| Objective: To observe the development of the offspring’s palatine embryonic process,feeding pregnant mice with zinc-deficient feed,and detect the expression changes and differences of Sp5 in the development of zinc-deficient and non-zinc-deficient palate tissues,initially explore the role of Sp5 in the development of palate.Methods: The SPF grade C57BL/6 pregnant mice were randomly divided into two group,normal zinc group and zinc deficiency group.The normal zinc group and the zinc deficiency group were fed free zinc diet(zinc containing 30mg/kg)and zinc-deficient feed(zinc containing 30mg/kg)form ED0.5-ED15.5.ED12.5,3pregnant mice in both two group were selected to take whole blood to measure the changes in serum zinc concentration,and then every 3 pregnant mice were killed in both two group on ED12.5,ED13.5,ED14.5,ED15.5 respectively.The embryo heads were took out,and the head specimens were prepared,sliced and then subjected to hematoxylin-eosin(HE)staining to observe the developmental status of embryonic palatal processes of fetal mice at different stages,and immunohistochemical staining was used to detect Sp5 expression changes and differences.ED17.5,6 pregnant mice in each group were killed,the occurrence of cleft palate was observed,and the incidence of cleft palate was counted.Results:(1)ED17.5,42 fetal mice were obtained in the normal zinc group and no individuals whit cleft palate;35 in the zinc deficiency group,13 showed cleft palate,and the incidence of cleft palate was 37.1%.(2)ED12.5,the serum zinc concentrations in the normal zinc group and zinc deficiency group were 9.4±0.781μmol/L and 3.23±0.231μmol/L,respectively,which were statistically significant(P<0.05).(3)The HE staining showed: the palatal developed normally in control group,the ED12.5 and ED13.5 palatine processes grew vertically downward,the ED14.5 tongue fell and the palate process lifted,and the ED15.5 bilateral palate process was fused at the midline.Part of the remaining upper belt of the middle crest is visible;the ED12.5and ED13.5 palatal processes of the zinc deficiency group was thin,the tongue of ED14.5 is not lowered,and the palatal process cannot be lifted.Although the ED15.5palatal process is lifted up,the bilateral palatal processes cannot touch and fuse.(4)The immunohistochemical staining showed: Sp5 is expressed in the nucleus and nucleus cytoplasm of mesenchymal and epithelial cells.In the normal zinc group,Sp5 was positively expressed,and the staining of ED14.5 and ED15.5 was deeper;the zinc deficiency group SP5 was strongest expression in ED14.5,and the expression of ED15.5 was lower.The staining of each day except for ED15.5 was darker than that of the normal zinc group.The average optical density values of the two groups were significantly different in ED12.5,ED13.5,ED14.5 and ED15.5(P < 0.05).The expression of Sp5 in normal zinc group was lower in ED12.5,ED13.5,and ED14.5.Compared with zinc deficiency group,but ED15.5 expression is stronger than zinc deficiency group.The mean optical density values in the normal zinc group were different from each other(P<0.05),there was no significant difference between ED13.5 and ED15.5 in the zinc deficiency group(P>0.05),and the other pairwise comparisons are statistically significant(P<0.05).The expression of Sp5 in the normal zinc group was the strongest in ED15.5,showing a gradual upward trend;The strongest expression of Sp5 in the zinc deficiency group was in ED14.5,showing a trend of rising and then descending.Conclusion: Sp5 was expressed in mesenchymal and epithelial cells from sprout to fusion period of development of palata,the expression of Sp5 during the cleft palate of mice caused by zinc deficiency was significantly higher than that of the control group,especially stronger in the vertical growth and uplift stages.It could be assumed that Sp5 was related to the formation of cleft palate. |
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