression of nitric oxide synthase (eNOS and iNOS) on palcenta.wich was belief to find out the factors of fetal abnormalities on endocrine.By experimental observation of the above indicators, does it affect the placental endocrine function of pregnant mice infected with Toxoplasma gondii toxicity,and analy the mechanism of endocrine.Methods:①Establish a mouse model infected with Toxoplasma gondii on the second trimwster: BALB/C virgin mice of weight (20-22)g on 10 weeks, was together with male miace,and then 100 of pregnant mice were randomly divided into 5 groups (A,B,C,D,E group), 20 mice in different groups.On the 8th day of pregnancy, the mice of A group were injected i.p. with 0.2 ml PBS, and B-E groups 0.2ml PBS contained respectively 25,50,100,200 tachyzoites.Pregnant mice were observed health status with vertical hair,fatigue,and other abnormalities camponotus;Pregnant mice were killed 5 in each group on 12th, 14th, 16th and 18th of pregnancy and find Toxoplasma gondii toxicity in peritoneal fluid under the microscope;Each dose group of pregnant mice were killed,and observed health status and weight of pregnant mice, live births of fetal,placental weight were observed to the toxicity of placenta.②On the base of animal modle,40 pregnant mice were distributed into two average groups randomly in the both normal control group and the infected group. On the 8th day of pregnancy, the mice of the nomal group were injected i.p. with 0.2 ml PBS and the infected group for 0.2 ml PBS contained 50 tachyzoites. 5 mice of each group were killed on the 12th, 14th, 16th and 18th day of pregnancy respectively, and collect maternal serum;The half of placenta made into homogenate and others were embedded in wax;The concentration of HPL of the palcenta was detected by radioimmunoassay (RIA),acetylcholine by Colorimetric alkaline hydroxylamine, and the expression of EGF by immunohistochemistry to observe transport of substances in the placenta.③The concentration of IGF-II was detected by ELISA and the expression of 11β-HSD2 to observe effects on fetal development with Toxoplasma gondii;④The concentration of CRH was detected by ELISA in the serumof mice and the expression of eNOS and iNOS by immunohistochemistry to observe the effect on delivery.Results:①Body weight of pregnant mice in the control group increased with days of pregnancy,and abnormal phenomenons of hair shaft,burnout, arched back was not found. Pregnant mice in the infected group had different degrees of vertical hair, fatigue, arched back of pregnant mice with the wormthe number of days of pregnancy prolongation of different degrees of vertical hair, fatigue, arched back, less active and so on,body weight was first increased and then decreased with days of pregnancy.Peritoneal fluid from pregnant mice collected or transferred species were seen to find T. gondii tachyzoites.The survival rate of fetal was up to 97% in A group,and that of B and C groups was lower than A group,but had live fetal on 12th, 14th, 16th and 18th day of pregnancy.Howere, there were no survival fetal in D and E group of late pregnancy,so they were not used to following experiment.The survival rates of fetals were commpared between B and A group and between C and A group respectively on the same days of pregnancy.The result was showed that the difference of B group on 18th of pregnancy was only significant (P<0.05);and there were significant differences on the same days of pregnancy in B group except on 12th of pregnancy. Comparing placental weight,the differences were statistically significant on the different days of pregnancy (FA=53.731, FB=16.319, FC=7.621, FD=11.336, FE=3.367, P<0.05).There was only significant differences on the same pregnant daysof 16th between A and B group and on the same pregnant days of 14th,16th and 18th between A and C group(P <0.05).②The concentration of placenta lactogen was increasing in the normal control group with the days of pregnancy, while roaring at first and decreasing later in the infected group. The concentration of acetylcholine and epidermal growth factor were decreaseing with prolonging days of pregnancy in both the control group and the infected group,but droped seriously in the infected group.On the different pregnant days, there was significant difference in the both groups (HPL:FNormal control group=3.260, FInfected group=5.573;EGF:FNormal control group=6.343 ,FInfected group=23.366;Ach:FInfected group=20.933,P<0.05), and there was no significant difference in the control group(Ach: FNormal control group =3.091, P=0.57).Comparing the means of two groups, the difference of placenta lactogen and acetylcholine was statistically significant on 16th and 18th of pregnancy (P<0.05),and the difference of expression of epidermal growth factor was statistically significant on the 14th, 16th and 18th of pregnancy(P<0.05).③The results of IGF-II test showed that the concentration was increased with the increase of the pregnant days in normal control group,but increased at first and then decreased in the infected group.On different gestational days of both groups the difference was statistically significant(FNormal control group =11.763, FInfected group =39.137,P<0.05).Compared on the same pregnant days between the two groups, the differences werestatistically significant (P <0.05). The expression of 11 -HSD2 showed that they were increased with the increase of the pregnant days in normal control group,but the trend of increasing in the infected group was less than that of normal control group.On different gestational days of normal control group the difference was statistically significant in normal control group(FNormal control group =4.319, P=0.021)and there was no difference in the infected group(FInfected group=0.313, P=0.816).Compared on the same pregnant days between the two groups, the differences were statistically significant except that on 12th of pregnant days (t12d=2.066, P=0.242; t14d=3.264, P=0.011; t16d=2.625, P=0.030;t18d=3.274,P=0.011 ).④Immunohistochemical localization of the two groups showed, eNOS was mainly expressed in placental syncytiotrophoblast cells, and vascular endothelial cells so on. iNOS mainly was expressed in the cytoplasm of macrophages on the placenta.The expression of eNOS and iNOS was increased with prolonged pregnancy in the control group, and have no significant change in the number of days,while it was significantly increased in infected group.On different days of pregnancy compared with normal control group,there was not statistically significant (eNOS: Fnormal control group=2.390, P=0.107; iNOS:Fnormal control group=0.816, P=0.504).But there were many significant difference in the infected group (eNOS:Finfected group=11.398,P <0.05; iNOS:Finfected group =610.230,P=0.000);On the same number of days of pregnancy comparison, expression of eNOS were in the 16d and 18d of pregnancy significantly (P <0.05), while expression of iNOS in the 12d, 14d, 16d, 18d were statistically significant (P <0.05). The concentration of CRH increased gradually with days of pregnancy and was(0.60 0.18)ng/ml on 16th of pregnant days in normal control group.While the concentration of CRH was (0.16 0.89)ng/ml on 14th pregnancy of days,(6.30±1.24)ng/ml on 16th pregnancy of days,and reduced to the initial level on 18th in the infected group.On different days of pregnancy,the difference was statistically significant (Fnormal control group=31.48,Finfected group=20.154, P<0.05). Comparing the two groups the same number of days of pregnancy, except for the first 12d,the remaining days of the pregnancy,the differences were statistically significant (t12d=0.763,P=0.467;t14d=2.56,P=0.018;t16d=9.580, P=0.000; t18d=3.878,P<0.05).Conclusions:①The dose of 50 Toxoplasma gondii tachyzoites was infecting mice of the second trimester because of higher survival rate of fetal and light toxicity and so on.②By the animal model of pregnant mice,the concentration of HPL,Ach and the expression of EGF were lower in the infected group than that in normal control group,which led to effect transport of substances,example glucose,amino acids and others.Insufficient supply of nutrients were given which was one of the mechanisms poor fetals development.③By the animal model of pregnant mice, the concentration of IGF-Ⅱand the expression of 11β-HSD2 were abnormal,which led to damage the glucocorticoid barrier.It may be intrauterine growth retardation of one of the reasons.④When pregnant mice infected Toxoplasma gondii, levels of eNOS and iNOS increased on the the placenta, which led to excessive production of NO.And NO could cause the increase of placental vascular resistance, result in reduced blood flow to the placenta, fetal lacked of hypoxia.It caused the concentration of CRH increased compensatorily to alleviate hypoxia of the fetus and losed that in the end.
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