剂量和给药频率相关的地塞米松诱导的小鼠腭裂发生率差异与凋亡及BMP-2之间的关系

发布时间：2018-03-13 08:34

本文选题：地塞米松　切入点：腭裂　出处：《吉林大学》2014年硕士论文　论文类型：学位论文

【摘要】：研究背景：腭裂是我们在临床工作中最常见的颅面先天畸形之一，现如今已被认识到可能是由多种母亲的某些基因及所生活的环境因素所导致的，在世界各地每出生500至2500名新生儿中就有1名患儿出现唇腭裂，致病率达到0,2%-0.04%，这给患儿在日常的进食、与人交流等基本生活方面带来障碍，还会在患儿成长过程中带来极大的心理影响。在近20年的研究报告显示地塞米松是一种导致胚胎畸形较强的药物，但究竟是一次性大剂量给药产生畸形率高还是小剂量多次给药所产生的畸形率高并没有明确的解释。许多国内外学者在研究中发现地塞米松主要作用于腭突间充质细胞，在腭突发育过程中可抑制EPM细胞的增殖，但细胞的凋亡是否也与腭裂的形成存在某种关系尚不清楚，所以本实验通过建立动物模型观察不同剂量地塞米松诱导小鼠致畸率的高低及与细胞凋亡的关系。实验目的：探讨小鼠腭裂与不同剂量地塞米松的关系及在腭裂形成过程中与BMP-2和细胞凋亡的相关性。模型的建立和方法：随机选取生活环境相近并且健康的近交系小鼠，雌雄比例按2:1于晚18:00合笼饲养，所有小鼠均在光照及黑暗时间12h的同样环境下饲养。并于次日早7:30检查雌性小鼠的阴栓，见阴栓之日起为孕0d（0GD），并依次称体重作标记，于10GD上午7:30再依次称体重，若体重显著增加2g以上者可认为妊娠阳性。并取妊娠阳性小鼠随机选取45只分为三组，每组15只放于同笼饲养并做好标记分别为小剂量组于妊娠后11.5d、12.5d、13.5d连续三天腹腔注射6mg/kg地塞米松；大剂量组于妊娠后11.5d腹腔内一次性大剂量注射50mg/kg地塞米松；空白对照组孕鼠在正常情况下饲养，不给予任何特殊处理。所有小组均在14.5d以脱臼法依次处死，每组小孕鼠均在处死后立即放置于冰块上，并剖腹取出胎鼠，并观察各组孕鼠的上颚的大体形态及常规HE染色病理切片、免疫组化、透射电镜扫描。结果：各组小鼠在饲养过程中均未出现疾病或死亡现象。经过肉眼观察可得出小剂量组共怀孕175只孕鼠，其中发生腭裂胎鼠72只，未发生腭裂胎鼠103只，平均体重是0.31克，腭裂发生率是41.14%；大剂量组共怀孕103只，其中发生腭裂的胎鼠63只，未发生腭裂的胎鼠40只，平均体重是0.43克，腭裂发生率是61.17%；空白对照组共怀孕139只，其中发生腭裂的胎鼠是7只，平均体重是0.54克，腭裂发生率是5.04%，三组小鼠称重后之间进行统计学分析小鼠平均体重和腭裂发生率P 0.05，有统计学差异。经HE染色光镜下可看到腭裂胎鼠的上腭正中位置有一条纵行走向边缘不规整的裂隙，分析后可见大剂量组腭裂发生率明显高于小剂量组；经过TUNEL染色观察腭裂小鼠腭突出现大量凋亡细胞，经免疫组化观察三组BMP-2变化可看出不同剂量之间并没有明显差异，但表达情况均较空白对照组表达减少，并且在透射电镜下可见腭裂胎鼠的头部的腭突间充质细胞形态及细胞器结构与空白对照组明显不同。结论： 1.一次大剂量注射地塞米松较多次少量注射地塞米松更易导致腭裂的发生。 2.剂量依赖性的腭裂发生率变化与腭突间充质细胞的凋亡水平密切相关 3.在地塞米松引起的腭裂胎鼠腭突处，BMP-2的表达较正常发育的小鼠明显减少。 4.不同剂量地塞米松引起的腭裂腭突的BMP-2的表达未见明显差异。
[Abstract]:Background: cleft palate is the most common in the clinical work of the craniofacial congenital malformation of now has been recognized to be caused by a variety of genes and mother living environment factors, born in the world every 500 to 2500 births in 1 children with cleft lip and palate appear, pathogenicity the rate of up to 0,2%-0.04%, this to the children in the daily diet, and communicate the basic life difficult, but also bring great psychological impact on children in the growth process. In the nearly 20 years of research report that dexamethasone is a cause of fetal abnormalities strong drugs, but whether it is a single large dose of administration produce deformity rate high or low dose of medicine repeatedly to the deformity rate produced by high and there is no clear explanation. Many domestic and foreign scholars have found that dexamethasone acts mainly on palatal mesenchymal cells in the study, in the palatal hair The proliferation of EPM cells can be inhibited during the course of growth. However, whether the cell apoptosis is related to the formation of cleft palate is not clear. Therefore, the animal model was established to observe the relationship between the teratogenic rate and the apoptosis of mice induced by different doses of dexamethasone.
Objective: To investigate the relationship between cleft palate and different doses of dexamethasone and the correlation between BMP-2 and apoptosis during the formation of cleft palate.
The establishment of the model and method: randomly selected similar living environment and healthy inbred mice, male and female ratio of 2:1 in the late 18:00 cages, all the mice were raised in the same environment light and dark time 12h. And on the next day at 7:30 in the morning to check the vaginal plug in female mice, vaginal plug date pregnant 0d (0GD), and in turn the body for markers in 10GD at 7:30 in the morning and then weighed, if the body weight increased significantly more than 2G can be considered positive and positive pregnancy. Pregnant mice were randomly selected 45 rabbits were divided into three groups, 15 rats in each group to put in the same cage and make mark were small dose to pregnant 11.5d, 12.5d, 13.5d for three consecutive days intraperitoneal injection of 6mg/kg dexamethasone; high dose group in pregnancy after 11.5d intraperitoneal single dose injection of 50mg/kg dexamethasone; control group of pregnant rats reared under normal conditions, without any special treatment. All groups were killed in the order of 14.5d at the time of dislocations. Every group of small pregnant mice was placed on ice on the ice immediately after they were executed, and the fetuses were taken out of the abdominal cavity. The gross morphology of the upper jaw and routine HE staining pathological sections were observed, and immunohistochemistry and transmission electron microscope scanning were used.
Results: the mice in the breeding process were not found in the phenomenon of disease or death was observed by naked eyes. The small dose group were pregnant and 175 pregnant rats, the occurrence of cleft palate 72 rats, 103 rats without cleft palate, the average weight is 0.31 grams, the incidence of cleft palate is 41.14%; the large dose group were pregnant 103, the occurrence of fetal cleft palate 63 rats, without the occurrence of cleft palate in fetal rats was 40, the average weight is 0.43 grams, the incidence of cleft palate was 61.17%; control group were pregnant and 139, which occurred in cleft palate of fetal rats is 7, the average weight is 0.54 grams, the incidence of cleft palate was 5.04%, three mice weighing statistical analysis of incidence rate of P 0.05 and the average weight of the mice after cleft palate, there were significant differences. After HE staining under light microscope can see the fetal palate cleft palate with a longitudinal center position toward the edge of irregular fracture analysis, visible after high dose group of cleft palate The incidence rate was significantly higher than the low dose group; after TUNEL staining of palatal cleft palate mice appear a large number of apoptotic cells by immunohistochemical observation of three groups of BMP-2 changes can be seen and there is no significant difference between different dose, but the expression was compared with blank control group was decreased, and the transmission electron microscope of cleft palate mice head palate process of mesenchymal cell morphology and cell structure and blank control group were significantly different.
Conclusion:
1. a large dose of dexamethasone injected with dexamethasone a few times is more likely to cause cleft palate.
The changes in the incidence of 2. dose dependent cleft palate are closely related to the apoptosis level of the palatine interprocess mesenchymal cells
3. in the palatine process of the cleft palate mouse induced by dexamethasone, the expression of BMP-2 was significantly lower than that in the normal developing mice.
4. there was no significant difference in the expression of BMP-2 in cleft palate and palate process caused by different doses of dexamethasone.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R782.22

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