| Objective:(1)The pregnant rats were exposed to hyperthemia from eighth to thirteenth days of gestation to explore the effects of heat exposure on the embryo,HSP70 expression and the apoptosis of placental cells during the second trimester of pregnancy.(2)Overexpression and interference expression adenovirus vector of HSP70 in SD rats were built to detect the effect of adenovirus vector on the HSP70 expression of placental tissue and plasma.(3)The HSP70 expression in pregnant rats was induced or interfered with,and then exposed to heat in the second trimester.The effects of HSP70 expression on heat exposure embryos and participating mechanism were studied.(4)High temperature and uterine hypoperfusion are important factors in the pathogenesis of FGR.The influence of heat exposure,uterine ischemia and uterine ischemia after heat exposure were observed and compared to explore the role and influence mechanism of heat exposure in the etiology of FGR.Methods:(1)Twenty-four SD pregnant rats were randomly divided into 2 groups.The control group: were fed during the whole pregnancy in(23±1?)environment;heat exposure group: In the early pregnancy,the rats were fed in(23±1?)environment,and these rats would be exposed to heat on the 8th and 14 th day of pregnancy,and then back to the(23±1?)environment.The rats' weight would be recorded on the 1st.8th,14 thand 21stday of the pregnancy.On the 21 stday,all the pregnant rats in the two groups were anaesthetized with 0.3% pentobarbital and treated with caesarean section.And 3ml blood was collected from the abdominal aorta.Measuring the number of survival,weight,sex,body length,tail length,malformation or not,placental weight and the number of stillbirths.The expression of HSF70,Bax,Bcl-2 of placentaswould be detected by Western blotting and HSP70 level in the plasma would be tested by ELISA.(2)12 pregnant SD rats were randomly divided into 3 groups.In the 8th day of the pregnancy,the rats in the control group would be injected with empty adenovirus vector.The rats in the expression group were injected with HSP70 recombinant overexpression adenovirus vector.The rats in the interference group were injected with HSP70 interference expression adenovirus vector,each dose was 2.5*109PFU/kg.Then the three groups of pregnant rats were placed in(35±1?C)temperature and then were exposed to the heat environment.All the pregnant rats were killed after 4 days of virus infection.The expression of HSP70 protein would be detected by Western blotting and the level of plasma HSP70 in the rats would be measured by ELISA to verify the effect of the expression or expression of adenovirus vector of HSP70.(3)30 SD pregnant rats were randomly divided into 3 groups.On the 8thday of pregnancy,the rats in the control group were injected with empty adenovirus vector,expression group were injected with HSP70 recombinant overexpression adenovirus vector,the interference vector group were injected with HSP70 interference expression adenovirus vector,each dose was 2.5*109 PFU/kg.Then all the rats went through heat exposure and went back to the(23±1?C)environment on the 14 th day of pregnancy.On the 21 stday,all the pregnant rats in the three groups were anaesthetized with 0.3% pentobarbital and treated with caesarean section.And 3ml blood was collected from the abdominal aorta.Measuring the number of survival,weight,sex,body length,tail length,malformation or not,placental weight and the number of stillbirths.The expression of HSF70,Bax,Bcl-2 of placentas would be detected by Western blotting and HSP70 level in the plasma would be tested by ELISA.The relationship between the development of fetal rats,the placenta and plasma HSP70 expression was analyzed.(4)36 SD pregnant rats were randomly divided into 3 groups.Heat exposure group(group ?): the rats were fed in the(23±1?)environment during the early pregnancy.and these rats would be exposed to heat on the 8th and 14 th day of pregnancy,and then back to the(23±1?)environment.The uterine ischemia group(group?): the rats were fed in the(23±1?)environment during the whole pregnancy.On the 14 thday of pregnancy,the rats went through the ligation of bilateral uterine arteriovenous.The uterine ischemia after heat exposure group(group ?): the rats were fed in the(23±1?)environment during the early pregnancy and went through heat exposure on the 8thand 14 thday of pregnancy,and ligated bilateral uterine arteriovenous.Then return to the(23±1?)environment.On the 21 st day,the pregnant rats in this group were treated with caesarean section.And 3ml blood was collected from the abdominal aorta.Measuring the number of survival,weight,sex,body length,tail length,malformation or not,placental weight and the number of stillbirths.The expression of HSF70,Bax,Bcl-2 of placentas would be detected by Western blotting and HSP70 level in the plasma would be tested by ELISA.The relationship between the development of fetal rats,the placenta and plasma HSP70 expression was analyzed.Results:(1)The first results: For the rats in the heat exposure,the weight and the maternal weight was gained less than the control group,and the differences were statistically significant(P < 0.05);The weight,body length and tail length were less than the control group,and the differences were statistically significant(P < 0.05);sex ratio is higher than the control group,the difference was statistically significant(P< 0.05);the expression of HSP70 and the ratio of Bax/Bcl-2 were higher than control group,the differences were statistically significant(P < 0.05);plasma HSP70 level is higher than the control group,there was statistical significance and the difference(P < 0.05).(2)Results of the effect of adenovirus vector: the level of HSP70 expression vector group of circulating HSP70 was higher than the control group and interference vector group,and the differences were statistically significant(P < 0.05),level of plasma HSP70 in interference of HSP70 expression was lower than the control group,there were significant differences(P < 0.05);the expression of placental HSP70 was higher than that in the control group and the interference vector group,and the differences were statistically significant(P < 0.05),the expression of placental HSP70 interference expression vector group was lower than those in the control group,therewere significant differences(P < 0.05).(3)The second result: the rats in the group of over expression of HSP70 have a lower rate of fetal mortality than those in control group and interference group,and the differences were statistically significant(P < 0.05),there was no significant difference between control group and interference group in terms of fetal mortality(P > 0.05);offspring sex ratio in the over expression group is the highest,the interference expression group has the lowest sex ratio,and the gender differences of pregnant rats among the three groups were statistically significant(P < 0.05);Placental weight of the rats in the HSP70 over expression group is higher than that of control group and interference group,and the differences were statistically significant(P < 0.05);the placental weight of the rats in the interference group is lower than that of the control group,the difference was statistically significant(P < 0.05);the levels of expression of placenta HSP70 among three groups are different: the over expression group has the highest level,the second is the control group,the interference expression group has the lowest level,and the differences among three groups were statistically significant(P < 0.05);the ratio of Bax and Bcl-2(Bax/Bcl-2)in the interference expression group was the highest among the three groups.The second is in the control group and the over expression group was the lowest,and their comparison between groups were statistically significant(P < 0.05);the plasma HSP70 level in the over expression group is lower than the control group and interference expression group,and the differences were statistically significant(P <0.05),but the difference between interference expression groups and the control group had no statistical significance(P > 0.05).(4)The third Results: the weight and maternal weight gained on the 21 st day of pregnancy in group ? was less than the other two groups,and the differences were statistically significant(P < 0.05).The differences between group ? and group ?were not statistically significant(P > 0.05);Compared to the other two groups,group? has the highest rate of fetal mortality(P < 0.05);The placental weight of the rats in group ? was much lower than that of the other two groups,and the differences were statistically significant(P < 0.05);The differences of the placental HSP70 expression level among three groups have no statistical significance(P > 0.05);theratio of Bax and Bcl-2(Bax/Bcl-2)is highest in group ?.Group ? has the second high ratio and the group ? has the lowest,and the comparison between groups was statistically significant(P < 0.05).The differences of plasma HSP70 level between group ? and group ? have no significant difference(P > 0.05),but plasma HSP70 level in group ? is higher than that in the other two groups,and the differences were statistically significant(P < 0.05).Conclusion:(1)Heat exposure(35±1?)in the second trimester of pregnancy can adversely affect the growth and development of offspring.(2)pAD-CMV-HSP70 and U6-shRNA-loop-anti-shRNA-teminator-UBC-eGFPHSP70 can be used to improve or interfere with the expression of HSP70 in placenta of pregnant rats.(3)The induction of heat exposure in pregnant rats with HSP70 high expression during the second trimester can contribute to the development of the placenta and reduction of fetal mortality.The interference of heat exposure of pregnant rats HSP70 expression in the second trimester can affect the development of the placenta(4)There is a close relationship among HSP70 expression in placenta and fetal mortality.It's assumed that HSP70 can protect placental growth and improve embryo survival rate.(5)The uterine ischemia after heat exposure in the second trimester of pregnancy may seriously affect the development of placenta and embryo.Different from the HSP70 in the cell,the increase of HSP70 level in maternal circulation will have an adverse effect on embryo development. |
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