叶酸对维甲酸诱导的腭裂小鼠的腭突间充质细胞增殖影响的研究

发布时间：2019-01-22 12:16

【摘要】：背景及目的：先天性唇腭裂是人类最常见的先天性畸形之一，在世界及我国都有较高的发病率，非综合征性唇腭裂（NSCL/P），非综合征性唇腭裂（NSCL/P），是指不伴发全身其他系统的器官畸形，且不属于各种综合征的唇裂、唇裂合并腭裂的先天性发育畸形的总称，占所有唇腭裂畸形的85%-95%。叶酸为维生素B复合体之一，临床普遍采用其预防先天性非综合征性唇腭裂（NSCL/P）及脊柱裂等，但其对可能致病因素的拮抗作用的机制及量化分析尚未得到确切结论。本研究应用BALB/C小鼠作为实验动物，探索维甲酸诱导其发生腭裂的致畸条件，并检测和分析其在叶酸干预下腭裂的胎鼠EPM细胞的增殖情况，探讨叶酸作为保护因素对NSCL/P的作用机制，为叶酸用于预防NSCL/P提供一定的依据。方法： 1、在孕鼠孕期第10天（GD10）时，按孕鼠体重50mg/kg管饲维甲酸，诱导胚胎腭裂模型，GD17剖宫取胎鼠，观察腭裂情况，得出致畸率。 2、分别在GD14、GD17处死孕鼠，剖宫取胎鼠，应用不完全消化法提取GD14、17的胚胎腭突间充质细胞(EPM cells)进行培养，制取细胞爬片，对细胞角蛋白、波形蛋白进行免疫细胞化学染色，，鉴定细胞并计算EPM cells阳性率。 3、在GD14、17的胚胎腭突EPM细胞中加入叶酸浓度为10μg/mL、20μg/mL、40μg/mL、80μg/mL的培养基，对照组培养基不含叶酸。采用四甲基偶氮唑盐（3-(4,5-dimethythiazol-z-yl)-2,5-dipheny tetrazolium bromide, MTT）法检测叶酸浓度的GD14、17的胚胎腭突EPM细胞连续8天的增殖能力，绘制细胞增殖曲线，对各组的增殖能力进行比较。结果： 1、在GD10管饲孕鼠50mg/kg维甲酸可致胎鼠腭裂畸形； 2、不完全消化法提纯EMP细胞纯度达到98%； 3、GD14的EMP细胞增殖受抑制，20μg/mL、40μg/mL浓度叶酸可拮抗这种抑制作用。结论： 1、过量维甲酸可致小鼠腭裂畸形，腭突间充质细胞增殖受抑制是其致畸机制之一； 2、叶酸可以拮抗维甲酸对小鼠GD14EPM细胞增殖的抑制作用。
[Abstract]:Background and objective: congenital cleft lip and palate is one of the most common congenital deformities in human beings. There is a high incidence of non-syndromic cleft lip and palate (NSCL/P) and non-syndromic cleft lip and palate (NSCL/P) in the world and in China. It refers to the cleft lip that does not accompany other system of the whole body, and does not belong to all kinds of syndrome cleft lip, cleft lip with cleft palate congenital development malformation, account for all cleft lip and palate deformity 85-95. Folic acid, one of the vitamin B complexes, is widely used to prevent congenital non-syndromic cleft lip and palate (NSCL/P) and spina bifida in clinic. In this study, BALB/C mice were used as experimental animals to explore the teratogenic conditions of retinoic acid induced cleft palate, and to detect and analyze the proliferation of fetal EPM cells induced by folic acid. To explore the mechanism of folic acid as a protective factor on NSCL/P, and to provide some basis for folic acid to prevent NSCL/P. Methods: 1. At the 10th day of pregnancy (GD10), the fetal cleft palate model was induced by feeding retinoic acid with 50mg/kg tube according to the body weight of pregnant rats. The fetal mice were removed by GD17, and the cleft palate was observed and the teratogenic rate was obtained. 2. The pregnant rats were killed in GD14,GD17, the fetal mice were taken from the uterus, the (EPM cells) of the embryonic palatine process mesenchymal cells of GD14,17 were extracted by incomplete digestion method, and the cell climbing slices were prepared, and the cytokeratin was obtained. Vimentin was stained by immunocytochemistry to identify the cells and calculate the positive rate of EPM cells. 3. The culture medium with folic acid concentration of 10 渭 g 路mL ~ (-1) or 20 渭 g 路mL ~ (-1) or 40 渭 g / mL ~ (-1) or 80 渭 g/mL was added to EPM cells of embryonic palatal process of GD14,17. The medium of control group was free of folic acid. The proliferation of EPM cells in embryonic palatine process of folic acid concentration GD14,17 was detected by 3- (4- (4-) -dimethythiazol-yl) -2- 5-dipheny tetrazolium bromide, MTT) assay for 8 days, and the cell proliferation curve was plotted. The proliferative ability of each group was compared. Results: 1) pregnant rats fed with 50mg/kg retinoic acid in GD10 tube could cause cleft palate malformation, 2the purity of EMP cells purified by incomplete digestion was 98%. 3The proliferation of EMP cells of GD14 was inhibited, and folic acid at 40 渭 g/mL concentration of 20 渭 g / mL could antagonize the inhibition. Conclusion: 1. Excessive retinoic acid can induce cleft palate malformation in mice, and the inhibition of proliferation of mesenchymal cells in palatal process is one of its teratogenic mechanisms; 2, folic acid can antagonize the inhibitory effect of retinoic acid on the proliferation of mouse GD14EPM cells.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R782.2

【相似文献】