| OBJECTIVE: To explore the expression pattern of transcription factor T-homeobox gene5（TBX-5）in the hearts of fetal rats whose mothers with gestational diabetes mellitus, to find outthe relationship between myocardial apoptosis and the fetal heart abnormalities caused by GDM,and elaborating the potential mechanisms of the heart developmental defect in the fetals withdiabetic mothers.METHODS:120Sprague-Dawley female rats were used in this experiment. Timed mating wascarried out by placing2female rats with one male rats in a cage overnight. The female ratswere determined by vaginal smears after pregnancy. There were114female rats succesfullypregnant, which were randomly divided into four groups: GDM group（group A）, insulin group（group B）, citric group(group C), and normal control group(group D).GroupA、B、C each has30pregnant rats, group D has24pregnant rats. There were3subgroups in each group:12days,15days, and19days after pregnant. In group A、B、C, there were10rats in each subgroup, whilein group D, there were8rats for each. Pregnant rats in group A and B were injected with STZ atbelly cavity, and subcutaneous injecting of insulin to control blood glucose when their bloodglucose exeed8.12mmol/L after injection of STZ. Rats in group C were injected with PBS in thesame position. Rats in group D were without any treatment.72hours after administration, ratswere detected blood glucose and body weight every2days. Pragnant rats in each group wereanaesthetized with chloral hydrate on12th,15th,19thdays after pregnant, and embryo heartswere collected after caesarean section. To observe pathological changes of myocardium by HEstaining, and to understand the localization and quantitative expression level byimmunohistochemistry and Western blotting assay. The expression of TBX5messengerribonucleic acid (mRNA) in heart tissues were analyzed by fluorescent real-time PolymeraseChain Reaction(qRT-PCR). We use TUNEL staining to detect myocardial apoptosis of fetal rats.RESULT:1. General growth of pregnant rats: rats of group A appear significantly diabete symptoms,suchas polydipsia, polyphagia, and loss of weight. Their average blood glucose (12.053.32mmol/l) were significantly higher than the other three groups (6.833.31mmol/l、5.650.81mmol/l、5.570.61mmol/l), P <0.01. The average blood glucose of group A and Bboth increased significantly on3thday after pregnancy, compared with group C and D, thedifferences were statistically significant, P<0.05. While on the6th,9th,12th,15th,19thdaysafter pregnancy, the blood glucose level of group B were in the normal range, with nosignificantly different among group C and D. It suggests that the glucose level of group Bwas well controled. There was no significant difference between the average glucose ofgroup C and D, P>0.05. At three time points, four groups weights showed an increase, butgroup A were lower than the other three groups,the difference were statistically significant,P <0.01. By2test, the mother rats of group A abortion rate inreased significantly than otherthree groups, with statistical difference, P <0.05.2. The general development of embryos: The liquefactive embryos,stillborn, and malformationincidences of group A were higher than the other three groups,with statistical difference, P<0.05. There was significant difference at the liquefactive embryo rate between group B andgroup D, P<0.05, while the incidences of stillborn and malformation were not statisticallydifferent between these two groups. There were varying degrees of fetal cardiacmalformations, such as cardiac hypertrophy, ventricular septal defect(VSD), expandedventricular cavity. The cardiac malformation rate of group A(18.18%) was significant higherthan the other three groups(2.21%,1.35%,1.53%), with statically significant, P<0.05. theincidence of cardiac malformation among group B, C, D were not statisticallydifferent,P>0.05.3. Semi-quantitative expression of TBX5protein:①TBX5was mainly expressed in the nucleiof myocytes. During the3time points, TBX5expressed most in12thday after pregnancy, itexpressed more or less the same in15thand19thdays after preganancy, but both weresignificantly less than it expressed in12thday. In group A,the expression of TBX5inmyocardial cells were decreased on12th,15th, and19thdays after pregnancy, and thedifference was significant compared with the other groups, P＜0.05.②In group A, theexpression of TBX5protein in malformation hearts were significant lower than the normalones, P<0.05. 4. Detecting the quantitative expression of TBX5protein in heart tissue by Western blotting:on12th,15th,19thdays after pregnant, the expression level of TBX5in group A was obviouslylower than in other groups, P<0.05. there were no significant difference among group B, C,D, P>0.05.5. The expression of TBX5mRNA in the heart of group A were significantly lower than otherthree groups on the12th,15th, and19thdays after pregnant by qRT-PCR technique(P<0.05).6. Detecting the myocardial apoptosis by TUNEL staining: on the12th,15th,19thdays afterpregnant, the number of apoptosis cardiomyocytes in group A was much more than group B,C, D, with statistically significant, P<0.05. there were no significant difference among groupB,C,D on the three time points, P>0.05.CONCLUSIONS:1. Fetus of GDM mothers have higher incidence of heart defect. Strict control of blood glucoseby insulin could prevent cardiac malformation in fetus with GDM mothers. STZ mayinfluence rats embryonic development at a certain extent,but it has no harm in heartdevelopment.2. The expression of TBX5protein in malformation hearts were significantly lower than thenormal ones. On the12th,15th,19thdays after pregnant, the expression of TBX5protein andmRNA in gestational diabetes fetal hearts were significantly decreased, suggesting thatdecreased expression of TBX5may be the potential mechanism of maternal diabetes-inducedcongenital heart defects.3. The number of apoptosis cardiomyocytes in the heart tissue of GDM mother rats wassignificantly increased, suggesting that increasing cardiomyocytes apoptosis may be involvedin the procedure of gestational dabetes-induced congenital heart abnormalities. |
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